Electrochemical studies were performed to explore electron transfer (ET) between human white blood cells (WBC) and carbon fiber electrodes (CFE). Currently, an active area of research involves encouraging ET between microbes and various electrodes in a biofuel cell (BFC). ET between microbes and electrodes are thought to occur i) directly through plasma membrane-bound electron transport chain proteins; and/or ii) indirectly through the release of metabolic products or biomolecules near the electrode surface. An important motivation of this research is the need for alternative long lasting power sources for implantable diagnostic and therapeutic devices. A particular interest is reducing the size and weight of implantable devices. Currently employed internal batteries largely contribute to both. BFCs are promising prospects as they couple the oxidation of a biofuel (such as glucose) to the reduction of molecular oxygen to water. Both glucose and oxygen are abundantly present within our bodys cells and tissues. The goal of this project is to explore the feasibility of utilizing WBCs (a human cell model) to generate electricity by fostering direct or indirect ET between these cells - or more specifically, between the metabolic processes of these cells - and the anode of a BFC. ET from the metabolic processes of whole cells to electrodes had, to the best of our knowledge, only previously been demonstrated for microbes. The electrochemical activities of WBCs isolated from whole human blood by red blood cell (RBC) lysis, peripheral blood mononuclear cells (PBMCs) isolated on a Ficoll-Paque gradient, as well as cells from a BLCL cell line and two leukemia cell lines (K562 and Jurkat) were all investigated by incorporation of the cells into the anode compartment of a proton exchange membrane fuel cell (PEMFC). Cyclic voltammetry was employed as an electrochemical technique to investigate the ET ability of the cells, as it can reveal both thermodynamic and kinetic information regarding oxidation-reduction processes at the CFE surface. The results of our studies demonstrate that upon activation, biochemical species, such as serotonin, are released by PBMCs, which may become irreversibly oxidized at the electrode surface.

STUDY DESIGN: The progression of intervertebral disc degeneration (IDD) follows anterolateral stab of adult rabbit lumbar discs by 16-gauge hypodermic needle to a limited (5-mm) depth was studied for up to 24 weeks using magnetic resonance imaging (MRI). OBJECTIVES: To develop a slowly progressive, reproducible rabbit model of IDD suitable for studying pathogenesis and pathopysiology of intervertebral disc degeneration. Moreover, to improve the MRI methods and achieve improved MRIs a custom quad coil tuned and matched for 3.0T was developed. Higher SNR achievable with this coil allowed the acquisition of higher resolution images. METHODS: Part1 - The L2-L3, L3-L4, and L4-L5 lumbar intervertebral discs of 18 skeletally mature female New Zealand White (NZW) rabbits were stabbed by 16-gauge hypodermic needle to a depth of 5-mm in the left anterolateral annulus fibrosis (AF). Serial MRI scans of the stabbed discs and intact L1-2 and L5-6 control discs were performed at 3, 6, 12, and 24 weeks post surgery and compared with preoperative MRIs. Development of the quad coil at 3.0T was competed with reference phantom to demonstrate the advantages over the 5 inch surface coil at 1.5T. To further illustrate the coils advantages in-vivo rabbit MRIs were obtained with the custom coil and compared to the 5 inch surface coil at 1.5T RESULTS: The stabbed discs exhibited a progressive decrease in MRI Index (the product of nucleus pulposus (NP) area and signal intensity from T2-weighted midsagittal plane images) starting at 3 weeks post stab and continuing through 24 weeks, with no evidence of spontaneous recovery or reversal of MRI changes. In addition, the constructed quad coil at 3.0T demonstrated the ability to obtain high resolution scans with SNR comparable to the 5 inch surface coil at 1.5T. CONCLUSIONS: Stabbing the anterolateral AF of adult rabbit lumbar discs with a 16-gauge hypodermic needle to a limited (5-mm) depth results in a number of slowly progressive and reproducible MRI changes over 24 weeks that show a similarity to changes seen in human IDD. This model would appear suitable for studying pathogenesis and pathophysiology of IDD and testing safety and efficacy of novel treatments of IDD.

While documented risk factors (e.g., hypertension, diabetes, etc.) for atherosclerosis are systemic in nature, atherosclerotic plaques appear in a heterogeneous distribution in the vasculature. This heterogeneity is thought to be related in part to the fact that the plaques tend to develop in areas of disturbed blood flow such as bifurcations and curvatures. Moreover, the coronary arteries, which also experience the added mechanical deformations of cyclic flexing, stretching, and twisting due to their tethering to a beating heart, are particularly susceptible to atherogenesis. This suggests that both fluid-induced (shear) and deformation-induced (mural) stress contribute to location specific susceptibility or protection from disease. We hypothesized that local variations in shear and mural stress associated with dynamic motion of arterial segments influence the distribution of early markers of atherogenesis. To test this hypothesis, we utilized our unique, well-established, and validated ex vivo vascular perfusion system in a combined experimental / computational study. Pairs of freshly-harvested porcine arterial segments were perfused ex vivo under normal hemodynamic conditions. One of the paired segments was exposed to coronary levels of either cyclic axial stretching, flexure, or twist. Post-perfusion tissue processing provided the extent and spatial distribution of early markers of atherogenesis, including endothelial permeability, apoptosis, and proliferation. Finite element analysis and computational fluid dynamics techniques were used to estimate the mural and shear stress distributions, respectively, for reconstructed models of each experimentally perfused segment. Quantitative correlations between biological marker and mechanical stress distributions were determined using multiple linear regression analysis. Vessel segments exposed to cyclic axial stretch and flexure showed significant increases in both permeability and apoptosis. In addition, we demonstrated that all three deformations generated complex, non-uniform distributions of both biologic endpoints and mechanical stresses. These distributions displayed a high degree of specimen-to-specimen variability which was attributed to the highly variable vessel geometries. Several specific mechanical stress measures, both mural and shear, were shown to be associated with cellular atherogenic marker distribution. Future work should be aimed at more fully elucidating the molecular mechanism linking mechanical stress in the tissue to these cellular based responses.

Current challenges in vascular medicine (e.g., bypass grafting, stenting, and angioplasty.) have driven the field of vascular regenerative medicine. Bone marrow-derived mesenchymal stem cells (BMMSCs) are adult stem cells which may be a suitable cell source for vascular regenerative medicine applications. While it is well known that BMMSCs readily differentiate into musculoskeletal cells, recent studies have provided evidence for their differentiation into smooth muscle cells (SMCs) and endothelial cells (ECs). We and others have demonstrated the ability of the mechanical stimulus of cyclic stretch to drive BMMSC differentiation towards SMCs in vitro, but a rigorous, systematic analysis of other relevant forces is lacking. The working hypothesis that this work addressed is that mechanical stimuli relevant to the vasculature will guide BMMSC differentiation towards SMCs and ECs. To test this hypothesis, rat BMMSCs were exposed to physiologically relevant magnitudes and frequencies of a Mechanical Panel, which consisted of cyclic stretch, cyclic pressure, and shear stress, each applied in parallel to subcultures of BMMSCs. Quantitative changes in morphology, proliferation, and gene and protein expression were assessed to determine the differential effect of each stimulus in a dose- and frequency-dependant manner. Next, we investigated the importance of the duration of applied stimulation to BMMSC differentiation as well as tissue commitment (i.e., cell plasticity) following mechanical stimulation. Our results demonstrate that mechanical stimulation differentially altered BMMSC morphology, proliferation, and gene and protein expression towards the cardiovascular lineage while limiting expression for other lineages including bone, fat, and chondrocyte. This was particularly evident for cyclic stretch, which caused an elongated, spindle-shape and expression of the SMC proteins alpha-actin, calponin, and myosin heavy chain. Furthermore, we found that cyclic pressure and shear stress tended to increase endothelial gene expression when these stimuli are applied to confluent BMMSCs. While our findings as a whole tended to support our hypothesis, our data indicate that SMC protein expression is more readily increased by mechanical stimulation, and is highly variable, even without associated changes in gene expression. Future work employing systems biology approaches that take into consideration the resulting transcriptional and proteomic changes in BMMSCs from these mechanical stimuli will be necessary to more accurately identify how mechanical stimulation can be used as a tool for regenerative medicine.

A novel framework for medical image analysis, known as Shells and Spheres, has been developed by our research lab. This framework utilizes spherical operators of variable radius, centered at each image pixel and sized to reach, but not cross, the nearest boundary. Statistical population tests are performed on the populations of pixels within adjacent spheres to compare image regions across boundaries, delineating both independent image objects and the boundaries between them. This research has focused on developing the Shells and Spheres framework and applying it to the problem of segmentation of anatomical objects. Furthermore, we have rigorously studied the framework and its applications to clinical segmentation, validating and improving our n-dimensional segmentation algorithm. To this end, we have enhanced the original Shells and Spheres segmentation algorithm by adding a priori information, developing techniques for optimizing algorithm parameters, implementing a software platform for experimentation, and performing validation experiments using real 3D ovine cardiac MRI data. The system developed provides automated 3D segmentation given a priori information in the form of a trivial 2D manual training procedure, which involves tracing a single 2D contour from which 3D algorithm parameters are then automatically derived. We apply this system to segmentation of the Right Ventricular Outflow Tract (RVOT) to aid in research toward the creation of a Tissue Engineered Pulmonary Valve (TEPV). Experimental methods are presented for the development and validation of the system, as well as a detailed description of the Shells and Spheres framework, our segmentation algorithm, and the clinical significance of this work.

Current artificial lungs, or membrane oxygenators, have limited gas exchange capacity due to their inability to replicate the microvascular scale of the natural lungs. Typical oxygenators have a surface area of 2 4 m2, surface area to volume ratio of 30 cm-1, and gas diffusion distances of 10 30 microns. In comparison, the natural lungs have a surface area of 100 m2, surface area to volume ratio of 300 cm-1, and diffusion distances of only 1 2 microns. Membrane oxygenators also suffer from biocompatibility complications, requiring systemic anticoagulation and limiting length of use. The goal of this thesis was to utilize microfabrication and tissue engineering techniques to develop biohybrid artificial lung modules to serve as the foundation of future chronic respiratory devices. Microfabrication techniques allow the creation of compact and efficient devices while culturing endothelial cells in the blood pathways provide a more biocompatible surface. Soft lithography techniques were used to create 3-D modules that contained alternating layers of blood microchannels and gas pathways in poly(dimethylsiloxane) (PDMS). The blood microchannels were fabricated with widths of 100 microns, depths of 30 microns, and inter-channel spacing of 50 microns. The diffusion distance between the blood and gas pathways was minimized and a surface area to blood volume ratio of 1000 cm-1 was achieved. The gas permeance of the modules was examined and maximum values of 9.16 x 10-6 and 3.55 x 10-5 ml/s/cm2/cmHg, for O2 and CO2 respectively, were obtained. Initial work examining thrombosis in non-endothelialized modules demonstrated the need for endothelial cells (ECs). Several surface modifications were explored to improve EC adhesion and growth on PDMS. Finally, endothelial cells were seeded and dynamically cultured in prototype modules. Confluent and viable cell monolayers were achieved after ten days. The work described in this thesis provides a strong foundation for creating more compact and efficient biohybrid artificial lungs devices.

Introduction: Vascular cells communicate through gap junctions, which are formed by connexin (Cx) proteins. Cx43 is expressed in both endothelial and smooth muscle cells. Studies have demonstrated alterations in gap junctions with atherosclerosis and hypertension, diseases that involve changes in mechanical forces. However, regulation of arterial gap junctions by mechanical forces has not been well understood. Methods: In the present study, ex vivo perfusion culture of rabbit thoracic aortas was used to investigate the regulation of Cx43 by pressure magnitude and pulsatility. After culturing for 6 or 24 h, the Cx43 protein and mRNA levels were detected by Western blot and real-time PCR, respectively. The Src inhibitor PP1 or NADPH oxidase inhibitor apocynin was added to the culture medium to study the molecular mechanisms in some experiments. Results: (1) An increase in the steady pressure level (from 80 to 150 mmHg) significantly increased both mRNA and protein levels of Cx43 at 6 h, which were blocked by PP1. High steady pressure also upregulated Cx43 mRNA at 24 h, although the Cx43 protein levels were similar. This pattern of steady pressure-induced regulation of Cx43 was not altered by the presence of pressure pulsatility or flow levels. (2) Cyclic stretch, elicited by pulsatile perfusion (mean: 80 mmHg, pulse: 30 mmHg, 192 cycles/min), decreased Cx43 protein for both 6 and 24 h, compared with steady stretch controls (mean: 80 mmHg, pulse: 0 mmHg). Concomitantly, levels of active and total Src were reduced by cyclic stretch at 24 h. PP1 in steady perfusion culture or apocynin in pulsatile perfusion culture eliminated the observed differences in Cx43 protein between cyclic and steady stretch. In addition, apocynin elevated active and total Src in aortas under cyclic stretch at 24 h. The ratio of active to total Src was not significantly altered in any case. Conclusions: Both pressure magnitude and pulsatility regulates Cx43 expression. High pressure upregulates Cx43 mRNA and is time-independent. High pressure upregulates Cx43 protein and is time-dependent. Cyclic stretch downregulates Cx43 protein and is time-independent. Src and NADPH oxidase may be involved in the signaling pathway.

This work involves three topics that advance the functionality of an elbow simulator in the Orthopaedic Biomechanics Laboratory at Allegheny General Hospital. To draw clinically and scientifically meaningful conclusions from future cadaver studies conducted with the simulator, its design must be validated and the accuracy of the data collection methods demonstrated. The simulator was designed to offer physiologically-correct adjustable moment arms throughout the elbows range of motion. To validate this, muscle moment arms were measured in three cadaver elbow specimens. Flexion-extension moment arms were measured at three different pronation/supination angles: fully pronated, fully supinated, and neutral. Pronation-supination moment arms for four elbow muscles were measured at three different flexion-extension angles: 30°, 60°, and 90°. The numeric results compared well with those previously reported. The biceps and pronator teres flexion-extension moment arms varied with pronation-supination position, and vice versa. This represents the first use of closed-loop feedback control in an elbow simulator, one of the first reports of both flexion-extension and pronation-supination moment arms in the same specimens, and demonstrates the adjustability of the moment arms that the elbow simulator can produce. Towards accurate motion analysis of the radial head, two areas were investigated. The first identified the phenomena of camera-switching, which occurs in motion analysis when data from one or more cameras is temporarily excluded from the computation of a markers three-dimensional position. Tests with static markers showed that camera-switching could cause up to 3.7 mm of perceived movement. The second area of investigation set the stage for future studies with cadaver elbows. A protocol was developed to quantify both the travel of the native radial head, radial head implants, and the finite helical axis during pronation-supination movement. The tracking of implant motion employs a unique circle-fitting algorithm to determine the implants center. A video-based motion analysis system was used to collect marker position coordinates actuated by a precision micrometer table. MATLAB code was designed and implemented to compute both the radial head position and finite helical axis from these data. Immediate future work will use these algorithms to evaluate radial head implants in comparison to the native radial head.

Cells interact with mechanical environments by mechanotransduction, a cellular process that converts mechanical signals into biochemical signals. Umbrella cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are coordinated and the mechanotransduction pathways involved are not well understood. By manipulating different forces and force parameters applied on umbrella cells, the responses of electrophysiological parameters (TEV, TER, and Isc) and apical membrane capacitance (1µF  1cm2 membrane surface area) are monitored through the modified Ussing chamber system. Stretch of the umbrella cells result in an acute change of electrical parameters, but not hydrostatic pressure. Further, the stretched response is sensitive to force direction, indicating that stretch of apical membrane causes umbrella cell TEV hyperpolarization, TER decrease, Isc increase, and apical membrane exocytosis, while stretch of basolateral membrane causes opposite effects, and this observation can be modeled mathematically. Stretch speed, which is defined by the filling rate, is further defined to play the key role in modulating the degree and time course of stretched umbrella cell responses, suggesting a mechnosensory function of umbrella cells. Use of channel blockers and openers established that the stretch of apical membrane is likely dependent on cation transport pathway, while stretch of basolateral membrane is dependent on K+ transport at the basolateral surface of the cells, indicating distinctive apical and basolateral membrane requirements for umbrella cell mechanotransduction. These results indicate that mechanotransduction in umbrella cells depends on the sequential activity of its distinct apical and basolateral membrane domains, which act in a collaborative manner to regulate apical membrane dynamics.

Left Ventricular Assist Devices (LVADs) are becoming a more widely accepted form of treatment for patients suffering from end stage heart failure. First generation LVADs attempted to mimic the physiology of the native heart by generating pulsatile blood flow. Second- and third-generation turbodynamic LVADs are much smaller than pulsatile LVADs, but alternatively generate non-physiologic continuous flow. A vast amount of time and resources have been spent on optimizing the hemodynamics LVADs. One area that has changed very little, however, is the apical cannula- an inflow conduit that is inserted into the apex of the left ventricle (LV) allowing blood to be drawn from the LV chamber by the LVAD so that the heart can be effectively unloaded. Current inflow cannulae are often straight rigid tubes which extend far into the LV, and are susceptible to becoming displaced during the implantation of the device or post-operatively. A malpositioned cannula may cause reduced flow, it may interfere with ventricular anatomy, and it may generate areas of stagnation that can form thrombus. Additionally, the advent of continuous flow LVADs brought about a new problem where the negative pressures generated within the ventricle by the LVAD can cause the chamber to collapse. A sub-optimally placed cannula may increase the likelihood of these suction events. An ongoing collaborative effort at the University of Pittsburgh and Carnegie Mellon University has resulted in the design of a more robust low-profile cannula. The design is intended to minimize variations in pump output by reducing the cannulas ability to be malpositioned. This reduces interactions with ventricular anatomy, and is more likely to generate hemodynamically favorable flow. This design is also intended to reduce the likelihood of ventricular collapse across the spectrum of physiologic conditions by providing mechanical support for the ventricular wall. In-silico and ex-vivo studies, including in-situ visualization of ventricular suction in an arrested ovine ventricle, demonstrated that the novel cannula design is indeed a more robust design, and more compatible with ventricular anatomy. Further development of the flared inflow cannula is warranted, as is the study of its interaction with the left ventricle under sub-optimal conditions.