Date of Award
PhD (Doctor of Philosophy)
Professor Sally-Ann Cryan
Professor Fergal J. O'Brien
This work is funded under the PRTLI Cycle 5 & co-funded through the European Regional Development Fund, part of the European Union Structural Funds Programme 2007-2013
Respiratory Tissue Engineering, Collagen, Biomaterials, Bilayered, Epithelial, Co-Culture
Today, lung disease and major airway trauma are a significant global healthcare concern with limited treatment options. Incurable airway diseases such as asthma, chronic obstructive pulmonary disease, cystic fibrosis and idiopathic pulmonary fibrosis contribute to an enormous clinical and socioeconomic burden. At the core of many of these debilitating conditions, epithelial cell dysfunction and persistent inflammatory damage to respiratory tissue play a central role in their pathophysiology. In order to identify new therapies that can cure these diseases and repair or replace damaged tissue, physiologically-representative in vitro models must be developed for improved drug development, in addition to new surgical interventions for extensive lung tissue injury. Tissue engineering strategies have the potential to provide such complex in vitro models as well as next generation biocompatible tissue replacement treatments.
The overall goal of this PhD project was to develop a novel tissue-engineered 3D in vitro model of the tracheobronchial region with potential applications in respiratory drug development and respiratory tissue regeneration. Specifically, this thesis sought to investigate the potential of collagen-glycosaminoglycan (CG) scaffolds as a 3D substrate for the growth and differentiation of a bronchial epithelial cell line and to develop a novel bilayered CG scaffold as an in vitro co-culture model for both a bronchial epithelial cell line and primary tracheobronchial epithelial cells. A final objective was to manufacture an all-trans retinoic acid-eluting bilayered scaffold as a platform technology for tracheal tissue regeneration.
This thesis initially investigated the ability of a fully-porous collagen-chondroitin-6-sulphate scaffold to support the growth and differentiation of the Calu-3 epithelial cell line under two sets of respiratory culture conditions: air-liquid interface (ALI) culture and liquid-liquid interface (LLI) culture. Scaffolds not only supported cell growth, but also had a direct influence on increasing epithelial mucin secretion when compared to culture on standard polymeric cell inserts at an ALI. The scaffold was verified as a suitable substrate for a novel tracheobronchial in vitro model, although the formation of a robust ciliated epithelial barrier was not possible on the porous biomaterial. Accordingly, the thesis next focused on the manufacture of a bilayered scaffold structure that mimicked tracheobronchial tissue architecture and composition. This bilayered collagen-hyaluronate (CHyA-B) scaffold was composed of a thin, densely-packed film top-layer for epithelial monolayer culture and a porous submucosal layer for 3D co-culture with lung fibroblasts. The scaffold design succeeded in resolving the major limitation of the fully-porous biomaterial by facilitating the formation of a confluent and continuous Calu-3 cell monolayer with suitable epithelial barrier integrity. Furthermore, this cell barrier was ciliated, pseudostratified in morphology and maintained enhanced mucin secretion, with organotypic localisation above a submucosal analogue of co-cultured fibroblasts and scaffold.
This study validated the CHyA-B scaffold as an innovative platform technology to generate a physiologically-representative 3D tracheobronchial in vitro model. In order to apply this novel 3D culture system as an organotypic physiological representation of the tracheobronchial region, the next stage of the project progressed to using a primary tracheobronchial epithelial cell co-culture with lung fibroblasts. As well as supporting Calu-3 epithelial cells, the CHyA-B scaffold also supported the growth and differentiation of primary tracheobronchial epithelial cells in the successful development of an organotypic 3D co-culture model with the formation of a ciliated pseudostratified epithelium that secreted mucus and exhibited a physiologically-relevant barrier integrity.
Having developed the CHyA-B scaffold as a 3D in vitro co-culture model with primary epithelial cells and lung fibroblasts, the final study in this thesis investigated the potential of the scaffold as a platform technology for tracheal tissue regeneration. For this application, all-trans retinoic acid (atRA) was incorporated into the film layer of the scaffold as a potential enhancer of rapid functional epithelialisation of the CHyA-B scaffold that is critical for tracheal implants. This atRA-CHyA-B scaffold was successfully manufactured and displayed stable retention of the drug in the film layer prior to its release in physiological buffer. The drug-loaded film layer of this scaffold enhanced mucociliary gene expression of tracheobronchial epithelial cells and with future studies, the atRA-CHyA-B scaffold can potentially pioneer the development of a novel and biocompatible device to address a currently unmet clinical need in tracheal replacement.
In conclusion, this thesis has successfully developed a bilayered collagen-glycosaminoglycan scaffold with applications in both airway modelling and tissue regeneration. This scaffold holds potential as a biofabricated template that provides a physiologically-relevant 3D in vitro model to develop novel therapeutics, perform toxicological analysis of inhalable formulations and generate more sophisticated disease models for understanding and treating respiratory disease. Finally, this scaffold can also be applied as a novel technology with enhanced functional epithelialisation for tracheal tissue regeneration as an advanced medical device that can potentially overcome the limitations of current tracheal implants.
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O'Leary C. The Development of Bilayered Collagen-GAG Scaffolds for Applications in Airway Modelling and Tissue Regeneration [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2016.
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