3D Bioprinted Respiratory Tissue Scaffolds for Disease Modelling Applications
Date
2024-07-02
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-8758-6133
Type
Thesis
Degree Level
Doctoral
Abstract
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.
Description
Keywords
Tissue engineering, bioprinting, respiratory tissue
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Biomedical Engineering
Program
Biomedical Engineering