Improvements in Pulmonary Tissue Engineering: Toward Functional Tracheal and Lung Replacements
Tissue engineering offers a uniquely powerful solution for the regeneration of damaged or diseased tissues, in the form of personalized living replacements. Building such replacements, however, requires a deep appreciation for the biological, chemical, and engineering aspects that govern their function. Currently, the generation of biomimetic trachea and lung replacements is hampered by incomplete recapitulation of certain of these critical aspects, which have been addressed through the work in this thesis. In Chapter 2, I reviewed all reported clinical applications of engineered tracheal grafts to replace long-segment, circumferential damage from 1898 to 2018. From these reports, I identified the current gold-standard in clinical tracheal replacement to be the Leuven protocol, based on patient survival and follow-up time. By collating graft-related causes of mortality, I generated a list of clinical care priorities and critical design criteria that will inform future efforts to generate engineered tracheal replacement grafts. In Chapter 3, I addressed the biomechanics of engineered tracheal grafts, which is a leading cause of tracheal graft failure. I evaluated 3D bulk mechanical properties of native and decellularized tracheas, isolating behaviors of each structural component of the trachea (cartilage, smooth muscle, and connective tissue). I then correlated mechanical deviations from native trachea, to structural changes to the extracellular matrix with decellularization treatment. Taken together, decellularized tracheal grafts possessed significantly impaired mechanical properties and protein structures compared to native, which should discourage their application clinically. In Chapter 4, I evaluated platform-specific effects on the differentiation of a novel population of pharmacologically expanded, primary basal cells (peBC). This work determined that artificial culture platforms, including air-liquid interface (ALI) and organoid cultures, impart non-physiologic artifacts on cellular response in these systems. Conversely, decellularized trachea and lung scaffolds impart region-specific differentiation cues on cultured peBC, which generate more physiologic cellular outcomes. This work represents the first published evaluation of engineered tissues by single-cell RNA sequencing (scRNAseq). In Chapter 5, I leveraged scRNAseq methodologies from Chapter 4 to evaluate SARS-CoV-2 viral infection dynamics in an ALI model of human proximal epithelium. This work identified a novel mechanism of viral entry via ciliated cells, which further elucidated a mechanism of enhanced system vulnerability on infection. In Chapter 6, I characterized the immune landscape of the postnatal developing rat lung by scRNAseq. I identified 26 distinct cell types and elucidated patterns of immune cell colonization, differentiation, and maturation. I also identified putative roles for certain immune cell types in regulating and contributing to the developing lung matrix morphology. In Chapter 7, I leveraged peBC in whole-lung engineered cultures to enhance epithelial barrier function, in co-culture with endothelium, fibroblasts, and/or pulmonary macrophages. I also further improved engineered lung culture paradigms to enhance tissue homogeneity and maturation. By scRNAseq evaluation of engineered lung epithelium, I found that peBC differentiate away from a proximal epithelial phenotype, and rather than gaining canonical distal epithelial character, gains a novel phenotype of regenerative, barrier-forming epithelium that resembles that observed in various disease states. Taken together, the work in this thesis represents significant strides toward generating functional engineered tracheal and lung replacements.