The prevalence and devastating nature of cardiovascular diseases has led to many advancements in the therapies used to treat the millions of patients that suffer as a result of these conditions. As coronary artery disease (CAD) is the most common of these cardiovascular conditions, it is a major focus of research among the medical industry. Although lifestyle changes and drug therapies can treat early CAD, more advanced cases often require more definitive interventions. In conjunction with angioplasty, stenting of an occluded vessel has shown significant success in preventing restenosis. However, as with nearly every therapeutic process in the medical field, several complications have arisen in stented patients that pose a need for further improvement of the devices. As a result, the stent industry is constantly striving towards improving the characteristics and outcome of their product and with these efforts comes the need for extensive testing and research. Continuous improvement and innovation in the field of tissue engineering has brought about the possibility of creating laboratory grown tissue engineered vascular grafts (TEVGs) for the purpose of replacing and/or bypassing damaged or occluded regions of the vasculature. By employing the techniques used to produce TEVGs, a blood vessel mimic (BVM) bioreactor system has been developed with the intent of using the resulting construct as a model for testing the cellular response of a human blood vessel to an intravascular device such as a stent. This would allow gathering of more significant data in the early stages of device development and may reduce the overall costs and time required to refine a design. Although the BVM system has previously been used to cultivate viable constructs that were subsequently used to observe the response to a deployed stent, the flow conditions within the original design are not representative of the physiologic conditions in a native vessel. This aspect of the original system presented a need for development in order to be considered by researchers as an accurate in vitro representation of the target vessels in which the stents are used. One of the primary concerns of this environment is creating and maintaining physiologic flow conditions that will represent those present in native vessels in order to facilitate cells sodded on the construct to grow as they would under native conditions. The two key aspects of flow are pulsatility and wall shear stress. Studies in this thesis were carried out to determine the best and most feasible methods for implementing appropriate levels of pulsation and wall shear stress in the previously established BVM bioreactor system with the intention of maintaining the original system’s simplicity and high throughput potential. Pulsatile flow was created by elevating backpressure in the BVM chamber while using a different pump head and pump tubing. Wall shear stress was adjusted by altering the viscosity of the perfusate and flow rate through the system. Both pulsatile flow and shear stress were established without any major changes to the overall configuration of the system. Pulsatile pressures of ~80 mmHg and wall shear stress forces of ~6.4 dyn/cm2 were established with minimal alteration to the original system. Pulsatility was created by using a 3-roller peristaltic pump head in place of the originally specified 8-roller head to create pulses that were then regulated with backpressure created by restricting down stream flow. Increasing the viscosity and corresponding flow rate allowed for instigation and control of wall shear stress at the inner wall of the BVM graft. Although the resulting protocols presented here require refinement for ultimately successful implementation, they are important underpinnings that will facilitate the eventual development of an ideal BVM system that is highly suitable for use as a high-throughput intravascular device testing model.

Introduction: Diabetes Mellitus is a metabolic disorder that affects a person’s ability to either produce insulin (Type I diabetes mellitus) or properly use insulin (Type II diabetes mellitus) in order to maintain adequate blood glucose levels. The most severe diabetic complications arise due to hyperglycemia – a state of extremely high blood glucose levels – such as, coronary artery disease (CAD), in which coronary stent therapy is a popular method of treatment. However, research has shown a high rate of in-stent restenosis in diabetic patients with CAD, most likely due to activation of cellular adhesion molecules on endothelial cells exposed to the hyperglycemic environment. Blood vessel mimics (BVMs) have been researched as viable options for in vitro studies on vascular stents; thus, it would be beneficial to create an in vitro diabetic BVM for stent manufactures to evaluate and determine the root cause of the high failure rate of stents in the diabetic population. In addition, a diabetic BVM would help manufactures optimize coatings or stent configurations for diabetic patients. Methods: The purpose of this thesis was to take the initial steps towards the goal of a diabetic BVM. The first aim was to establish a procedure of developing glycemic cell media solutions of various glucose concentrations, and to establish a feasible method of monitoring the glucose concentration of the solutions. Glycemic cell media solutions were developed and their glucose concentrations were evaluated with a blood glucose meter (specifically, the Aviva Accu-Chek blood glucose meter) or visual blood glucose test strips (Glucoflex R visual blood glucose test strips). The second aim was to ensure that the developed glycemic cell media solutions could be monitored in a cell culture environment over time, and to determine if the hyperglycemic conditions induced any change to endothelial cells. Bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) were used to evaluate glucose consumption and cell morphology. Glucose concentration of the cell media was recorded to evaluate glucose consumption, and the cells were evaluated under a microscope in order to determine cell morphology and an increase in cell death. Results & Conclusions: Data accumulated from the first set of experiments confirmed that glycemic cell media solutions can be developed by adding Sigma G6512 D-(+)-glucose to base cell media. Additionally, the Aviva Accu-Chek blood glucose meter recorded the most accurate and precise glucose concentrations of the various glycemic cell media solutions compared to the Glucoflex-R blood glucose visual test strips. Lastly, the series of experiments with BAECs and HUVECs confirmed that the glycemic cell media solutions could be effectively monitored over time, and that these conditions evoked higher glucose consumption by the endothelial cells compared to the normal glycemic cell media solutions. Additionally, neither glycemic environment evoked significant cell death. These results met the aims of this thesis, and therefore provide the foundation for further development of a diabetic BVM.

Intravascular devices, such as stents, must be rigorously tested before they can be approved by the FDA. This includes bench top in vitro testing to determine biocompatibility, and animal model testing to ensure safety and efficacy. As an intermediate step, a blood vessel mimic (BVM) testing method has been developed that mimics the three dimensional structure of blood vessels using a perfusion bioreactor system, human derived endothelial cells, and a biocompatible polymer scaffold used to support growth of the blood vessel cells. The focus of this thesis was to find an in-house fabrication method capable of making cellular scaffolding for use in the BVM. Research was conducted based on three aims. The first aim was to survey possible fabrication methods to choose a technique most appropriate for producing BVM scaffolding. The second aim was to set up the selected fabrication method (electrospinning) in-house at Cal Poly and gain understanding of the process. The third aim was to evaluate consistency of the technique. The work described in this thesis determined that electrospinning is a viable fabrication technique for producing scaffolding for BVM use. Electrospun scaffolding is highly tailorable, and a structure that mimics the natural organization of nano sized collagen fibers is especially desirable when culturing endothelial cells. An electrospinning apparatus was constructed in house and a series of trial experiments was conducted to better understand the electrospinning process. A consistency study evaluated scaffold reproducibility between different spins and within individual spins while setting a baseline that can be used for comparison in future work aimed at electrospinning.

Coronary artery disease affects millions of people and the ability to detect and treat the disease is advancing at a rapid rate. As a result, the development of intravascular technologies is the focus of many medical device manufacturers. Specifically, coronary stent implantation is being performed in an increasing number of patients and a number of new stent designs have been introduced to the market, resulting in the need for improved preclinical testing methods. An in vitro tissue engineered “blood vessel mimic” (BVM) system has previously been established and its feasibility for the initial testing of newly emerging intravascular technology has been demonstrated. There are limitations that exist with this original design, however, and the focus of this thesis was to both improve and expand upon the original model. Therefore, research was conducted based on two specific aims. The first aim was to develop a more ideal BVM system to accommodate a wider range of stent lengths and diameters, while allowing for easy graft insertion and seal-ability. The second aim was to develop next generation BVM systems,focused on future needs and technology, such as long, angulated and bifurcated geometries. The work described in this thesis demonstrates that a BVM chamber can be created which has the advantages of easy graft insertion and seal-ability, as well as the ability to accommodate varying sizes of vessel scaffolds, all while maintaining the needs of a tissue engineering bioreactor system. The next generation BVM systems presented demonstrate that the BVM concept can be expanded to meet the needs of long, angulated and bifurcated geometries. Overall, the work in this thesis describes the design and optimization of an in vitro blood vessel mimic bioreactor system for the evaluation of intravascular devices, specifically coronary stents, in simple and complex vessel geometries.

Cerebral aneurysms occur due to the ballooning of blood vessels in the brain. Rupture of aneurysms can cause a subarachnoid hemorrhage, which, if not fatal, can cause permanent neurologic deficits. Minimally invasive neurovascular devices, such as embolization coils and flow diverters, are methods of treatment utilized to prevent aneurysm rupture. The rapidly growing market for neurovascular devices necessitates the development of accurate aneurysm models for preclinical testing. In vivo models, such as the rabbit elastase model, are commonly chosen for preclinical device testing; however, these studies are expensive, and aneurysm geometries are difficult to control and often do not replicate the variety of geometries found in clinical cases. A promising alternative for preclinical testing of neurovascular devices is an aneurysm blood vessel mimic (aBVM), which is an in vitro tissue-engineered model of a human blood vessel composed of an electrospun scaffold with an aneurysm geometry and human vascular cells. Previous work in the Cal Poly Tissue Engineering Lab has established a process for creating different aneurysm scaffolds based on the shape of different geometries, and this work aimed to further advance these aneurysm geometries in order to enhance the versatility of the in vitro model. The overall goal of this thesis was to customize the aBVM model through variations of different dimensions and to validate the scaffold variations for neurovascular device testing. First, a literature review was performed to identify critical ranges of aneurysm neck diameters and heights that are commonly seen in rabbit elastase models and in human clinical settings in order to set a foundation for creating new geometries. Based on the results, aneurysm geometries with varying neck sizes and heights were modeled and molded, and scaffolds were fabricated through electrospinning. Methods were developed to characterize scaffolds with internal measurements through imaging techniques using a scanning electron microscope. To validate these scaffolds for use as aBVMs for neurovascular device testing, constructs were created by dual-sodding human endothelial cells and smooth muscle cells into scaffolds with varying neck sizes. Finally, flow diverters were deployed in constructs with varying neck sizes in order to evaluate feasibility and initial healing. Customized aneurysm scaffolds can eventually be used with a variety of device studies for screening of neurovascular devices or as a predecessor for in vivo preclinical testing.

No / MicroRNAs are a relatively new class of small, noncoding RNA species that represent a cornerstone of cell biology, with diverse roles ranging from embryonic development to aging. miRNAs function to regulate posttranscriptional gene expression, are critical to the normal function of cells, and as such are frequently dysregulated during disease processes. In this chapter, we discuss the biogenesis and mechanism of action of miRNA and their role in cancer initiation, promotion, and progression. In addition, we discuss the most recently identified dual roles of miRNA in epigenetic gene regulation; how they are both regulators and regulated. Finally, we discuss the emerging roles of miRNA as epigenetic anti-cancer therapeutics, the current research examining inhibition of oncogenic miRNAs, and studies now establishing the potential of replacing lost, tumor-suppressive miRNA.

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Material jetting (MJ) is an additive manufacturing (AM) process that involves the selective jetting of a liquid material into the shape of a layer and subsequent solidification, often via ultraviolet (UV) irradiation, in a layer-wise fashion. The MJ process has the potential to emerge as a robust fabrication method: the inherent, facile, multi-material capability in a high-resolution process should distinguish the technology as a competitive, multi-functional, manufacturing process. However, it is mainly constrained to prototyping use, limited by both material and process constraints. This research expands material and process knowledge by characterizing the multi-material process-structure-property relationships in photopolymer-based MJ, which provides a basis for advancing the capability of MJ to fabricate accurate and consistent multi-material parts for functional applications. One of the challenges for advancing MJ is the general lack of processable materials. For example, MJ is increasingly being used for fabricating anatomic models for use as pre-procedural planning or medical student trainee tools, but commercial MJ elastomers are unable to mimic human tissues' mechanical properties, which limits the instructional value of printed anatomic models. By combining photo-curing and non-curing materials, a cardiac tissue-mimicking material was achieved and integrated into a fully-printed heart model used to practice the transseptal puncture procedure. Several mechanical properties of this multi-material combination were evaluated to facilitate quicker screening of future tissues that would be desired to be mimicked. Also impeding technological advancement of MJ systems is a lack of understanding the effects of indiscriminate UV exposure on material properties. Depending on factors such as part design and build layout, an indiscriminate UV toolpathing strategy poses the risk for providing inconsistent UV dosing to parts and causing unintended variations in mechanical performance. Experiments were conducted to quantify these effects, and an empirical model was developed to predict the accumulated exposure parts receive. A connection was then made between accumulated exposure received by material voxels and final part properties, where it was observed that overexposure effects exist, and are largely dependent on material, build layout, and toolpathing. This work will lead to improved design guidelines and process modifications to ensure consistency of UV dosing and achieve desired mechanical performance. This knowledge will enable future photopolymer AM systems to account for potential overcuring effects toward fabricating repeatable and reproducible functional products. Finally, documented in this work are efforts toward expanding the knowledge about the use of AM to safely produce personal protective equipment during the COVID-19 pandemic. Amid prospects of large-scale, distributed production of respirators via AM, the lack of filtration efficiency testing generated concerns about the respirators' effectiveness. The goal of this work was to measure particle transmission through respirators fabricated with powder bed fusion and fused filament fabrication processes and compare their performance to that of cloth masks and standardized N95 respirators. Through systematic post-processing, the connection between printed respirator deficiencies and changes in filtration efficiency were discerned. Identifying the system-level quality control challenges responsible for the respirator failure modes highlights some the current limitations in AM for fabricating functional parts. The findings will assist future efforts toward both creating enhanced designs and optimizing printer parameters, ultimately working toward qualifiable, end-use parts. / Doctor of Philosophy / The material jetting (MJ) additive manufacturing (AM) process operates in a similar fashion to inkjet printing. For MJ of photopolymer materials, liquid droplets are selectively deposited onto a build plate, and an ultraviolet (UV) light bulb provides the energy to solidify the droplets into a three-dimensional layer by curing the materials. Droplets are then deposited on top of these solidified droplets to fabricate a part layer by layer. Multiple materials and colors can be jetted simultaneously within a single part layer. If these materials exhibit different mechanical behavior, such as one material being rigid and another being flexible, a printed part could have regions with different material properties, as well as intermediate gradients of these properties. The MJ process offers high resolution, smooth surface finishing, a large build volume, and the opportunity to print multiple parts in one build. However, the process is mainly limited to prototypes and non-functional applications. One of the challenges for advancing MJ is the general lack of processable materials. In the medical field, surgeons are increasingly looking to MJ to fabricate physical, patient-specific models to assist in pre-surgical planning and to serve as practice models for medical student trainees. In particular, a printed cardiovascular model was sought to enable the practice of the transseptal puncture procedure; however, the available materials were not able to mimic the heart tissue. In this work, a non-curing liquid was patterned into an elastomer to soften the material and attain tissue-mimicking performance for a model to practice the transseptal puncture procedure. By characterizing this expanded material space, this work enables the potential for mimicking a broader spectrum of tissues in future anatomic models. Another aspect limiting widespread functional use for MJ is the lack of understanding how UV exposure affects material performance. For the MJ process, the UV light is on the same assembly as the printheads and remains on throughout the duration of a print, which means that the amount of administered energy is not consistent across the build plate. If, for example, parts have different heights, the shorter part will finish printing first and receive excess UV exposure, which has been shown to alter the mechanical performance for some materials. A model was developed to predict the accumulated exposure received by parts of different materials and build scenarios. Observed changes in mechanical properties could then be connected to specific instances of overexposure. With this knowledge, future strategies can be implemented to achieve consistency of UV exposure and thus better ensure reliable, functional parts. Additionally presented in this work is a study involving the use of AM to safely produce personal protective equipment for COVID-19 relief efforts. During the initial stages of the pandemic, AM was sought to address respirator shortages; however, there were no studies measuring printed respirators' effectiveness. By measuring particle transmission through respirators fabricated with a variety of AM processes, it was found that even when N95 filters were inserted, printed respirators were not able to consistently filter 95% of virus-sized particles, even with modifications. The quality control challenges for the AM processes identified in this study will assist future efforts in part design and printer parameter optimization to work toward accurate and qualifiable products.