A new detection reagent that could possibly augment or replace antibodies research and diagnosis methods are aptamers. Aptamers are ssDNA, RNA or polypeptide constructs that function like active antibodies. Antibodies and aptamers both specifically bind to selected target molecules, and as such they enable the detection or targeting of the presence or absence of a specific antigen. In order to ensure that ssDNA aptamers perform similarly to antibodies, anti-VEGF-A polyclonal antibody and anti-VEGF-A ssDNA aptamer were evaluated against vascular endothelial growth factor A (VEGF-A) using Surface Plasmon Resonance (SPR). It was hypothesized that the anti-VEGF-A aptamer had the same, if not better, binding kinetics than the anti-VEGF-A polyclonal antibody, and as such offers an ideal replacement for use in in field, real-time testing assays. SPR revealed that both the polyclonal antibody and ssDNA aptamer bound the target antigen, VEGF-A. Additionally, from the SPR kinetic analysis, the anti-VEGF-A aptamer had KD values of 20-28 nM and the anti-VEGF-A antibody had KD values of 16-127 uM. The binding efficacy of the aptamer was several orders of magnitude better than that of the antibody. The aptamer was also stable in solution for a longer amount of time than the antibody, which denatured in solution after two weeks.

Transfemoral amputees around the world experience increased difficulty in climbing stairs due to lack of muscle, balance, and other factors. The loss of a lower limb greatly diminishes the amount of natural force generation provided that is necessary to propel oneself up stairs. This study investigated possible solutions to the problem of stair ascension for transfemoral amputees by the means of designing and developing an externally attachable device to a prosthesis. The number of amputations from military service has greatly increased since 2008, which shows there is a clear need for assistive devices (Wenke, Krueger, & Ficke, 2012). With the number of amputations rising and no current externally attachable products on the market to aid in stair ascension for transfemoral amputees, the need for this specific device has become more prominent. Research, previous work, and preliminary testing provided a basis for design and development of a new prototype. Bench top testing was conducted to review concepts in the prototype and provide data for further modifications. Results from testing of previous work, as well as testing of new concepts and modifications, provided a framework for designing a new externally attachable device for assistance in stair ascension. A new prototype was then designed, manufactured, and tested with bench models as well as real-time testing with amputees. Success of the device’s performance was based on bench top results and feedback from amputees, noting both the advantages and shortcomings of the new prototype. Testing provided results and feedback that the device was well built and functioned properly, but did not perform satisfactorily, particularly in the categories of force generation and balance.

Cerebral ischemia leading to an ischemic stroke is a possible complication of a transcatheter aortic valve replacement (TAVR) procedure. This is because embolic debris can become dislodged and travel through the aortic arch, where they either continue to the descending aorta and join the systemic circulation or travel into the cerebral vasculature through the three daughter vessels that branch off the top of the aortic arch. These three vessels are the brachiocephalic artery, the left subclavian artery, and the left common carotid artery. These three vessels lead either directly or indirectly to the cerebral vasculature, where the diameter of vessels become very small. If a large enough embolus travels into the cerebral vasculature, it can become stuck in the small cerebral vessels, blocking blood flow and cutting off the supply of oxygen to brain cells. The purpose of this study is to expand upon previous work in order to 1) create a more accurate physics simulation of blood and debris flow through the aortic arch 2) report on embolic debris distribution through the aortic arch and 3) analysis on which physical parameters affect embolic debris distribution. The physical parameters analyzed were particle diameter and particle density. This study was performed by creating a finite element model in COMSOL Multiphysics™ using a SolidWorks model of an aortic arch, with dimensions taken from a patient’s CT scan. Computational fluid dynamics was performed using a pulsatile pressure waveform throughout the aortic arch with a non-constant viscosity model. Once the velocity profile through the aortic arch matched with value ranges from literature, the particle tracing study was implemented. Both a pulsatile pressure waveform and a constant pressure model were analyzed, as well as a constant viscosity model and a non-constant viscosity model. The pulsatile pressure waveform influenced particle distribution and is recommended for future studies since this model leads to pulsatile flow, which is representative of flow through the aorta. It was seen that the non-constant viscosity model did not have a large effect on the velocity profile, but more than doubled the surface average value of viscosity. It also had an effect on the particle distribution through the aortic arch. Small diameter emboli were more likely to flow into the descending aorta, the brachiocephalic artery, and the left subclavian artery; larger emboli were more likely to flow into the left common carotid. Lower density emboli were more likely to flow into the descending aorta and the brachiocephalic artery. Averaging all densities and sizes, it was determined 44.8% of emboli flow into the three daughter vessels, but ultimately only 30.61% of emboli flow into the cerebral vasculature and have the potential to cause an ischemic stroke.

Tooth cusp radius of curvature (RoC) has been hypothesized to play an important role in food item breakdown, but has remained largely unstudied due to difficulties in measuring and modeling RoC in multicusped teeth. We tested these hypotheses using a parametric model of a four cusped, maxillary, bunodont molar in conjunction with finite element analysis. When our data failed to support existing hypotheses, we put forth and tested the Complex Cusp Hypothesis which states that, during brittle food items breakdown, an optimally shaped molar would be maximizing stresses in the food item while minimizing stresses in the enamel. After gaining support for this hypothesis, we tested the effects of relative food item size on optimal molar morphology and found that the optimal set of RoCs changed as relative food item size changed. However, all optimal morphologies were similar, having one dull cusp that produced high stresses in the food item and three cusps that acted to stabilize the food item. We then set out to measure tooth cusp RoC in several species of extant apes to determine if any of the predicted optimal morphologies existed in nature and whether tooth cusp RoC was correlated with diet. While the optimal morphologies were not found in apes, we did find that tooth cusp RoC was correlated with diet and folivores had duller cusps while frugivores had sharper cusps. We hypothesize that, because of wear patterns, tooth cusp RoC is not providing a mechanical advantage during food item breakdown but is instead causing the tooth to wear in a beneficial fashion. Next, we investigate two possible relationships between tooth cusp RoC and enamel thickness, as enamel thickness plays a significant role in the way a tooth wears, using CT scans from hundreds of unworn cusps. There was no relationship between the two variables, indicating that selection may be acting on both variables independently to create an optimally shaped tooth. Finally, we put forth a framework for testing the functional optimality in teeth that takes into account tooth strength, food item breakdown efficiency, and trapability (the ability to trap and stabilize a food item).

This research developed methods for culturing self-assembling capillaries in an injectable gel as a potential method for vascularizing tissue-on-a-chip models to mimic physiological drug delivery. Additionally, a mathematical model was developed as a tool for understanding nutrient delivery and comparison of potential delivery systems. Organs-on-a-chip provide novel platforms for studying biology and physiology in 3D, allow exploration of tissue engineering on a manageable scale, and serve as models for drug screening and drug-delivery testing. Methods were first developed for co-culture of endothelial cells and fibroblasts (3T3s or HDFs) in 2D, evaluating culture time, seeding density and ratio of HUVECs and fibroblasts, and immunostaining with a HUVEC-specific marker. Cells formed large sheets with no signs of vessel formation in 2D; therefore, the setup was translated to 3D culture to further induce stress and release of angiogenetic factors, using fibrin gel to suspend cells in 3D. After 9 days of culture, HUVECs had extensive network formation with a high degree of complexity in the experimental cell ratios (especially with 5:1 HUVECs:HDFs). Therefore, these parameters can be used as a starting point for further development of vascularized tissue constructs. A mathematical model was also successfully developed to assess the impact of cell concentration, consumption, and mode of nutrient delivery on 3D cellular constructs which can be used to predict the spatial distribution of glucose over time. Although the model shows flow introduced through a device is sufficient to maintain nutrient levels for cell growth, developing perfusable capillaries is still a critical part of creating physiologically representative tissues.

Ion sensitive field effect transistors (ISFETs) are semiconductor sensors that have the capability to determine the selected concentration of a specific ion in a solution. Most modern ISFETs utilize their ion selective properties for glucose monitors for diabetics. However, in this thesis, the ISFET fabricated is for the selective detection of K+. The goals of this thesis are to develop a functioning ion-selective polymer membrane, manufacture a working FET device, and implement the two aspects together into a working bench-top K+ selective ISFET device. Properties of a polymer composed of 33 wt.% polyvinyl chloride (PVC) 66 wt.% dioctyl sebacate (DOS) and 1 wt.% valinomycin applied to an ion-sensitive electrode (ISE) were investigated. The membrane generated a sensitivity value of -9.864E-08 Ω/log10(CK). Though this data set was affected by both the maximum resolution of the I-V curve tracing device and the thin-membrane effect. Selectivity tests following the IUPAC two-solution method in the presence of Na+ as the interfering ion, provided selectivity values of 0.228 and 0.443 with higher ratios of primary ion to interfering ion resulting in higher selectivity coefficients. Additionally, utilizing an illumination test, dielectric constants of 17.71 and 10.88 were calculated dependent on the amount of solvent used during formulation. Fabrication of the FET device also resulted in developments in metal contact materials, nitride film processing, and physical vapor deposition (PVD) processes. With further improvements, it is possible to fabricate a biocompatible, wearable K+-selective monitor for continuously testing dialysis patients.

Snoring is a sleep disruption that can lead to obstructive sleep apnea (OSA), which interrupts breathing by obstructing the airway. Injecting a protein crosslinker, such as genipin, into the soft palate could decrease the severity of snoring and OSA by stiffening the soft palate. Equine soft palates modeled human palates due to a high incidence of awake snoring and apnea. The pilot in vivo study treated six horses with two 100 mM injections of the buffered genipin reagent. The efficacy phase horses underwent respiratory audio recordings to document snoring changes using Matlab and ImageJ in the time and frequency domains. Histological analysis was completed on the safety phase palates post treatment. All horses were successfully treated with the genipin injections. At least one horse showed high frequency amplitude reductions, and all horses had low frequency amplitude reductions, correlating to a reduction in palatal displacement and snoring loudness. One efficacy horse appears to have been completely cured. The histological analysis presented tissue damage, mucosal tissue damage, and mild inflammation due to palate expansion and errant injections. Different injection volumes and techniques should be investigated next. Applying this treatment to human studies for snoring and OSA applications is the ultimate goal.

Muscle mechanical behavior potentially plays an important role in some of the most common bladder disorders. These include overactive bladder, which can involve involuntary contractions during bladder filling, and impaired contractility or underactive bladder, which may involve weak or incomplete contractions during voiding. Actin-myosin cross-bridges in detrusor smooth muscle (DSM) are responsible for contracting and emptying the bladder. The total tension produced by muscle is the sum of its preload and active tensions. Studies suggest that actin-myosin cross-links are involved in adjustable preload stiffness (APS), which is characterized by a preload tension curve that can be shifted along the length axis as a function of strain history and activation history. DSM also exhibits length adaptation in which the active tension curve can exhibit a similar shift. Actin-myosin cross-bridges are also responsible for myogenic contractions in response to quick stretch of DSM strips and spontaneous rhythmic contractions (SRC) that may occur during bladder filling. Studies show that SRC may participate in the mechanical regulation of both APS and length adaptation. However, the mechanical mechanisms by which actin-myosin interactions enable this interrelated combination of behaviors remain to be determined and were the primary focus of this dissertation. The objectives of this study were to: 1) provide evidence to support the hypothesis that a common mechanism is responsible for SRC and myogenic contraction, 2) develop a sensor-based mechanical model to demonstrate that SRC in one cell is sufficient to trigger stretch-induced myogenic contraction in surrounding cells and propagate the contraction, and 3) develop a conceptual model with actin-myosin cross-bridges and cross-links that produces the coupled mechanical behaviors of APS, SRC, and length adaptation in DSM. Improved understanding of bladder biomechanics may enable the identification of specific targets for the development of new treatments for overactive and underactive bladder.

The recreation of the complicated tissue microenvironment is essential to reduce the gap between in vitro and in vivo research. Polysaccharide-based hydrogels form excellent scaffolds to allow for three-dimensional cell culture owing to the favorable properties such as capability to absorb large amount of water when immersed in biological fluids, ability to form “smart hydrogels” by being shear-thinning and thixotropic, and eliciting minimum immunological response from the host. In this study, the biodegradable shear-thinning polysaccharide, gellan-gum based hydrogel was investigated for the conditions and concentrations in which it can be applied for the adhesion, propagation and assembly of different mammalian cell types in an unmodified state, at physiological conditions of temperature. Cell studies, to show successful propagation and assembly into three-dimensional structures, were performed in the range of hydrogels which were deemed to be optimum for cell culture and the cell types were chosen to represent each embryonic germ layer, i.e., human neural stem cells for ectoderm, human brain microvasculature cells for mesoderm, and murine β-cells for endoderm, along with a pluripotent cell line of human induced pluripotent stem cells, derived from human foreskin fibroblasts. Three-dimensional cell organoid models, to allow for gellan gum based bioprinting, were also developed using human induced pluripotent stem cells and human neural stem cells.

Statistical data from clinical studies indicate that the death rate caused by heart disease has decreased due to an increased use of evidence-based medical therapies. This includes the use of magnetic resonance imaging (MRI), which is one of the most common non-invasive approaches in evidence-based health care research. In the current work, I present 3D Lagrangian strains and torsion in the left ventricle of healthy and isoproterenol-stimulated rats, which were investigated using Displacement ENcoding with Stimulated Echoes (DENSE) cardiac magnetic resonance (CMR) imaging. With the implementation of the 12-segment model, a detailed profile of regional cardiac mechanics was reconstructed for each subject. Statistical analysis revealed that isoproterenol induced a significant change in the strains and torsion in certain regions at the mid-ventricle level. In addition, I investigated right ventricular cardiac mechanics with the methodologies developed for the left ventricle. This included a comparison of different regions within the basal and mid-ventricular regions. Despite no regional variation found in the peak circumferential strain, the peak longitudinal strain exhibited regional variation at the anterior side of the RV due to the differences in biventricular torsion, mechanism of RV free wall contraction, and fiber architecture at RV insertions. Future applications of the experimental work presented here include the construction and validation of biventricular finite element models. Specifically, the strains predicted by the models will be statistically compared with experimental strains. In addition, the results of the present study provide an essential reference of RV baseline evaluated with DENSE MRI, a highly objective technique.