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The primary cause of death for cancer patients stems not from the primary tumor, but from the spread of the disease to distal sites. Once the cancer has become detectable past the point of origin, tumor burden in vital organs is typically too high, limiting the efficacy of current treatment options. Development of novel therapies to target disseminated cancer cells prior to the onset of extensive metastatic burden could improve patient outcomes. Focal therapy, or energy-based ablation, has been successfully used to treat solid tumors, but has yet to be effectively applied toward disseminated cells. Microporous polymer scaffolds have previously been developed to capture metastatic breast cancer cells in vivo, and have been shown to reduce tumor burden in vital organs and improve survival. The purpose of this dissertation is to further improve the therapeutic potential of these biomaterials by destroying recruited cancer cells. Chapter 1 discusses in further detail how these scaffolds recruit metastatic cancer cells and how different types of focal therapy have been used to treat solid tumors in the clinic. To demonstrate that focal therapy could be used to kill cells within the scaffold, metal disks were integrated into polymer scaffolds to serve as a heat source through induction heating for focal hyperthermia. It was shown in Chapter 2 that these modified scaffolds could be applied successfully to kill infiltrating cells in vivo. The release of cancer-specific antigens following ablation of metastatic cells could be utilized to induce an anti-cancer systemic immune response. Utilizing melanocytes because of their well-defined tumorigenic antigens, collaborative work demonstrated that lysates generated from ablation via irreversible electroporation (IRE) resulted in the greatest amount of T cell activation in vitro in comparison to focal hyperthermia or cryogenic ablation. Thus, for potential future combinatorial applications with immunotherapy, Chapter 3 investigated methods to introduce IRE to polymer scaffolds to treat disseminated cells. To accomplish this, metal mesh electrodes were integrated on either side of polymer scaffolds to create a near-uniform electric field distribution. These modified scaffolds were shown to be capable of complete ablation of infiltrating cells upon IRE in vivo in some mice, although further refinement is required to eliminate the variability in the response. The amount of antigen release from the treatment of recruited cells may be insufficient to induce a robust immune response following IRE treatment, and the local delivery of adjuvants may be required. Chapter 4 explored how polymer scaffolds could be coupled with inducible release of small molecules after electroporation. Scaffolds were modified to facilitate conjugation of inducible drug carriers and properties affecting IRE-mediated release were investigated to identify optimal conditions. In Chapter 5, polymer scaffolds were evaluated for recruitment of melanoma cells in both experimental and spontaneous metastasis models such that future studies of the application of combined focal and immunotherapy to disseminating tumor cells via biomaterial scaffolds could be performed in mouse models in which defined antigens could be used to track the efficacy of treatment. Finally, recommendations for future studies were discussed in Chapter 6. Altogether, the results of these studies represent an advance in the development of treatments that can target metastatic cancer, for which there are currently few effective therapeutic options.

Partial or complete tears of the anterior cruciate ligament (ACL) can greatly afflict quality of life and often require surgical reconstruction with autograft or allograft tissue to restore native knee biomechanical function. However, limitations exist with these treatments that include donor site pain and weakness found with autografts, and longer "ligamentization" and integration times due to the devitalization of allograft tissue. Alternatively, a tissue engineering approach has been proposed for the fabrication of patient-specific grafts that can more rapidly and completely heal after ACL reconstruction. Electrospun micro-fiber networks have been widely utilized as biomaterial scaffolds to support the growth and differentiation of mesenchymal stem cells toward many tissue lineages including ligament. However, these micro-fiber networks do not possess suitable sizes and shapes for a ligament application and cannot support cell infiltration. The objective of this work was to develop techniques to 1) rapidly cellularize micro-fiber networks, 2) assemble micro-fiber networks into cylindrical composites, 3) provide cues to mesenchymal stem cells (MSCs) to guide their differentiation toward a ligament phenotype. The cellularization of micro-fiber networks was performed utilizing a co-electrospinning/electrospraying technique. Cells deposited within a cell culture medium solution remained where they were deposited and did not proliferate. The inclusion of space-filling hydrogel network such as collagen was necessary to reduce the density of the micro-fiber network to facilitate spreading. However, it became apparent that the incorporation of significant collagen phase was necessary for long-term MSC survival within the micro-fiber network. Next, two approaches were developed to fabricate large cylindrical, composites. The first approach utilized a co-electrospinning/electrospraying technique to generate micro-fiber/collagen composites that were subsequently rolled into cylinders. These cylindrical composites exhibited greater diameters and water weight percentages as collagen content increased. However, the high micro-fiber content of these composites was inhibitory to cell survival. In the second approach, thin layers (~5-10 fibers) of aligned electrospun PEUR fibers were encapsulated within a collagen gel and subsequently rolled the composites into cylinders. These sparse-fiber composites were nearly 98% by weight water and confocal imaging revealed the presence of sparse fiber layers (~5 fibers thick) separated by approximately 200 μm thick collagen layers. We hypothesize that the proliferation and migration of MSCs within these micro-fiber/collagen composites may not be restricted by the presence of a dense, non-manipulatable electrospun fiber network present in traditionally rolled fiber composites. Simple model platforms were then developed to study the influence of sparse micro-fibers on MSCs differentiation within a collagen hydrogel. MSCs in the presence of the softest (5.6 MPa) micro-fibers elongated and oriented to the underlying network and exhibited greater expression of scleraxis, and α-smooth muscle actin compared to the stiffest (31 MPa) fibers. Additionally, preliminary results revealed that the incorporation of fibroblast growth factor-2 and growth and differentiation factor-5 onto micro-fibers through chemical conjugation enhanced expression of the ligamentous markers collagen I, scleraxis, and tenomodulin. In conclusion, micro-fiber/collagen composite materials must possess sufficient space to support the infiltration and differentiation of MSCs. The strategies described in this document could be combined to fabricate large, micro-fiber/collagen composites that can support cell infiltration and provide relevant cues to guide the formation of an engineered ligament tissue.