3D Bioprinted Respiratory Tissue Scaffolds for Disease Modelling Applications

Respiratory tissue engineering (RTE) aims to develop functional tissue constructs for regenerative or modelling applications by using engineering approaches. Among these approaches, the recently emerging technique of bioprinting is promising as it allows for the repeatable creation of hierarchical cell-containing structures, thus providing the ability to create functional tissue constructs/ models. However, there are still challenges in the use of this approach in RTE, primarily related to generating physiologically relevant constructs that recapitulate the complexity of native tissues. Aspects including biomaterial selection, incorporating accurate biomechanical stimuli, and providing natural biochemical signals are all different facets requiring consideration in increasing the physiological relevance of bioprinted respiratory tissues. Based on the promise of RTE, this thesis aims at developing novel in vitro respiratory tissue constructs by means of bioprinting. To address research issues in the field of RTE, four specific objectives are set in this thesis including, (1) synthesis and characterization of an optimal bioink, (2) incorporation of biomechanical stimuli mimicking the native respiratory environment, (3) incorporation of biochemical stimuli through use of a nanoparticle-controlled release system, and (4) proof of concept application of the developed constructs in disease modelling. Objective (1) involves the investigation and synthesis of bioinks from hydrogels and characterization of the bioinks in terms of mechanical properties, printability, and biocompatibility. Alginate was selected as the base material due to its lack of biotoxicity and its ability to undergo ionic cross-linking, which allows for a high degree of printability; however, alginate expresses negligible cell-adhesion motifs. As collagen type I is the primary protein found throughout the connective tissue of the respiratory tract, its addition increases biocompatibility and cell adhesion. After synthesis, rheological characterization was used to inform selection of printing parameters and printability was assessed to ensure consistent structures that closely recapitulated the design could be created. Bulk compression testing was carried out to determine the compressive modulus, while tensile testing of printed scaffolds was used for determination of the 3D printed lattice properties. These mechanical properties were compared to that of native respiratory tissues to determine similitude. Finally, human pulmonary fibroblast proliferation and viability within the materials was assessed to ensure biocompatibility. The cumulation of all of these results was then used to select the most promising alginate/collagen biomaterial for further use in creation of a respiratory tissue construct. Work then continued in Objectives (2) and (3) to increase the physiological relevance of the engineered construct through two different pathways. First, a bioreactor mimicking the pressure changes and airflow conditions of the human lung was developed and tested to determine the effect that biomechanical stimulus had on cell growth within the construct. Conditions recapitulating shallow, normal, and heavy breathing were tested to determine the effect on degradation, tensile properties, and human pulmonary fibroblast and bronchial epithelial cell proliferation and viability. These experiments provided insight into the influence of mechanical stimulus on cell growth and ECM production, with normal breathing conditions leading to an increase in cell proliferation. Second, a nanoparticle system for controlled release of growth factor was developed and tested to determine the effect of including relevant biochemical stimulus had on cell development within the bioprinted construct. For investigation into biochemical stimulus, a chitosan-coated alginate nanoparticle system was synthesized using an emulsion technique. These particles were loaded with growth factor aimed at stimulating epithelial growth. Initially, release kinetics of the particle system were tested comparing coated/uncoated and static/dynamic conditions. Rheology and printability of the bioink containing the loaded particles was tested along with tensile properties of the printed scaffolds. Finally, the bioactivity of the loaded nanoparticles was assessed to determine the functionality of the controlled release system. Although cell proliferation appeared unaffected, confocal imaging demonstrated an increase in the formation of an epithelial barrier layer. Finally, in Objectives (4) the application of the designed constructs, including both biomechanical and biochemical stimulus, in disease modelling was then investigated. The bioink used was varied slightly through the addition of gelatin and characterized accordingly in terms of rheology, mechanical properties, printability, and biological properties. Following this, structures containing human pulmonary fibroblasts and monocytes were printed before seeding with human bronchial epithelial cells. These structures were cultured at an air-liquid interface before being infected with an influenza A virus. Cell viability, metabolism, and chemokine release were measured to determine the ability of these constructs to function as a disease model. This thesis presents comprehensive work on the creation of bioprinted respiratory tissue scaffolds for disease modelling applications. This work may pave the way to improving disease modelling and therapeutic screening pathways by providing a humanized intermediary between 2D and animal models.

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