Engineered in vitro models for studying pulmonary fibrosis and infectious disease
fibrosis resulting in scar formation can be caused by exposure to aerosolized
environmental contaminants, radiotherapy, chemotherapy and infectious disease (e.g.,
Tuberculosis). Idiopathic Pulmonary Fibrosis (IPF) is a particularly severe form of
pulmonary fibrosis of unknown etiology and has a median life expectancy of 3 years after
diagnosis. Treatments for IPF are limited ... read more(pirfenidone and nintedanib) and most lead
candidate drugs identified in pre-clinical animal studies have failed human clinical
trials. While animal models are useful for delineating fibrotic disease pathways, they
do not accurately represent human IPF disease. Significantly, animal models (e.g.,
bleomycin) of fibrosis do not reproduce human fibroblastic foci (Hum-FF) formation,
widely accepted as the pathological hallmark of human IPF disease. Human fibroblastic
foci (Hum-FF) are important prognostic markers and both FDA approved treatments
primarily target signaling pathways associated with myofibroblast cells found within
these sites. Hence, replicating Hum-FF using model systems has the potential to improve
our understanding of human IPF disease progression and treatment. Collagen-type I
hydrogels, polyacrylamide hydrogels and fibrosis-on-chip systems have been used to model
Hum-FF formation. However, current iterations of these systems are unable to
successfully replicate the 3D complexity and biochemical composition of Hum-FF tissue.
Thus, there is a need for advanced engineered in vitro models recapitulating 3D Hum-FF
morphology with the capacity to apply biomechanical stimuli and mechanical tunability.
We hypothesized that an engineered 3D fibroblastic focus (Eng-FF) model generated using
silk fibroin dityrosine crosslinked hydrogels seeded with human pulmonary cells in a
bioreactor would provide suitable tissue systems to model Hum-FF. The current
dissertation evaluated silk dityrosine crosslinking in the presence of dopants (e.g.,
phenol red) towards establishing key variables, parameters required for 3D pulmonary
cell encapsulation. Results from these studies showed that tyrosine containing amino
acids and dopants including phenol red covalently crosslinked with the silk hydrogel
framework, modulating crosslinking reaction times and hydrogel elastic moduli. The
conclusion of this study marked identification of key parameters required for pulmonary
cell encapsulation/culture and the fabrication of cytocompatible phenol-red tyrosine
crosslinked hydrogels that could be used for in vitro pH sensing applications.
Collagen-type I is the major ECM protein found within mature Hum-FF and dityrosine
crosslinked silk-collagen-type I hydrogels were fabricated towards modeling Hum-FF.
Characterization of pulmonary fibroblast encapsulated silk-collagen-type I hydrogel
systems showed reduced fibroblast mediated collagen contraction compared to
collagen-type I hydrogels, providing stable substrates for long-term in vitro culture of
pulmonary cells. Silk-collagen-type I hydrogel systems exhibited superior mechanical
tunability with the capacity to represent distinct normal and fibrotic disease states.
Using customized Flexcell Tissue Train bioreactors and cellular seeding regime the
Hum-FF pathology was successfully reproduced, and a thick stromal pulmonary fibroblast
layer separated airway epithelial and microvascular endothelial layers. Fibroblasts and
collagen fibers within Eng-FF tissues underwent parallel alignment with increase in
culture period, accurately replicating Hum-FF cellular morphology. Eng-FF tissues can be
used to model myofibroblast differentiation and long-term cultures resulted in a
proto-myofibroblast phenotype and robust myofibroblast differentiation was induced by
exogenous TGF-β1 cytokine. Pirfenidone abrogated TGF-β1 induced
myofibroblast differentiation better than nintedanib at tested concentrations and Eng-FF
tissues supported evaluation of myofibroblast phenotype following anti-fibrotic drug
treatments. Eng-FF tissues could be used to model different facets of IPF disease and
proof-of-concept studies showed replication of epithelial injury with the facile
addition of bleomycin, and cellular recruitment to the FF could be studied by the
perfusion of cells through the hydrogel microchannel. Towards future work, preliminary
results were obtained towards fabricating silk-ECM hydrogels incorporating lung ECM
proteins from normal and pathological disease states including ECM proteins from human
IPF lungs and murine tuberculosis granulomas. In summary, the current dissertation
evaluated tyrosine crosslinking of dopants and ECM proteins with silk fibroin proteins
towards creating in vitro culture systems for modeling normal lung physiology and
disease. The dissertation affirms the feasibility of utilizing silk-ECM hydrogels for
modeling lung disease with the successful creation of a 3D in vitro engineered
fibroblastic focus model using human cells that reproduces Hum-FF pathology and cellular
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Biomedical Engineering.
Advisor: David Kaplan.
Committee: Gordana Vunjak-Novakovic, Lauren Black III, Gillian Beamer, and Bree Aldridge.
Keywords: Biomedical engineering, Bioengineering, and Biology.read less