Zhang, MiqinErickson, Ariane Elizabeth2018-11-282018-11-282018Erickson_washington_0250E_19232.pdfhttp://hdl.handle.net/1773/43090Thesis (Ph.D.)--University of Washington, 2018Biomaterial scaffolds are an essential element in tissue engineering (TE), providing an extracellular matrix (ECM) substitute for cell attachment, proliferation, and differentiation at the site of a tissue defect. Designed to support a variety of tissues, biomaterial scaffolds, once used exclusively for TE, have now emerged as a promising tool for disease modeling. Scaffolds recapitulate ECM structural cues and more accurately represent native cell behavior relative to monolayer culture. In cancer research applications, mimicking native cell behavior could result in a better understanding of mechanisms underlying tumor progression leading to development of more effective and targeted anti-metastatic therapeutics. This dissertation presents novel two-dimensional (2D) and three-dimensional (3D) chitosan-based biomaterial scaffolds for osteochondral tissue engineering and glioblastoma cancer research. Chitosan is a biocompatible, biodegradable, and non-toxic natural polymer with a proxy glycosaminoglycan structure. However, chitosan is prone to swelling and mechanical weakness. When combined with anionic polymers, cationic chitosan can form a polyelectrolyte complex (PEC) to improve mechanical stability while preserving biocompatibility. This dissertation will explore electrospinning and thermally induced phase separation (TIPS) techniques for chitosan-based scaffold fabrication, highlighting the opportunities and challenges of 2D and 3D scaffolds in mimicking native ECM. First, the development of pseudo-2D nanofiber substrates is explored using a high-throughput centrifugal electrospinning (HTP-CES) system. The HTP-CES is a high-yield production method resulting in a large number of highly aligned nanofiber samples. Compared to conventional electrospinning techniques, nanofibers produced with the HTP-CES exhibited both superior alignment and enhanced diameter uniformity. Further, the research explored nanofiber diameter tunability by varying the spinneret needle diameter, establishing a concave correlation between the needle diameter and resultant nanofiber diameter. The HTP-CES system shows potential for scaled up production of highly aligned nanofibers with tunable diameters to meet the needs of various engineering and biomedical applications. Next, highly aligned chitosan-polycaprolactone (C-PCL) nanofibers fabricated with the HTP-CES were employed to study the influence of topography and biochemistry on human glioblastoma multiforme (GBM) cell motility. GBM is a highly invasive form of brain cancer. GBM tumor recurrence and lethality are attributed to diffuse cancer cell invasion into adjacent healthy brain tissue and influenced by topographical cues associated with the brain parenchyma. In this research, we fabricated highly reproducible C-PCL nanofibers coated with hyaluronic acid (HA), a glycosaminoglycan commonly found in the brain, to mimic the structure and biochemistry of native brain tissue. We cultured human GBM-derived cells (U-87 MG) on uncoated and HA-coated C-PCL nanofibers. Elongated cell morphologies occurred along the nanofiber length on all nanofiber substrates. Regardless of coating, cells on nanofibers were more resistant to the therapeutic alkylator temozolomide (TMZ) than cells grown in adherent polystyrene plates. Cell migration captured by time lapse imaging revealed the influence of the HA coating as cells migrated the farthest and the fastest on nanofibers coated with 0.5% HA. These results indicate that HA-coated nanofibers are a promising substrate to characterize GBM migration and investigate novel anti-metastatic therapies. After evaluation of GBM motility on 2D nanofiber substrates, we investigated the influence of biomechanical cues on GBM tumor sphere progression in 3D porous scaffolds. Tumor matrix stiffness is implicated in the regulation of cell proliferation, drug resistance, and reversion to a more invasive phenotype. Understanding the relationship between stiffness and cell behavior is vital to develop appropriate in vitro tumor models. We fabricated chitosan-hyaluronic acid (CHA) polyelectrolyte complex (PEC) scaffolds with varying stiffness, encompassing healthy and tumorous brain tissue to evaluate the effect of scaffold stiffness on human glioblastoma (U-87 MG) cell behavior. After 12 days of culture, we observed larger tumor spheroids and an increased resistance to TMZ-induced cell death in scaffolds with higher stiffness. Moreover, the stiffer 8% CHA scaffolds exhibited an increase in expression of drug resistance and invasion-related genes compared to 2D monolayer culture. These results indicate that CHA scaffolds enhance tumor cell malignancy, providing a valuable in vitro microenvironment for studying tumor progression and screening anti-cancer therapies. Finally, we developed a 3D bilayer porous scaffold for osteochondral tissue regeneration. Osteochondral defects result from damage to the articular cartilage and subchondral bone. When left untreated, osteochondral defects can lead to osteoarthritis and decreased quality of life. Due to the gradient osteochondral tissue, multiphasic scaffolds in which different layers represent different microenvironments, are a promising treatment approach, yet stable joining between layers remains challenging. We fabricated a bilayer scaffold using thermally induced phase separation (TIPS) where the cartilage region was optimized for HA content and stiffness and the bone region was defined by higher stiffness and osteoconductive hydroxyapatite (HAp) content. The bilayer scaffold displayed seamless interfacial integration and a mechanical stiffness gradient similar to that in the native osteochondral microenvironment. Co-culture with chondrocyte-like (SW-1353 or mesenchymal stem cells) and osteoblast-like cells (MG63) displayed cell proliferation and invasion to the interface, along with increased expression of relevant gene markers indicating the potential of this bilayer scaffold for osteochondral tissue regeneration.application/pdfen-USnonecancer tumor modelschitosanelectrospinningglioblastomascaffoldtissue engineeringMaterials ScienceNanotechnologyMaterials science and engineeringThesis