%0 PDF %T Engineered in vitro models for studying pulmonary fibrosis and infectious disease %A Sundarakrishnan, Aswin. %D 2018-10-09T07:38:27.197-04:00 %8 2018-10-09 %R http://localhost/files/d504rx79v %X Abstract: Pulmonary 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 (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 phenotype.; 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. %[ 2022-10-11 %9 Text %~ Tufts Digital Library %W Institution