Theses and Dissertations

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Open Access
A nanoparticulate-reinforced hyaluronan copolymer hydrogel for intervertebral disc repair
(Colorado State University. Libraries, 2011) Yonemura, Susan S., author; James, Susan P., advisor; Bailey, Travis S., committee member; Kisiday, John D., committee member; Wheeler, Donna L., committee member
Degenerative disc disease (DDD) is an inevitable consequence of aging, commonly resulting in low back pain (LBP). Current clinical treatment options for disc degeneration exist at two extremes: conservative management or extensive surgical intervention. Given the economic impact of lost productivity and disability associated with low back pain, there is significant interest in earlier, less invasive intervention. Biomimetic disc replacement and regenerative therapies offer an attractive alternative strategy for intervertebral disc repair, but materials employed to date have not exhibited a successful combination of mechanical and biological properties to achieve viable solutions. The composite material developed and characterized in this work consisted of a novel hyaluronan-co-poly(ethylene-alt-maleic anhydride) (HA-co-PEMA) hydrogel matrix reinforced with nanoparticulate silica; the hydrogel matrix provided a compliant hydrated matrix conducive to integration with the surrounding tissue while the nanoparticulate reinforcement was manipulated to mimic the mechanical performance of healthy ovine nucleus pulposus (NP) tissue. HA-co-PEMA was formed via an esterification reaction between a hydrophobically-modified HA complex and PEMA, and candidate formulations were characterized by chemical, thermal, and physical means to select an appropriate base hydrogel for the reinforced composite. Three grades of commercially-available fumed silica, varying by degree of hydrophobic surface modification, were evaluated as nanoparticulate reinforcement for the composite materials. Mechanical testing of two reinforced composite formulations (620-R and 720-R) emphasized dynamic shear properties and results were directly compared to ovine nucleus pulposus (NP) tissue. The complex shear modulus (G*) for 620-R ranged from 1.8±0.2 KPa to 2.4±0.3 KPa over the frequency range 0.1 Hz < f < 10 Hz, while G* for 720-R varied from 4.4 ± 0.5 KPa to 6.1 ± 0.6 KPa over the same frequency range. Ovine NP tissue tested using identical methods exhibited G* of 1.7 ± 0.2 KPa at 0.1 Hz up to 3.8 ± 0.5 KPa at 10 Hz. Thus, the complex shear moduli (G*) for 620-R and 720-R effectively bracketed G* for NP over a physiologically-relevant frequency range. Subsequent in vitro cytotoxicity and biocompatibility experiments suggest that the 720-R formulation warrants consideration for future in vivo modeling.
Open Access
Effects of walking speed on knee joint loading estimated via musculoskeletal modeling
(Colorado State University. Libraries, 2012) Haight, Derek Joseph, author; Browning, Ray, advisor; Reiser, Raoul, committee member; Puttlitz, Christian, committee member; Greene, David, committee member
Walking is the most common form of physical activity and is assumed to incur a relatively small risk of musculoskeletal injury. However, walking related- musculoskeletal injuries, particularly at the knee joint, are not uncommon in individuals who walk for exercise. Surprisingly, there is scant data regarding how walking conditions (e.g. speed, grade, surface) affect loads (i.e. contact forces) across lower extremity joints. Studies to date have used proxy measures of joint loading, primarily net muscle moments (NMM); however the validity of these proxy measures to estimate joint contact forces (JCF) is not well established. The purpose of this study was to estimate knee JCFs during slow, moderate and fast walking and to examine the validity of NMMs to estimate JCFs. We hypothesized that both knee JCFs and sagittal plane NMMs would increase with walking speed, but that the increases in NMMs would be much greater than the increases in axial JCFs. We collected kinematic and kinetic data as ten adults (mass = 67.2 (12.0) kg, mean (SD)) walked on a dual-belt force measuring treadmill at 0.75, 1.25, and 1.50 m•s-1. An OpenSim three-dimensional musculoskeletal model with 23 degrees of freedom and 92 muscle actuators was scaled to each subject. We calculated NMMs and muscle forces via inverse dynamics and static optimization, respectively, for 5 gait cycles per subject at each speed. We determined knee JCFs from the vector sum of the joint reaction force and individual muscle forces crossing the knee joint, in the tibial reference frame. During weight acceptance in early stance, axial and anterior-posterior knee JCFs increased by ~30% and 175%, respectively as walking speed increased from 0.75 m•s-1 to 1.50 m•s-1. At the same point in the gait cycle, peak sagittal plane extensor NMM increased by over 200% (P<0.001) as speed increased. The modest differences in axial knee JCFs with walking speed, suggest that slower speeds may not reduce joint loading substantially. Additionally, our results suggest that NMMs are not a good proxy measure of axial JCFs and that detailed musculoskeletal models should be used to quantify the effects of walking conditions on joint loading.
Open Access
Titania nanotube arrays: interfaces for implantable devices
(Colorado State University. Libraries, 2012) Smith, Barbara Symie, author; Popat, Ketul, advisor; Gonzalez-Juarrero, Mercedes, committee member; Prasad, Ashok, committee member; Dasi, Lakshmi Prasad, committee member; Dow, Steven, committee member
For the 8-10% of Americans (20-25 million people) that have implanted biomedical devices, biomaterial failure and the need for revision surgery are critical concerns. The major causes for failure in implantable biomedical devices promoting a need for re- implantation and revision surgery include thrombosis, post-operative infection, immune driven fibrosis and biomechanical failure. The successful integration of long-term implantable devices is highly dependent on the early events of tissue/biomaterial interaction, promoting either implant rejection or a wound healing response (extracellular matrix production and vasculature). Favorable interactions between the implant surface and the respective tissue are critical for the long-term success of any implantable device. Recent studies have shown that material surfaces which mimic the natural physiological hierarchy of in vivo tissue may provide a possible solution for enhancing biomaterial integration, thus preventing infection and biomaterial rejection. Titania nanotube arrays, fabricated using a simple anodization technique, provide a template capable of promoting altered cellular functionality at a hierarchy similar to that of natural tissue. This work focuses on the fabrication of immobilized, vertically oriented and highly uniform titania nanotube arrays to determine how this specific nano-architecture affects skin cell functionality, hemocompatibility, thrombogenicity and the immune response. The results in this work identify enhanced dermal matrix production, altered hemocompatibility, reduced thrombogenicity and a deterred immune response on titania nanotube arrays. This evidences promising implications with respect to the use of titania nanotube arrays as beneficial interfaces for the successful implantation of biomedical devices.
Open Access
Compartmentalization of membrane proteins by the actin cytoskeleton
(Colorado State University. Libraries, 2013) Higgins, Jenny, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Bamburg, James, committee member; Azimi-Sadjadi, Mahmood, committee member
Acting as the point of contact for the outside world, the plasma membrane is crucial for cellular signaling events. Proper organization of membrane components is necessary to accomplish this task. Although a number of experiments have demonstrated the compartmentalization of lipids and proteins on the plasma membrane, direct observation of the mechanisms by which the organization occurs has been challenging, in part due to the imaging restrictions of a diffraction-limited system and the dynamic nature of the membrane compartmentalization. Using photoactivated localization microscopy (PALM), a superresolution technique, we have captured the dynamics of compartments formed by the cortical actin cytoskeleton. Live human embryonic kidney (HEK293) cells were imaged with a temporal resolution of 2 s and a spatial resolution of 40 nm. The actin cytoskeleton forms compartments with a mean area of 2.3±0.3 μm2 that are partially outlined by actin bundles. When the PALM images of actin were combined with single particle tracking of membrane proteins, we directly observed the cytoskeleton acting as a barrier to the diffusion of Kv2.1 and Kv1.4, two voltage-gated potassium channels. In addition, we used a novel compartment detection and tracking algorithm to show that Kv2.1 and Kv1.4 channels avoid actin when changing compartments. This work represents the first direct observations of individual membrane protein interactions with barriers formed by the actin cytoskeleton.
Open Access
A Haversian bone model of fracture healing in a simulated microgravity environment
(Colorado State University. Libraries, 2015) Gadomski, Benjamin C., author; Puttlitz, Christian M., advisor; Browning, Raymond, committee member; Donahue, Tammy, committee member; Heyliger, Paul, committee member
Ground-based models of weightlessness and microgravity have provided valuable insights into how dynamic physiological systems adapt or react to reduced loading. Almost all of these models have used rodent hindlimb unloading as the means to simulate microgravity on isolated physiological systems. Unfortunately, results derived from rodent studies are significantly limited when one tries to translate them to the human condition due to significant anatomical and physiological differences between the two species. Therefore, it is clear that a novel animal model of ground-based weightlessness that is directly translatable to the human condition must be developed in order for substantial progress to be made in the knowledge of how microgravity affects fracture healing. In light of this, four specific aims are proposed: (1) develop a ground-based, ovine model of skeletal unloading in order to simulate weightlessness, (2) interrogate the effects of the simulated microgravity environment on bone fracture healing using this large animal model, (3) develop a computational model of weightbearing in ovine bone under different experimental conditions in order to characterize the loads experienced by the fracture site, and (4) develop countermeasures that enhance bone fracture healing in the presence of simulated microgravity. Successful completion of this project will substantially elevate the understanding of how fracture site loading affects the subsequent healing cascade in the presence of microgravity and will form the foundation for designing future rehabilitation protocols to facilitate bone healing during long-duration spaceflight.
Open Access
Influence of adipose-derived mesenchymal stromal cells on osteosarcoma minimal residual disease
(Colorado State University. Libraries, 2015) Aanstoos-Ewen, Megan, author; Ehrhart, Nicole, advisor; Kipper, Matthew, committee member; Dow, Steven, committee member; Custis, James, committee member
Introduction: Mesenchymal stromal cells (MSCs) have been shown to improve bone integration and healing in several preclinical studies and have therapeutic potential in limb salvage following massive bone loss due to tumor resection. However, MSCs have also been shown to promote primary and pulmonary metastatic tumor growth when injected in the presence of gross tumor or when co-injected with tumor cells in rodent models. While these results raise concerns about the safety of using MSCs in sarcoma patients, MSCs are unlikely to be utilized in a clinical setting when gross tumor is present. The objective of this dissertation project was to develop murine models of minimal residual osteosarcoma following primary tumor removal then to utilize these models to determine whether the administration of adipose-derived MSCs with or without chemotherapy treatment in a minimal residual disease setting would promote either pulmonary metastatic osteosarcoma progression or local disease recurrence. We hypothesized that surgical site or intravenous administration of MSCs will influence either osteosarcoma pulmonary metastatic burden or local disease recurrence in a minimal residual disease setting. Materials & Methods: Two syngeneic, orthotopic models of luciferase-expressing osteosarcoma were developed. In the first model, tumor-bearing mice underwent a coxofemoral amputation and were followed to assess development of pulmonary metastases. In the second model, a femorotibial amputation was performed in order to develop a model of consistent local tumor recurrence. In this model, all gross tumor was removed, however, microscopic tumor remained at the surgical margin. In this dissertation project, three principle projects were completed to test our hypothesis. The first project explored the use of MSCs delivered either to the surgical site or intravenously to ascertain their influence on pulmonary disease burden. A follow-on pilot explored concurrent MSC and chemotherapy treatment on development of pulmonary disease. The second project evaluated the use of MSCs delivered either to the surgical site or intravenously on local recurrence of osteosarcoma at the surgical site. Gross recurrent tumor size was measured for comparison between treatment groups. The third project examined the use of cisplatin and MSCs on survival of mice following removal of primary osteosarcoma. Data were expressed in mean +/- SD or median with 95% CI. ANOVA test, Kruskal-Wallis test, Fisher’s Exact test, Welch’s test, t-test, and Mann Whitney test were used for statistical analysis. Significance was set at p<0.05. Results: Mice treated with intravenous MSCs had a faster time to first pulmonary metastatic disease detection than mice treated with MSCs injected into the surgical site or control mice (no MSCs) (p=0.022). No treatment effect was seen between groups with respect to time to tumor recurrence or size of recurrent tumor in the second study. Survival curves were significantly different when comparing cisplatin, cisplatin and MSC treatment, MSC alone treatment and untreated mice (p<0.001) as well as in pairwise comparisons. Mice treated with MSCs had a 73% chance of earlier death than untreated controls. Discussion/Conclusion: Intravenous administration of MSCs in a minimal residual osteosarcoma environment resulted in a faster time to first detection of pulmonary disease and in a higher chance of earlier death compared to untreated mice. However, administration of MSCs locally in a surgical site following sarcoma excision appears to be safe, even in the setting of known residual microscopic disease. Further, the use of cisplatin treatment appeared to ameliorate the effects of intravenous MSCs on survival. Based on these results, further study is warranted to evaluate the influence of intravenously administered MSCs on minimal residual pulmonary metastatic disease.
Open Access
Constitutive modeling of the biaxial mechanics of brain white matter
(Colorado State University. Libraries, 2016) Labus, Kevin M., author; Puttlitz, Christian M., advisor; Donahue, Seth, committee member; Heyliger, Paul, committee member; James, Susan, committee member
It is important to characterize the mechanical behavior of brain tissue to aid in the computational models used for simulated neurosurgery. Due to its anisotropy, it is of particular interest to develop constitutive models of white matter based on experimental data in order to define the material properties in computational models. White matter has been shown to exhibit anisotropic, hyperelastic, and viscoelastic properties. The majority of studies have focused on the shear or compressive properties, while few have tested the tensile properties of the brain. Brain tissue has not previously been tested in a multi-axial loading state, even though in vivo brain tissue is in a constant multi-axial stress state due to fluid pressure, and data from uniaxial experiments do not sufficiently describe multi-axial stresses. The main objective of this project was to characterize the biaxial tensile behavior of brain white matter via experimentation and constitutive modeling. A biaxial experiment was developed specifically for testing brain tissue. Experiments were performed at a quasi-static loading rate, and an Ogden anisotropic hyperelastic model was derived to fit the data. A structural analysis was performed on biaxially tested specimens to relate the structure to the mechanical behavior. The axonal orientation and distribution were measured via histology, and the axon area fraction was measured via transmission electron microscopy. The measured structural parameters were incorporated into the constitutive model. A probabilistic analysis was performed to compare the uncertainty in the stress predictions between models with and without structural parameters. Finally, dynamic biaxial experiments were performed to characterize the anisotropic viscoelastic properties of white matter. Biaxial stress-relaxation experiments were conducted to determine the appropriate form of a viscoelastic model. It was found that the data were accurately modeled by a quasi-linear viscoelastic formulation with an isotropic reduced relaxation tensor and an instantaneous elastic stress defined by an anisotropic Ogden model. Model fits to the stress-relaxation experiments were able to accurately predict the results of dynamic cyclic experiments. The resulting constitutive models from this project build upon previous models of brain white matter mechanics to include biaxial interactions and structural relations, thus improving computational model predictions.
Open Access
The kinetics of proteins on lipid bilayers
(Colorado State University. Libraries, 2017) Nepal, Kanti, author; Krapf, Diego, advisor; Peersen, Olve, committee member; Levinger, Nancy, committee member
Signaling molecules trigger downstream signaling pathways when they arrive at the plasma membrane. They have to be recruited to the plasma membrane by membrane targeting domains. Our experiments throughout focus on understanding kinetics of C2 domain's diffusion on the membrane. In contrast to trans-membrane proteins, interactions between these domains and the plasma membrane is found to be peripheral and transient. These proteins perform two dimensional diffusion on membrane surfaces and faster three dimensional diffusion in the bulk. We label proteins at the single molecule level and do single particle tracking. In addition to two dimensional surface diffusion, it is sometimes observed that they dissociate from the membrane and rebind at a another location of the membrane after a short journey in the bulk solution. The time averaged mean square displacement (MSD) analysis of individual trajectories is linear whereas ensemble average MSD is superdiffusive. The distribution of displacements fit to a Gaussian distribution followed by a long tail which is Cauchy's distribution. This long tail in cauchy's distribution is from the larger displacements caused by jumps of molecule to explore greater area for efficient target search. The second section of this thesis explored the effect of crowding agents on these proteins. Polyethylene glycol (PEG) is used here to simulate crowded cellular environment aiming to understand its effect on membrane targeting C2 domains as well as on the lipid bilayer. In this chapter, we recognized that a crowding agent like PEG plays a significant role in changing the trend on diffusion behavior of C2 domains. When the PEG concentration is increased, there is a decrease in the transition of molecule between the surface and the bulk phase. With the same series of PEG concentration, there is increase in population of immobile C2 domains and desorption time. But no such increasing or decreasing trend is seen on the lipid bilayer alone. Experiments were reproduced and imaged a number of times using total internal reflection (TIRF) and fluorescence recovery after photobleaching (FRAP) techniques. Lastly, a small part of my thesis also dealt with set of experiments done to monitor tethered particle motion of DNA as well as flow extension experiments on DNA and RNA using bright field microscopy. DNA/RNA had beads tethered to one end of the strand and other end to the cover glass. Primary results are presented.
Open Access
The development of hyaluronan enhanced expanded polytetrafluoroethylene and linear low density polyethylene for blood contacting applications
(Colorado State University. Libraries, 2019) Bui, Hieu T., author; James, Susan, advisor; Reynolds, Melissa, committee member; Popat, Ketul, committee member; Olver, Christine, committee member
Cardiovascular disease is the number one cause of death in high income, industrialized countries. Designing cardiovascular implants from synthetic polymers is a cost-effective solution to the growing demand for medical treatments such as heart valve replacements and cardiovascular bypass procedures. Synthetic polymers are often known for their tunability, durability, and low production cost. Unfortunately, these materials are also prone to induce thrombosis. Therefore, improving the blood compatibility of these polymers is still a major challenge in the biomedical field. This dissertation discusses the alteration of two synthetic polymers, linear low density polyethylene (LLDPE) and expanded polytetrafluoroethylene (ePTFE), using hyaluronan (HA) to improve their blood compatibility. HA, a naturally occurring polysaccharide in the human body, is known for its wound healing and anticoagulant properties. In this work, two unique methods were developed for HA enhancement of ePTFE (HA-ePTFE) and LLDPE (HA-LLDPE). This was a process driven research that aimed at designing HA-ePTFE and HA-LLDPE by analyzing the effect of different treatment parameters on the properties of the resultant materials. In the case of ePTFE, it was demonstrated that HA can be incorporated into vascular ePTFE grafts by exploiting the micro pores of the polymer and adjusting the spraying treatment. In the HA-LLDPE fabrication process, its parameters were varied to assess their effects on the interpenetrating polymer network (IPN) formation. Surface characterization such as water contact angle goniometry, infrared spectroscopy, and toluidine blue O (TBO) staining prove that HA treatment successfully changed the surface chemistry and increased the hydrophilicity of ePTFE and LLDPE. Thermal analysis and gas chromatography-mass spectrometry were used to quantify the effects of different treatment conditions on material properties. Tensile properties such as elastic modulus, tensile strength, yield stress and ultimate strain are unchanged by HA enhancement for both polymers. The biological results reveal that HA-ePTFE and HA-LLDPE are not cytotoxic and result in less blood clotting and platelet activation than ePTFE and LLDPE.
Open Access
Fabrication of natural and magnetic slippery surfaces
(Colorado State University. Libraries, 2019) Sutherland, Daniel James, author; Kota, Arun K., advisor; Popat, Ketul, committee member; Park, Juyeon, committee member
Liquid-infused porous surfaces (LIPS) are a type of low adhesion surfaces. They allow most common solids and liquids to slide off the surface easily. Because of this they are able to reduce the adhesion of food, ice, and even blood platelets to surfaces. As a result, LIPS have applications in reducing food waste, aircraft and powerlines deicing, and hemocompatable implants. However, most LIPS are made of toxic fluorocarbon materials. In this work, in order to eschew toxic fluorocarbon materials, we designed several LIPS made from natural hemp products. The first is an all-natural LIPS made from hemp fibers, which could help reduce or eliminate liquid food waste. The second is an aluminum LIPS that shows excellent anti-icing and de-icing properties. The third is a titanium nanotube LIPS that allows blood to slide off without impinging or clotting. This surface also shows excellent platelet reduction. Finally, we demonstrated a simple way of replicating this LIPS system on multiple metals including copper and steel. Further, we demonstrated a simple fabrication of LIPS on top of the fabricated texture atop a magnetic tape. Magnetic tapes are no longer widely used and they are extremely difficult to dispose of. In this magnetic tape-based LIPS, we were able to use an external magnet to manipulate droplets on the surface of the magnetic tape. This can lead to a microfluidic system with a repurposed substrate and precise manipulation of droplets using a magnetic field.
Open Access
Computational approaches to predict drug response to cytotoxic chemotherapy
(Colorado State University. Libraries, 2020) Mannheimer, Joshua D., author; Gustafson, Daniel, advisor; Prasad, Ashok, advisor; Krapf, Diego, committee member; Thamm, Douglas, committee member
Cancer is the second leading cause of death in the United States. Statistically, within a lifetime there is slightly above a one-third chance of developing some form of cancer and a one in five chance of dying from the disease. Thus, it is no hyperbole that the understanding and treatment of cancer is one of the most pressing issues in medical research of the current era. Cytotoxic chemotherapies are a class of anti-cancer drugs that are widely used to treat a number of cancers. While cytotoxic chemotherapies are extremely effective in treating a subset of individuals for some cancers, drug resistance resulting in failure of treatment is a prominent obstacle in many cancer patients. Precision medicine, a novel concept to the 21st century, is the application of disease treatments that are specifically tailored to an individual and the specific attributes of their disease. In oncology, precision medicine particularly refers to the use of gene expression and other biological factors to inform an individual's treatment. Because cancer and its response to treatment result from many complex biological interactions, computational methods have become an essential tool to identify the molecular signatures that are the basis for precision treatment. In this thesis, a systematic analysis of the computational approaches is performed to gain insight necessary for the development of novel computational approaches in precision medicine in cancer. Statistical learning models are a class of computational modeling methods that identify and extrapolate complex patterns from large amounts of data. Specifically, this involves applying statistical learning approaches on in vitro data from cell lines and patient tumor data to predict drug response, particularly for cytotoxic chemotherapies, with an emphasis on understanding the fundamental modeling principles and data attributes driving model performance. The first chapter serves as an introduction to chemotherapy and the advancements that have driven computational approaches to precision applications in cancer. The second chapter serves as a technical introduction to statistical learning models and approaches. In the third chapter a systematic assessment of linear and non-linear modeling approaches are applied to in vitro cell lines panel including the National Cancer Institute's 60 cancer cell lines (NCI60) and cell lines of Genomics of Drug Sensitivity in Cancer (GDSC) to predict drug response in several cytotoxic chemotherapies. With in-depth analysis it is shown that the relationship between tumor tissue histotype and drug response is the major driver of model performance and can be maintained in as little as 250 random genes. The fourth chapter utilizes statistical models to explore the influence of drug induced gene perturbations on drug response models in comparison with basal gene expression. The findings indicate that drug induced changes in gene expression are superior predictors of drug response. Second, it is demonstrated that Boolean network representation of gene interactions show distinct topological differences between drug induced changes in gene expression and basal gene expression. Finally, in the fifth chapter, drug induced gene changes demonstrating high levels of connectivity in the previously developed networks are applied to derive a basal gene expression signature to predict response to combined gemcitabine and cisplatin chemotherapy treatment in patients with bladder cancer. These models show that this derived signature performs better than a random cohort of genes and in some situations genes derived directly from basal gene expression.
Open Access
A pharmacokinetic investigation of chloroquine analogues in cancer autophagy modulation
(Colorado State University. Libraries, 2020) Collins, Keagan P., author; Gustafson, Daniel, advisor; Prasad, Ashok, committee member; Yao, Tingting, committee member; Tham, Douglas, committee member; Thorburn, Andrew, committee member
Hydroxychloroquine (HCQ) is currently being investigated for safety and efficacy as an autophagy inhibitor in Phase I/II cancer clinical trials. It is the only clinically-approved autophagy inhibitor for use in cancer clinical trials in the United States. HCQ is used in combination with other chemotherapeutics to augment their efficacy and has shown moderate success in treating patients with late stage cancers. While HCQ has a good safety index and shows promise as an addition to standard of care treatment regimens, it suffers from several critical pharmacologic shortcomings which we take steps to address herein. The primary issues with usage of HCQ addressed in this work are the metrics used to predict patient tumor concentration of the drug following various dosing regimens. HCQ pharmacokinetics (PK) are highly variable in patients, with no correlation between traditional plasma:tumor concentrations. The first step taken to address this problem is to characterize likely sources of interindividual variability in HCQ PK. To do this a physiologically-based pharmacokinetic (PBPK) model was developed to investigate absorption, distribution, metabolism, and toxicity (ADMET) factors relating to HCQ in a mathematical system representative of the human body. This model was developed based on physiological and biochemical parameters relevant to HCQ ADMET in mice and scaled to represent humans. The model was capable of simulating single and multiple dosing regimens in humans, that would be characteristic of a cancer clinical trial. PBPK modeling addressed variability that would be associated with the macrophysiologic scale, but intrinsic and extrinsic factors on the cellular scale needed to be further defined to strengthen the understand of HCQ PK. To investigate factors that affect cellular uptake and sub-compartment localization of HCQ, a base PK model of lysosomotropic agents like HCQ was applied. Model specific parameters were identified for a panel of four human breast cancer cell lines, and a majority of differences in cellular uptake of the drug could be attributed to differences in the relative lysosomal volume fraction of each cell line. The model was able to characterize HCQ PK under different extracellular pH conditions, and identified a positive-feedback loop, related to transcription factor EB (TFEB) activation. This feedback loop caused the cell to increase its lysosomal volume over time of exposure to HCQ, resulting in a continuous increase of HCQ concentration within the cell. Through sensitivity analysis of the model, acidic extracellular pH was identified as a critical limiting factor of HCQ uptake into cells – which is particularly important as the tumor microenvironment is physiologically acidic. HCQ concentrations in cells cultured in an acidic microenvironment are decreased up to 10-fold, which cannot be overcome without the aid of agents that neutralize this pH. Dimeric analogues of HCQ, Lys05 and DC661, have been reported to maintain potency in acidic conditions and so were investigated in a comparative context to HCQ. Lys05 and DC661 were found to behave similarly to HCQ pharmacokinetically – i.e. highly dependent on the lysosomal profile of the cell. These drugs exhibited similar kinetic uptake curves as HCQ, and also induced the lysosomal biogenesis PK feedback loop. Unlike HCQ, Lys05 and DC661 uptake was not completely inhibited by acidic extracellular pH, and they were able to maintain activity under these conditions. PK of these drugs was characterized in a murine model to investigate their potential as in vivo agents, suggesting they could maintain high concentrations for a longer duration than HCQ. Lys05 and DC661 share many pharmacologic similarities to HCQ, while not sharing significant shortcomings such as inactivity under acidic extracellular conditions suggesting they should be investigated for further application as next generation autophagy inhibitors.
Open Access
Atomic force microscopy: more than surface imaging
(Colorado State University. Libraries, 2020) Bishop, Terrance Tyler, author; Krapf, Diego, advisor; Prasad, Ashok, committee member; Van Orden, Alan, committee member
Atomic Force Microscopy (AFM) is a powerful imaging tool that has capabilities that go beyond the abilities of most other microscopes. Here, three examples of these capabilities were considered. First, the AFM was operated in an image generating mode to determine the surface heterogeneity of polysaccharide membranes. Second, the AFM was used to record force-indentation curves, these curves were fit with a Hertzian model to determine the stiffness of murine smooth muscle cells. Finally a approach for attaching 10 µm and 2 µm polystyrene beads to tip-less AFM cantilevers was proposed, and a viscoelastic contact model was tested to determine the viability of the created probes.
Open Access
Nitric oxide-releasing or generating surfaces for blood-contacting medical devices
(Colorado State University. Libraries, 2020) Zang, Yanyi, author; Reynolds, Melissa, advisor; Kipper, Matt, committee member; Li, Yan Vivian, committee member; Zabel, Mark, committee member
Medical device-induced thrombosis is a major complication that impairs the expected performance of blood-contacting medical devices. Traditional anticoagulation therapies are used to reduce thrombus formation; however, systemic anticoagulants such as heparin increase the risk of thrombocytopenia or even bleeding, which are detrimental to patients who already have injuries. To address these issues, surface modification has been widely studied to improve the performance of blood-contacting medical devices, ranging from biopassive surfaces to biomimetic surfaces. To date, such modifications are not sufficient to prevent blood clotting alone. Supplementary anticoagulation remains necessary to maintain clot-free surfaces. Nitric oxide (NO) is a well-known signaling molecule that has antiplatelet properties. Our approach is to use surfaces that can either release NO via NO donors or promote NO production via an NO catalyst. In this work, a NO-releasing polyelectrolyte multilayer coating effectively reduces platelet adhesion, platelet activation and delay blood clotting on titania nanotube array surfaces. In addition, NO-releasing polymeric surfaces mediate blood serum protein deposition in a manner that prevents platelet adhesion and platelet activation. However, the NO donors used in these two coatings are photo- and thermo- sensitive, and the NO release is limited by the amount of NO donor added to the coating. To overcome these shortcomings, a copper-based metal organic framework (MOF) was used to infinitely promote NO production from NO donors in the blood. The copper-based MOF polymer coating was successfully applied to the surfaces of extracorporeal life support catheters and circulation tubing via custom coating systems. These copper-based MOF-coating also exhibited inherent antibacterial properties under both static and dynamic flow conditions.
Open Access
Design and evaluation of an instrumented microfluidic organotypic device and sensor module for organ-on-a-chip applications
(Colorado State University. Libraries, 2020) Richardson, Alec Evan, author; Henry, Charles, advisor; Tobet, Stuart, advisor; Bark, David, committee member; Abdo, Zaid, committee member
Organ and tissue-on-a-chip technologies are powerful tools for drug discovery and disease modeling, yet many of these systems rely heavily on in vitro cell culture to create reductionist models of tissues and organs. Therefore, Organ-on-chip devices recapitulate some tissue functions and are useful for high-throughput screening but fail to capture the richness of cellular interactions of tissues in vivo because they lack the cellular diversity and complex architecture of native tissue. This thesis describes the design and testing of 1) a microfluidic organotypic device (MOD) for culture of murine intestinal tissue and 2) a microfluidic sensor module to be implemented inline with the MOD for real-time sensing of analytes and metabolites. The MOD houses full-thickness murine intestinal tissue, including muscular, neural, immune, and epithelial components. We used the MOD system to maintain murine intestinal explants for 72 h ex vivo. Explants cultured in the MOD formed a barrier between independent fluidic channels perfused with media, which is critical to recapitulating intestinal barrier function in vivo. We also established differential oxygen concentrations in the fluidic channels and showed that more bacteria were present on the tissue's mucosal surface when exposed to near-anoxic media. The sensor module is a reversibly sealed microfluidic device with magnetic connections that can withstand high backpressures. Further, electrodes housed in commercial finger-tight fittings were integrated into the sensor module in a plug-and-play format. Future work will include developing electrochemical/optical sensors for various biological compounds relevant to intestinal physiology. Ultimately, the MOD and sensor module will be implemented in long-term microbiome studies to elucidate the relationship among microbial, epithelial, neuro and immune components of the gut wall in health and disease.
Open Access
Clinical importance of autophagy dependency and inhibition in cancer treatment
(Colorado State University. Libraries, 2021) Van Eaton, Kristen M., author; Gustafson, Daniel L., advisor; Munsky, Brian, committee member; Thamm, Douglas, committee member; Thorburn, Andrew, committee member; Yao, TingTing, committee member
Autophagy, a lysosomal degradation recycling process, has a complex and context-dependent role in cancer. Certain cancers have been found to be inherently dependent on autophagy for survival regardless of the environment. Autophagy is also implicated as a mechanism of resistance to many chemotherapies. More autophagy dependent tumors are generally more sensitive to autophagy inhibition genetically and pharmacologically. Therefore, determining what tumors are autophagy dependent is important for selecting patients that are viable candidates for autophagy inhibition. Currently, autophagy inhibition is being tested in over 90 clinical trials using FDA- approved hydroxychloroquine (HCQ) alone or in combination with other therapies. However, responses have been variable, especially in trials where HCQ is used as a monotherapy. Further, the relationship between HCQ pharmacokinetics and pharmacodynamics is not well understood in patients. Pharmacokinetics of HCQ and one of its active metabolites DHCQ was assessed in non-tumor bearing mice. Both parent and metabolite were observed at clinically relevant concentrations after 72 hr and this corresponded with evident autophagy inhibition in various tissues, although autophagy inhibition was inconsistent across the mice. The pharmacokinetic data established 60 mg/kg as the human equivalent dose observed in patients based on HCQ exposure. Cellular responses to HCQ were assessed in 2D cell culture, 3D tumor organoids, and in vivo tumor xenografts using autophagy dependent and autophagy independent tumors. Overall, cellular responses were similar across the in vitro and in vivo methods. Autophagy was inhibited regardless of autophagy status, but autophagy dependent tumors had increased cell death and decreased cell proliferation at earlier time points and lower doses of HCQ, suggesting autophagy dependency matters for optimal results. Since autophagy inhibition was inconsistent in vivo, it is still important to determine better biomarkers and possibly consider using more potent autophagy inhibitors in the clinic. Since there have not been any major advancements in osteosarcoma survival over the past four decades, autophagy dependency was assessed in osteosarcoma. Osteosarcoma was found to be intermediately to very dependent on autophagy following a genetic screen. Further, initially autophagy dependent tumor cells were able to survive and adapt to autophagy loss. Not all tumor cells adapted in the same way nor were these autophagy deficient tumor cells more sensitive to standard osteosarcoma chemotherapy, highlighting the difficulty of determining what context autophagy inhibition should be used in the clinic. Since some autophagy inhibitors like HCQ are lysosomal inhibitors and do not specifically target autophagy alone, the results of these studies also emphasized the importance of understanding whether autophagy inhibition via lysosomal degradation or autophagy inhibition of the autophagic pathway itself is superior. Overall, these results indicate targeting autophagy in osteosarcoma is a promising therapy.
Open Access
Molecular diagnostic platforms for point-of-need pathogen detection
(Colorado State University. Libraries, 2021) Jain, Sidhartha, author; Henry, Charles S., advisor; Geiss, Brian J., advisor; Dandy, David S., committee member; Magzamen, Sheryl L., committee member
Rapid, accurate, reliable nucleic acid testing (NAT) platforms are essential in the diagnosis and management of diseases. The inherent complexity associated with NAT requires that such testing be performed in centralized laboratories by highly trained personnel. Modified molecular technologies that can be used at the point-of-care (POC) are needed to improve the turnaround times of results and lower the global burden of infectious diseases. To help address this urgent need, we have developed a nucleic acid sensor platform utilizing nuclease protection and lateral flow detection for rapid, point-of-need nucleic acid analysis. We have also improved the analytical performance of the assay by pairing it with isothermal padlock rolling circle amplification (RCA). RCA is one of the simplest and most versatile isothermal amplification techniques as it only requires one primer and a strand-displacing polymerase. Utilizing our rolling circle amplification lateral flow platform, we have developed assays for beta-lactamase resistance genes for antimicrobial resistance monitoring and severe acute respiratory virus coronavirus 2 (SARS-CoV-2). We have also explored the use of exponential isothermal amplification to further improve the assay limit of detection. We also propose a microfluidic device to rapidly detect the RCA amplicons. The device allows programmable sequential delivery of reagents to a detection region, reducing the number of user steps. With further development, such microfluidic devices can be used to develop fully integrated sample-to-result molecular diagnostic platforms that integrate sample pretreatment, amplification, and detection in an easy-to-use, point-of-need nucleic acid sensor platform. Chapter 1 presents a brief review of the nucleic acid testing landscape, the challenges associated with the development of point-of-need nucleic acid sensors and recent successes utilizing paper-based devices for fully integrated sample-to-result sensors. Chapters 2 and 3 discuss the development of the nuclease protection lateral flow assay and padlock probe-based rolling circle amplification lateral flow assay. Chapter 4 describes our work on the use of exponential RCA to improve the limit of detection of the SARS-CoV-2 assay. In Chapter 5, we present our work on a paper-plastic microfluidic device for the rapid detection of the RCA amplicon. We believe that such devices can be used for the development of integrated molecular diagnostic sensor platforms that can be used at the point-of-need in resource-limited settings.
Open Access
Biomimetic and antimicrobial surfaces for orthopedic implants
(Colorado State University. Libraries, 2021) Wigmosta, Tara, author; Kipper, Matt, advisor; Popat, Ketul, advisor; Giess, Brian, committee member; DeLong, Susan, committee member; Schenkel, Alan, committee member
The number of total knee and hip replacement surgeries is expected to continue to rise in the United States. As such, the number of revision surgeries is also expected to rise. The two most common causes of failure for these implants is aseptic loosening, caused by incomplete osseointegration, and infection. Therefore, preventing infection while increasing the osteogenic properties of the surfaces used in orthopedic implants could reduce the number of revision surgeries. It is the goal of this work to create nanostructured surfaces that both increase mineralization and antimicrobial properties of titanium surfaces commonly used in orthopedic implants. To accomplish this, chitosan/heparin polyelectrolyte multilayers (PEMs), with the addition of either bone morphogenetic protein 2 (BMP-2) or gentamicin, were adsorbed onto titania nanotubes. BMP-2 has been used in clinical applications to increase osseointegration in spinal fusions, and gentamicin is effective against the most common pathogens found in infected orthopedic implants. Both heparin and chitosan are biocompatible and have antimicrobial properties. BMP-2 has a binding site for heparin that increases BMP-2's half-life in vitro. The first chapter summarizes the motivation and previous strategies used to increase osseointegration and antimicrobial properties of nanostructured biomimetic orthopedic implant surfaces. The first chapter concludes with a shift in hypothesis testing, outlining three different hypotheses: 1) surface modification(s) increase cytocompatibility and the osteogenic properties of mammalian bone cells; 2) surface modification(s) reduce bacterial adhesion, proliferation, and infection rate, without decreasing cytocompatibility; and 3) surface modification(s) provide a favorable environment in which mammalian cells can beat bacterial cells and colonize the surface first, thus increasing the osteogenic and antimicrobial properties of the surface. The testing of these hypotheses are explored in chapters 2 through 4. The second chapter explores hypothesis 1) by testing if BMP-2 released from chitosan/heparin PEM coated titania nanotubes surfaces induce an osteogenic response from rat bone marrow cells. Chapter 3 explores hypothesis 2) by testing if iota-carrageenan/chitosan and pectin/chitosan PEMs have antimicrobial properties against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), and support rat bone marrow cell adhesion and proliferation. The last chapter explores hypothesis 3) by testing if gentamicin released from titania nanotubes coated with chitosan/heparin PEMs influences the "race to the surface" in favor of mammalian cells.
Open Access
Development of novel mechanical diagnostic techniques for early prediction of bone fracture healing outcome
(Colorado State University. Libraries, 2021) Wolynski, Jakob G., author; McGilvray, Kirk, advisor; Puttlitz, Christian, advisor; Heyliger, Paul, committee member; James, Susan, committee member; Wang, Zhijie, committee member
To view the abstract, please see the full text of the document.
Open Access
Vascular endothelium glycocalyx-mimetic surfaces designed for blood contacting devices
(Colorado State University. Libraries, 2021) Vlcek, Jessi, author; Kipper, Matthew, advisor; Reynolds, Melissa, advisor; Popat, Ketul, committee member; Olver, Christine, committee member
Each year millions of blood-contacting devices are used in clinical scenarios, with contact durations designed to range anywhere from hours to years. Current blood-contacting devices can perform their intended purposes well but require the assistance of systemic drugs to inhibit failure via unfavorable interactions between the material's surface and the surrounding biology. The drugs used to inhibit failure of these devices are associated with side effects that can cause increased morbidity of patients because of their systemic administration. Thus, there is a need to design materials that can inhibit thrombus, inflammation, and infection locally at the surface of a device for the device's lifetime. In this work bio-inspired surfaces were engineered to reduce unfavorable blood-material reactions. The success of the designed surfaces was tested by evaluating their cell-material interactions, whole blood interactions, enzymatic stability, and mechanical durability. The inspiration behind these surfaces is the vascular endothelial glycocalyx, which is the luminal side of blood vessels and inhibits blood coagulation during hemostasis. The vascular endothelial glycocalyx is a meshwork of glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins which are predominantly negatively charged that acts as mediator between the blood and the underlying tissue. The surfaces proposed in this work are made to mimic the glycocalyx in its topography and chemistry by adsorbing polyelectrolyte multilayers (PEMs) onto a substrate, and subsequently adsorbing PG mimics on top of the PEMs. There are two different PG mimics used for this project which are: polyelectrolyte complex nanoparticles (PCNs) and proteoglycan mimetic graft copolymers (GC); both of which will present either heparin (HEP) or chondroitin sulfate (CS). Some of the surfaces will also be made to release nitric oxide (NO) from the surfaces through a modified version of chitosan (CHIT). Additionally, further modifications were made to the PEM surfaces to make them more mechanically durable by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) crosslinking of the CHIT and hyaluronan (HA) layers, and the addition of an initial polydopamine (PDA) layer. In the first chapter of this work outlines the current approaches to blood-contacting materials and their limitations, along with the biological components and processes that they will need to interact. The second chapter evaluates PCN and PEM surfaces that do and do not release NO via their cell-material interactions with key cell types in the processes of thrombosis, inflammation, and infection. Chapter three examines the interactions between two different PG-mimics (PC or GCs) and enzymes when suspended in solution or adsorbed onto PEM surfaces. The fourth chapter includes an evaluation of the mechanical durability of PEM and mechanically improved PEM surfaces, and whole blood evaluations of PG, GC, and modified and unmodified PEM surfaces. Taken together, this work produces multiple bioinspired surfaces that have varying degrees of success in their blood compatibility and longevity.