The aims of this research were to investigate polymer fibre morphology, overall mat morphology, mechanical properties and general handling of the mats, and ideal mat thickness in order to fabricate a suitable substrate for potential use in cell transplantation for application as a permanent substrate for the treatment of dry age-related macular degeneration (AMD). Polystyrene (PS), poly(ethylene terephthalate) (PET) and polyurethane (PU) were electrospun to ascertain the ideal electrospinning parameters to reproducibly obtain fibres to construct a mat as a potential candidate for a replacement Bruch’s membrane (BM). After identifying the ideal spinning parameters, mats were fabricated, their fibre morphology, overall mat morphology, and handling during processing were examined. This allowed the shortlisting of PS and PET substrates, which were suitable to be taken forward for further testing and cell culture. PU was found to be unsuitable as it had a tendency to become entwined and stick to itself, which would destroy the gross mat morphology. Therefore PU was excluded from further testing. Further handling, both quantitative and qualitative, and thickness and porosity were tested for PS and PET mats. Electrospun PET demonstrated greater handling and durability properties compared to PS mats, which were more fragile. PET was able to withstand twisting, folding, and rolling, whereas PS could not undergo twisting and fell apart. PS mats were thicker and more porous compared to PET mats, which was attributed to the widely spaced placement of the larger PS fibres and the fluffy gross morphology of the PS mats, in comparison to the closer fibre placement of the smaller PET mats which had a smooth gross mat morphology. Considering this, PS mats were compressed and thickness and porosity was reduced, while maintaining its fibrous structure. However the compressed PS mats became extremely fragile and could not withstand much handling. Although PET mats were thinner than PS mats, it did not match the native BM thickness and so experiments in varying collection time during electrospinning to match the native BM thickness were undertaken. Tensile tests, thickness and porosity measurements showed that PET tensile properties, thickness, and porosity reduced with reduced collection time. For the purposes of surface treatment and cell culture, uncompressed mats collected for 60 minutes were used since sufficient PS fibres were able to be collected to form a mat that was able to withstand processing at this collection time. Effect of UV/ozone surface treatment was tested for both PS and PET mats. Treatment of both substrate types affected protein adsorption, with evidence of aminolysis observed on PET substrates. Short-term initial growth and survival of retinal pigment epithelial cells (RPE cells) on electrospun, surface oxidised PS and PET was investigated. Untreated PS did not support cell proliferation and although treated PS did, the resultant RPE cell morphology was undesirable, therefore was not taken forward to long term cell culture. Treated and untreated PET supported cell proliferation, and was taken forward to the long term culture study, where cells exhibited the desired monolayer morphology. In this work it has been demonstrated that electrospun PET may potentially be a suitable candidate as cell carrier substrate for subsequent implantation in application towards AMD treatment.

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Electrospinning has provided the opportunity to create extracellular matrix (ECM) mimicking scaffolds for the development of tissue-engineered constructs. Within Professor Kristen Cardinal’s Blood Vessel Mimic (BVM) Lab, at Cal Poly, there exists a constant demand for innovation and the expansion of polymer types and electrospinning capabilities for its BVM model. Along these lines, the BVM Lab has recently acquired two new electrospinning systems: the Spinbox, a commercially graded electrospinning system, and the Learn-By-Doing system, which was part of a recently completed thesis conducted by Jason Provol. Additionally, recently published literature has demonstrated polyvinyl alcohol (PVA) as a viable option for creating electrospun scaffolds in the nanometer range. These findings prompt interest in investigating this polymer type due to its potential for producing extremely thin fiber diameters. Therefore, the overall objective of this thesis was to enhance the electrospinning capabilities of the BVM Lab through the utilization of the water-soluble polymer, PVA and to comprehensively compare the three available electrospinning systems within the BVM Lab, for novel tissue engineering or classroom applications. The work performed in this thesis was structured around three main Aims. The first Aim of this thesis was to demonstrate the feasibility of using PVA to create flatsheet scaffolds using the Spinbox system. To achieve this, different PVA types with varying degrees of hydrolysis (DH) and molecular weight (MW) were spun to determine the most suitable PVA formulation. These experiments revealed that PVA with low DH and ultra-high MW was the most suitable for electrospinning. Subsequently, a formal Design of Experiments (DOE) was conducted to determine an effective parameter combination for Spinbox flatsheets. The DOE yielded a parameter combination with a voltage of 27 kV, a flow rate of 0.50 ml/hr, a gap distance of 17 cm, and a weight percentage of 10%. The selection of a PVA formulation with appropriate parameters in Aim 1 established the groundwork for accomplishing the objectives of Aim 2. Aim 2 sought to extend PVA’s electrospinning utility to other collector geometries across all three of the BVM lab’s electrospinning systems, while also comparing the usability, safety, and adjustability of each system relative to one another. This was the first time all 3 systems were directly compared. The results from Aim 2 demonstrated the reproducibility of tubular scaffolds on both the Custom and Spinbox systems, featuring nanoscale fibrous scaffolds, as well as on the LBD system with flatsheets. Furthermore, a qualitative comparison of the systems indicated that the Spinbox exhibited the highest degree of adjustability and safety among the electrospinners, albeit with the lowest relative degree of usability. Conversely, the LBD system demonstrated the highest usability or intuitiveness, while also being the most hazardous and least adjustable of the systems. The Custom system ranked in the middle for all three metrics. Finally, the successful creation of tubular PVA scaffolds led to Aim 3 of this thesis, which focused on evaluating the potential of PVA scaffolds in a bioreactor environment for research applications and devising an accessible classroom PVA protocol for teaching applications. To accomplish this final aim, the scaffolds produced in Aim 2 were characterized and evaluated based on their solubility in cell-media. Additionally, methods for enhancing water-resistance through methanol cross-linking were explored and assessed. The results indicated that cross-linking PVA with methanol could enhance water resistance, but additional treatment would be necessary for PVA to serve as a standalone vascular scaffold in BVMs. However, a PVA Lab protocol was successfully developed to facilitate classroom education, providing a tangible and immediately impactful outcome of this thesis.

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