Engineering Tunable Synthetic Biomaterials for Mechanistic and Therapeutic Lymphatic Vasculature Applications

Lymphatic vasculature pervades the human body and is responsible for extracellular fluid homeostasis, immune cell trafficking, and inflammatory responses. Despite the critical functions of this complex and nearly ubiquitous system, much remains to be understood. Discovery of the uniquely expressed receptors vascular endothelial growth factor receptor-3 (VEGFR-3) and lymphatic vessel hyaluronan endothelial receptor-1 (LYVE-1) in 1996 and 1999, respectively, enabled the identification of this secondary circulatory system, distinct from the blood circulatory system. Dysfunction of the lymphatic system is associated with a wide spectrum of disease states, including progression of neurodegenerative diseases, cardiovascular disease, metabolic syndrome, lymphedema, cancer metastasis, and wound healing. Despite an exponential increase in lymphatic-related research, the field is still nascent and limited understanding of development and disease has constrained the discovery and creation of therapeutic interventions for conditions related to lymphatic dysfunction. Controlling lymphangiogenesis, the formation of new lymphatic vessels, exhibits potential as a novel therapeutic strategy for the treatment of lymphatic-related conditions and could provide the first-long term treatment, replacing current treatments which are limited in effectiveness and longevity of relief. While this approach has widely been postulated as a novel treatment, its exploration has remained very limited, partly due to the unavailability of controllable matrix environments, challenges associated with 3-dimensional (3D) lymphatic vessel morphogenesis, and an incomplete understanding of the regulatory processes governing lymphangiogenesis.

To bridge this knowledge gap, the main goal of this dissertation was to develop a physiologically relevant, advanced model of lymphatic development for in vitro mechanistic investigations and in vivo regenerative medicine applications. Hyaluronic acid (HA) provides a unique advantage as a material for lymphatic tissue engineering applications due to LYVE-1 on LECs being a receptor for HA, and it was selected as the biomaterial of choice for the hydrogel-based models developed in this dissertation. HA-based hydrogels were used to investigate the role of mechanical and biochemical cues in regulating lymphatic vessel formation within a synthetic matrix, and to evaluate their potential for in vivo functionality to progress towards a viable lymphatic vessel transplant therapy. Both 2.5D and 3D models were developed with the ability to precisely tune mechanical properties of the matrix and pro-lymphangiogenic growth factors were incorporated. In the 3D model, parameters including polymer composition, crosslinking modality and degree, and peptide patterning added additional methods to tune lymphatic morphogenesis. This HA-based system preserved the expression of key lymphatic markers and demonstrated that matrix stiffness primes lymphatic tube formation. The individual contributions of matrix and biochemical factors were investigated, demonstrating the functionality of this system to probe specific pathways which are traditionally very difficult to study in animal models. Within two to three days after encapsulation within this synthetic matrix, self-assembled lymphatic tubes are observed. These human cell-laden matrices were also implanted into immunodeficient mice and demonstrate the ability to survive, integrate with the host vasculature, and continue to grow. Collectively, the studies presented in this dissertation present a novel system that can be used for both in vitro mechanistic insights and various approaches in tissue engineering.