Thesis (M.S.)--University of Wyoming, 2006. / Title from PDF title page (viewed on Nov. 14, 2007). Includes bibliographical references (p. 91-92).

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<p>Performance of biomedical implants has been hindered by fibrosis, infection, and deficient tissue integration due in part to the body's foreign body response. In addition, diabetes mellitus is affecting a greater number of people worldwide and in the United States. As the percentage of the population affected by diabetes increases, a larger fraction of these implanted devices will be placed in diabetic patients. Unfortunately, diabetes is often complicated by poor wound healing and a greater risk of infection, issues that could adversely affect proper acceptance of an implant. Diabetic animal models are useful in studying the response to infection as well as the tissue response to an implanted device. Therapies such as nitric oxide release have been applied to indwelling devices to mediate the foreign body response and improve the wound healing response around implants. Particularly relevant to diabetic patients are implantable glucose sensors, and so determining the diabetic tissue response to these devices is crucial to improving their lifetime and performance. </p><p>A novel outcome-based streptozotocin dosing regimen was developed to induce Type 1 diabetes in a rodent model. Male CD (Sprague-Dawley type) rats weighing 150-200 g were given three consecutive daily doses of 40 mg/kg streptozotocin (STZ) on Days 1, 2, and 3. On Day 5, tail vein blood glucose was checked. If blood glucose was not within the target diabetic range of 350-600 mg/dl, rats received an additional dose of STZ. This procedure was repeated every 48 hours until all rats achieved target hyperglycemia. Control rats were given similar doses of vehicle (saline/citrate), which had no effect on blood glucose. After the last injection of streptozotocin, two weeks were allowed to ensure the full effects of the diabetic state would be present at device implantation. Blood glucose was measured every 2 days for diabetics and 4 days for controls for the duration of the experiment. The developed diabetic model resulted in a stable hyperglycemia for the duration of the experiment (in some cases, up to 2 months). Animals also exhibited typical symptoms of diabetes, such as minimal changes in weight, excessive thirst, and polyuria.</p><p>Infection response in the presence of implanted devices was investigated in healthy and diabetic animal models. In the healthy animal model, control stainless steel compression plates and plates coated with a nitric-oxide releasing xerogel (20% AHAP) were attached to the femurs of 12 adult rabbits. Both femurs were inoculated with 3x106 CFU MSSA (methicillin-sensitive Staph aureus) for a period of 20 minutes before the surgical sites were sutured. After 7 days, the wound, device, and a portion of bone were cultured. A muscle biopsy was removed and homogenized to quantify bacterial infection. Since the microbiologic data were not normally distributed, they were compared using an unpaired Wilcoxon Rank Sum Test. No significant differences in bacterial burden were observed between the control and NO-eluting devices, however the study did find a high correlation of temperature of the adjacent muscle at implantation with the ensuing bacterial content. In the diabetic animal model, a novel dosing regimen of streptozotocin based on a target blood glucose of 350-600 mg/dl was used to induce type-1 diabetes. Stable hyperglycemia was maintained for 21 days. Two weeks after achieving the target hyperglycemia, stainless steel fracture plates were secured to each femur with stainless steel screws approximately 3 mm in length. The implant site on the right side of the animal was inoculated with 3 x 107 CFU of methicillin-sensitive S. aureus while the left side served as a control. After seven days, quantitative bacterial count was performed at explantation and no cross-over of bacteria was detected from the inoculated side to the non-inoculated side. Infection after S aureus inoculation in the presence of an implanted device was significantly higher in diabetic animals when compared to that of control animals (p = 0.0003, Wilcoxin Rank-Sum Test) supporting the hypothesis that diabetes adversely affects the ability to fight infection in the presence of an indwelling device in an animal model. There was not a significant difference detected in the infectious burden for the non-inoculated limb (left) between the diabetic and non-diabetic groups when compared using a Wilcoxon Rank-Sum Test (p = 0.0682), however this near-significance suggests that even in the absence of an introduced inoculum, diabetes increases infection susceptibility in the presence of an implant.</p><p>Nitric oxide (NO) release can be used to mediate the foreign body response around implanted devices. NO also has antibacterial properties that may enhance the body's immune response to implant-associated infection. Additionally, diabetic wounds are characterized by nitric oxide deficiency, and thus NO supplementation may promote better wound healing and implant acceptance in diabetics. The use of nitric oxide to modulate the tissue response to indwelling implants was explored in two studies. In vivo glucose recovery of subcutaneously implanted NO-releasing microdialysis probes was evaluated in a healthy rat model using saturated NO solutions that provided a steady release of NO. A constant NO flux of 162 pmol cm-2 s-1 was perfused through the probe membrane for 8 hours daily. The in vivo effects of increased localized NO were evaluated by monitoring glucose recovery over a 14-day period. Beginning at 7 days, significant differences in glucose recovery between the control and NO-releasing probes were observed. At the 14-day time point, histological analysis revealed decreased inflammatory cell density at the probe surface and a thinner collagen capsule. In the second study, polyurethane-coated wires with varying NO release properties were implanted subcutaneously in 17 Yorkshire piglets with time points of 3, 7, 21, and 42 days. To create the NO-releasing coating, the NO-releasing vehicle (i.e., PROLI/NO, AEAP3 or MPTMS nanoparticles) was dispersed into EtOH (2.5 mL) at concentrations of 36 or 72 mg/mL. This solution was then mixed with an equal volume of 50:50 wt% HPU/TPU (160 mg/mL total PU). Effects of NO release were analyzed using histological data. These data were analyzed using a non-parametric Wilcoxon rank-sum test. Coatings with short NO release durations (i.e., 24 h) failed to reduce collagen capsule thickness at 3 and 6 weeks. Longer release durations (3 and 14d) however significantly reduced collagen capsule thickness at longer timepoints. The acute inflammatory response was significantly affected by coatings with the longest duration and greatest dose of NO release. However these benefits were not realized at later timepoints, suggesting that NO must be actively released in order to influence inflammatory response.</p><p>The tissue response to percutaneously implanted glucose sensors was investigated in healthy and diabetic rats. A multi-dose regimen of streptozotocin was used to induce diabetes in experimental rats. Three types of functional, implantable glucose sensors, supplied by Medtronic® were used: SofTM sensor, EnliteTM sensor, and Enlite 2TM sensor. The sensors were percutaneously implanted in the rat dorsum subcutaneous space. MiniLinkTM transmitters were attached to the rats, permitting continuous glucose monitoring. At 3 days, 1 week, and 4 weeks, tissue directly adjacent to the sensors was evaluated for collagen encapsulation, density of any collagen encapsulation, inflammatory response as measured via inflammatory cell density, and microvessel density. These endpoints were evaluated histologically via Masson's trichrome, Hoechst, H&E, and CD31 staining. Additionally, continuous functional sensor data was evaluated for sensor accuracy, attenuation, and lag time. Histological analyses revealed few significant differences in collagen thickness among different sensors, in different tissue types, or over time. In general, Masson's trichrome-stained images seem to suggest a balance between collagen capsule formation and inflammatory cell density. As inflammation increased adjacent to sensors over time, collagen capsule thickness decreased somewhat and stabilized. Collagen capsule formation was most evident adjacent to the plastic tubing portion of the sensor whereas inflammation was greatest adjacent to the sensing electrode. Likewise, few significant differences in collagen density index (CDI) were observed among sensor types, tissue types, or over time. CDI remained relatively constant over time for all sensors. Analysis of inflammatory cell density in general revealed a greater inflammatory response adjacent to percutaneous Enlite sensors, though these results were not significant. Additionally, inflammatory cell density was generally greater adjacent to non-diabetic sensors, however this result was also not significant. Inflammatory cell density increased or remained stable over time for all sensor types, suggesting that the presence of percutaneously-implanted sensors produces a chronic inflammatory response that does not resolve. Vascularity adjacent to implanted sensors remained generally stable over time, sometimes decreasing but not significantly. At the 1-month timepoint, no significant differences in vasculature were seen among sensor types. A balance also appears to exist for microvessel and inflammatory cell densities. The non-diabetic percutaneous Enlite sensor had the greatest microvessel density at earlier timepoints, while also having the greatest inflammatory cell density. However, at later timepoints, microvessel density decreased somewhat as inflammation increased somewhat. Finally, analysis of sensor performance showed significant sensor failure at longer timepoints. Sensitivity decreased somewhat for all sensors except for the non-diabetic Enlite sensor, which in general had greater overall sensitivity in comparison to the non-diabetic Sof sensor. Lag time was relatively similar among all sensor types, tissue types, and over time. MARD values were considerably lower for diabetic sensors for the Day 1 bolus, but were generally similar for all sensors at the 1-week bolus. These results suggest that the diabetic foreign body response, while somewhat decreased, is not significantly different than that in non-diabetic tissue. In addition, the design of the EnliteTM and Enlite 2TM sensors promoted a more aggressive inflammatory response despite being smaller and more flexible in design. Most evident from the results was the presence of a chronic inflammatory response adjacent to the percutaneously-implanted sensors, which likely contributed to the high rate of sensor failure over time.</p> / Dissertation

<p> Cilia are cellular organelles that generate microfluidic flow at multiple sites in the body. They are important for health due to their critical roles in mucus clearance in the respiratory tract, circulation of cerebrospinal fluid in the ventricles of the brain, transport of ova in the Fallopian tubes, and left-right patterning of the body. Nonetheless, standards for basic mechanical phenotyping of cilia are still relatively undefined. The aim of this thesis is to develop an experimental and conceptual framework for comprehensive ciliary phenotyping. Towards that aim, we pursue three major lines of investigation involving ciliary physiology, pathophysiology, and measurement.</p><p> Our investigation into pathophysiology looks at the possibility of quantifying intermediate ciliary flow defects. Specifically, we investigate subtle changes to ciliary flow generated by genetic knockdown of ciliary proteins, alterations in chemical signaling, and changes to the viscous environment of ciliated surfaces. We additionally quantify the onset of ciliary flow in the context of normal development.</p><p> Secondly, we demonstrate the use of optical coherence tomography (OCT)-based velocimetry techniques for the measurement of cilia-driven fluid flow. In particular, we focus on a class of correlation-based techniques that utilizes the complex OCT signal to recover the total speed of fluid flow. We analyze and extend these techniques towards directional velocity measurements, and eventually towards quantification of the full three-dimensional, three vector component velocity flow field.</p><p> Finally, our investigation into ciliary physiology involves the development of a simplified model of ciliary function. Building on previous models of ciliated surfaces as shearing elements, we develop our "treamdill-in-a-pool" model of ciliated surface that also incorporates the dynamics of functional reserve and failure. These efforts motivate the measurement of three important physical parameters, flow rate, force, and mechanical power output, under conditions of increased viscous loading.</p><p> Building on these themes, we propose a comprehensive, optical-imaging based approach towards quantifying flow, force, and power in the context of ciliary performance and failure. We present a method of quantifying these mechanical properties not by direct measurement, but rather by inference from the fluid flow field that is generated by ciliary action. In all, we propose a new theoretical and experimental framework for biomechanical phenotyping of ciliated surfaces.</p>

Glioblastoma is a highly invasive and aggressive brain tumor which accounts for nearly 82% of all gliomas. Patients with glioblastoma typically have a poor prognosis, suffering recurrence 7 to 10 months from the conclusion of adjuvant therapy. One promising direction for improving the clinical care of cancer, in general, and glioblastomas, in particular, is the development of accurate and precise predictive mathematical models of tumor growth. Through the use of non-invasive imaging data, mathematical models can be parameterized by the unique characteristics of an individualâs tumor to provide a âforecastâ of future tumor growth and treatment response. However, there is currently a paucity of mathematical models that have been evaluated in a controlled setting where model predictions can be validated directly to experimental results. In this work, an experimental and modeling framework is developed in which imaging measurements acquired before and after treatment are used to inform individualized biophysical models of glioma growth and response to radiotherapy. For untreated tumor growth, this work demonstrated that the commonly used reaction-diffusion model poorly predicts in vivo glioma growth. Secondly, this work demonstrated that mechanobiological effects are a necessary component to brain tumor modeling. Thirdly, this work showed that a variable carrying capacity is needed to capture the intra-tumoral spatial heterogeneity. Finally, a model was developed incorporating rapid cell death and reduced cellular proliferation and was shown to accurately predict future tumor growth following whole brain radiotherapy. Together, these studies show the potential power that image driven individualized tumor âforecastsâ could have on improving the clinical care of cancer.

Insulin resistance together with insufficient insulin secretion leads to the development of type II diabetes mellitus. Glucose-stimulation of insulin secretion has been extensively studied, but other pathways that regulate insulin secretion are not as well understood. I investigated the signaling mechanisms involved in the inhibition of insulin secretion by dopamine, which is synthesized by pancreatic ?-cells and co-secreted with insulin. Previous research has shown that dopamine-inhibition of insulin secretion is mediated primarily by the D3 dopamine receptor (DRD3) even though the DRD2 receptor has been reported to be expressed in ?-cells. To further understand this dichotomy, I investigated the dynamic protein-protein interactions between the dopamine receptor subtypes and their heterotrimeric G-proteins using two-color fluorescence fluctuation spectroscopy (FFS). I characterized the use of two fluorescent proteins, mApple and EGFP, to measure dynamic heteromerization changes with FFS. Furthermore, I showed that to detect proper GPCR signaling, both the G? and G? subunits of the G?? complex must be overexpressed in the cell. Triple transfections of a dopamine receptor and G? and G? subunits each labeled with a different fluorescent protein resulted in plasma membrane localization of all three fluorescent proteins and permitted FFS evaluation of interactions between the dopamine receptor and G?? complex. Upon dopamine stimulation, I measured a decrease in protein-protein interactions between the D3 receptor and G?? complex, indicating activation of the D3 receptor. In contrast, no significant changes in protein interactions were measured between the D2 receptor and G?? complex after dopamine treatment. These results demonstrate that two-color FFS is a powerful tool to measure dynamic protein interactions in living cells, and show that preferential DRD3 signaling in ?-cells occurs at the level of G-protein release.

Purpose: To develop software that enables the rapid implementation of custom MRI spectrometers using commercially-available software defined radios (SDRs). Methods: The gr-MRI software package comprises a set of Python scripts, flowgraphs, and signal generation and recording blocks for GNU Radio, an open-source SDR software package that is widely used in communications research. gr-MRI Implements basic event sequencing functionality, and tools for system calibrations, multi-radio synchronization, and MR signal processing and image reconstruction. It includes four pulse sequences: a single-pulse sequence to record free induction signals, a gradient recalled echo imaging sequence, a spin echo imaging sequence, and a spin echo inversion recovery imaging sequence. The gr-MRI sequences were used to perform phantom imaging scans with a 0.5 Tesla tabletop MRI scanner and two commercially-available SDRs. One SDR was used for RF excitation and reception, and the other for gradient pulse generation. The total SDR hardware cost was approximately $2000. The frequency of radio desynchronization events and the frequency with which the software recovered from those events was also measured, and the SDRâs ability to generate frequency-swept RF waveforms was validated and compared to the scannerâs spectrometer. Results: Gradient echo and spin echo images geometrically matched those acquired using the scannerâs spectrometer, with no unexpected distortions. Inversion recovery images exhibited expected behavior as a function of inversion time. Desynchronization events were more likely to occur at the very beginning of an imaging scan, but were nearly eliminated if the user invokes the sequence for a short period before beginning data recording. The SDR was able to produce a 500 kHz bandwidth frequency-swept pulse with high fidelity, while the scannerâs spectrometer produced a waveform with large frequency spike errors. Conclusion: The developed gr-MRI software can be used to develop high-fidelity, low-cost custom MRI spectrometers using commercially-available SDRs.

The inability to identify the parathyroid glands is a significant challenge during endocrine procedures. Successful parathyroid and thyroid surgeries require careful resection of diseased tissue and preservation of normal tissues, but this is not always the reality. Inaccurate localization of parathyroid glands during these procedures may permanently prevent patients from achieving normal calcium levels after surgery. Current parathyroid detection methods cannot convey real-time information and are limited to localization of only diseased glands. There is, therefore, a large unmet need in endocrine surgery for a technique to find diseased and normal parathyroid glands during surgery. Previous studies have observed an intrinsic near-infrared (NIR) fluorescence signal in the parathyroid gland that is higher than the fluorescence of surrounding neck tissues. The goal of this dissertation is to develop NIR fluorescence spectroscopy and imaging into a reliable, real-time tool for parathyroid detection regardless of disease state. The clinical utility of NIR fluorescence spectroscopy was established over a diverse patient population. Studies show 97% accuracy in NIR fluorescence detection of the parathyroid glands with minimal effects from patient factors. Parathyroid imaging was achieved to replace point measurements acquired from spectroscopy with spatial images to show gland location. A novel Overlay Tissue Imaging System (OTIS) was developed to project fluorescence information directly on the patient in the surgeonâs line of sight. This imaging approach could replace traditional display monitors and reduce errors in image perception. Finally, the mechanism of the NIR fluorescence signal in the parathyroid was investigated. The endogenous NIR fluorophore in the parathyroid gland has an emission peak at a wavelength that has been thought to be devoid of autofluorescence. Studies revealed the biochemical behavior and location of the fluorophore. Ultimately this combination of studies lowers the barrier for clinical translation of the technology. Widespread adoption of NIR fluorescence detection of the parathyroid glands will greatly improve patient care by reducing harmful surgical complications.

<p> Anterior cruciate ligament (ACL) injuries are one of most common musculoskeletal injuries and negatively affect mobility and quality of life. ACL rupture requires reconstruction to repair ligament at an estimated cost of $1.5 billion/year. Current surgical solutions invariably involve either donor site morbidity with the use of autografts or the risk of disease transmission and immune rejection with the use of allografts. Successful reconstruction requires the presence of an intact interface between ligament and bone, a transitional tissue called the enthesis. The enthesis is critical for the safe and effective transfer of force from the stiff bone to the more compliant ligament by providing a gradual transition of mechanical and biochemical properties to prevent the formation of stress concentrations. A tissue engineered ligament containing mature entheses is a promising alternative to autografts and allografts, especially since this interface does not normally regenerate. Toward this end, this dissertation sought to improve engineered fibrin-based bone-to-bone ligaments previously developed by our lab and to demonstrate their utility in understanding physiological processes through three specific aims: 1) optimize the environment for <i> in vitro</i> ligament function, 2) induce the formation of a fibrocartilaginous interface, and 3) demonstrate the utility of engineered ligaments as a physiological model.</p><p> In Aim 1, the <i>in vitro</i> culture environment was investigated for engineered ligaments formed using human ACL fibroblasts. Using a DOE approach, we identified significant effects and interactions of soluble factors on the maximal tensile load (MTL) and collagen content of engineered human ACL. The DOE model was used to predict a maximal growth media which significantly improved the MTL and collagen content of engineered ligaments and can be combined with increases in the initial construct volume for 77% further improvement in MTL. In addition to the improvements in tissue function, these data suggest that a DOE approach can more efficiently optimize <i>in vitro</i> parameters including the dosage and timing of chemical and mechanical stimuli as well as any interactions.</p><p> Aim 2 presented two strategies to improve of the engineered enthesis. First, the local release of bone morphogenetic protein (BMP)-4 at the enthesis of engineered ligaments demonstrated improved interface strength as well as the transition of cells at the enthesis towards an unmineralized fibrocartilage phenotype. Second, engineered ligaments formed in a modular fashion improved the mechanical function and the morphology of the engineered enthesis including the development of cell and soft tissue integration into the mineral phase, a tidemark between mineralized and unmineralized tissue, and the presence of a dense band of extracellular matrix (ECM) at the soft tissue-mineral interface. Importantly, this is the first demonstration of the <i>in vitro</i> formation of a functional interface between engineered ligament and mineral in a complete bone-to-bone ligament unit.</p><p> Aim 3 demonstrated the use of our engineered ligament model as a physiological tool. During the estrogen surge in the menstrual cycle, there is an associated increase in the incidence of ACL ruptures as well as knee laxity. Using physiological levels of estrogen mimicking the estrogen surge <i>in vitro</i>, we determined that estrogen decreases the activity of the collagen crosslinker lysyl oxidase (LOX) with a subsequent decrease in tissue stiffness providing insight into why women have greater incidences of ACL rupture. We also examined the role of the exercise-induced biochemical environment on connective tissue using our <i>in vitro</i> model. Engineered ligaments cultured with serum obtained from human donors after exercise had significantly better mechanical strength and collagen content than those treated with serum obtained at rest. In 2D culture, we determined that this effect was likely a result of greater mTOR and ERK signaling.</p><p> In summary, the work in this dissertation has made great strides in developing a more mature engineered bone-to-bone ligament. We have optimized a growth factor environment for their <i>in vitro</i> culture and created the most advanced engineered enthesis to date. We have also used these engineered tissues as a platform to mechanistically study the influence of hormones on connective tissue. With further advances in our understanding of the <i> in vivo</i> development of ligaments and their entheses, our bone-to-bone engineered ligaments can be improved making them more suited for clinical applications and for probing physiologically processes in a more controlled environment.</p>