Urethral dysfunction is a common complication of many conditions including diabetes mellitus, spinal cord injury, vaginal childbirth, and pelvic trauma. Stress urinary incontinence (SUI) is the involuntary loss of urine due to the inability of the urethral sphincter to maintain a tight seal during the storage phase and is a condition that physically and emotionally affects millions of women. Currently treatments for SUI show limited effectiveness and/or complications. Regenerative medicine techniques may improve function and support of the diseased urethra. We hypothesized that exposure of bone marrow progenitor cells to mechanical stimuli would differentiate these cells to a contractile smooth muscle cell phenotype. This hypothesis was tested with the use of a bioreactor to aid in the differentiation via mechanical and/or chemical stimulation. In addition, an animal model of SUI was used to assess how the structural, functional and mechanical properties of the urethra were affected by placement of the tissue engineered urethral wrap (TEUW) around the native urethra. Our results indicate that bioreactor culture caused an up-regulation of myosin heavy chain, a contractile smooth muscle cell marker, with the highest expression seen after 10 days of culture with transforming growth factor beta. While some differentiation was observed, bioreactor conditions were not sufficient to create a fully functional TEUW. Exploration of stimulation regimens similar to what is seen by the urethra may cause further differentiation of these cells towards a smooth muscle phenotype. No differences between in-vivo groups were observed in leak point pressure results, but mechanical and pharmacological assessments indicated that the TEUW provided support to the urethra with responses more similar to controls than to SUI animals. Although ex-vivo results are promising, placement of the TEUW as a full wrap required separation of the urethra from the vagina, which appeared to cause remodeling and possibly scar formation. This separation may have contributed to the discrepancies seen between our in-vivo and ex-vivo data. Continued exploration of the benefits of the TEUW could include an animal model which receives the TEUW as only a partial wrap, attached to only the dorsal urethra, negating the need for tissue separation.

The remodeling response to extracellular matrix (ECM) scaffold materials such as porcine small intestinal submucosa (SIS) is characterized by intense mononuclear cell infiltration during the first 4 weeks post-implantation. Persistence of macrophages in wounds is typically diagnosed as chronic inflammation with downstream formation of scar tissue and/or foreign body reaction, but ECM scaffolds remodel into organized site-specific tissue. Macrophages can express either proinflammatory (M1) or immunomodulatory and tissue remodeling (M2) phenotypes. Processing methods used during the manufacturing of ECM scaffolds can influence macrophage phenotype and downstream remodeling outcome. In the first study, human monocyte-derived macrophages were cultured on SIS and carbodiimide (CDI) crosslinked SIS in 20% and 6% oxygen concentrations. Macrophage phenotype was evaluated by expression of M1 (CXCL10 and CCR7) and M2 (ARG-1, CCL13, CCL18, and MRC-1) gene markers, and secretion of CXCL10, CCL13, CCL18, and MMP9. Macrophages cultured on SIS expressed an M2 profile, while macrophages cultured on CDI-SIS expressed a mixed M1/M2 profile. No consistent patterns were observed when comparing oxygen concentrations. The second study used radioactive 14C-labeled scaffolds to measure ECM scaffold degradation in a rodent model of musculoskeletal reconstruction with and without the depletion of macrophages. Tissues were characterized by expression of M1 (iNOS and IFN-ã) and M2 (ARG-1 and IL-10) gene markers, and cell surface markers CD68 (pan-macrophage), CCR7 (M1), and CD163 (M2). Results showed that macrophages are required for early and rapid degradation of SIS scaffolds, and that CDI-SIS is resistant to macrophage-mediated degradation. Furthermore, depletion of macrophages resulted in an attenuated inflammatory response and slowed the rate of scaffold degradation. The third study determined the contractile response and histomorphologic appearance of tissue repaired with SIS, CDI-SIS, or autologous tissue at 26 weeks after implantation. Contractile properties and fatigue resistance of remodeled tissue and of contralateral native tissue were assessed using an in-situ methodology. Muscle fiber-type distribution, blood vessel density and distribution, and innervation were determined. The tissue repaired with SIS showed complete replacement by tissue that histologically and functionally resembled native muscle. CDI-SIS was characterized by chronic inflammatory response and produced little to no measurable tetanic force output.

Basement membranes (BMs) are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders. We introduce the inner limiting membrane (ILM), located at the retinal-vitreal junction, as a model system for studying the biophysical properties of BM. We also introduced atomic force microscopy techniques as important tools to investigate the ILM under physiologically relevant conditions. We were able to determine changes in both the thickness and elasticity of chick ILM during embryonic development. We also determined that BMs are much thicker in their native state than previously thought. Proteoglycans, specifically their heparan sulfate side-chains, were found to significantly contribute to ILM biophysical properties. The effects of aging on the composition, structure, and biophysical properties of the adult human ILM were investigated. A compositional shift in the ILM was associated with an increase in both the ILM thickness and elastic modulus during aging. The role of heparan sulfate molecules in the human ILM was determined to be similar to that of the chick ILM. The biophysical changes in mouse knockout models for congenital muscular dystrophy were also investigated. These models had protein knockouts that inhibit the proper formation of basement membranes. There were differences in the biophysical properties along with visually noted disruptions in the mouse inner limiting membranes due to improper formation of the BM. This study provided novel insight into the biophysical importance of BMs and the ability to study changes associated with a variety of biological conditions that are relevant to proper biological function, in addition to setting a baseline for the biophysical properties of BMs.

Quantitative analysis of the MRI structural and functional images is a fundamental component in the assessment of brain anatomical abnormalities, in mapping functional activation onto human anatomy, in longitudinal evaluation of disease progression, and in computer-assisted neurosurgery or surgical planning. Image registration and segmentation is central in analyzing structural and functional MR brain images. However, due to increased variability in brain morphology and age-related atrophy, traditional methods for image registration and segmentation are not suitable for analyzing MR brain images from elderly individuals. The overall goal of this dissertation is to develop algorithms to improve the registration and segmentation accuracy in the geriatric population. The specific aims of this work includes 1) to implement a fully deformable registration pipeline to allow a higher degree of spatial deformation and produce more accurate deformation field, 2) to propose and validate an optimum template selection method for atlas-based segmentation, 3) to propose and validate a multi-template strategy for image normalization, which characterizes brain structural variations in the elderly, 4) to develop an automated segmentation and localization method to access white matter integrity (WMH) in the elderly population, and finally 5) to study the default-mode network (DMN) connectivity and white matter hyperintensity in late-life depression (LLD) with the developed registration and segmentation methods. Through a series of experiments, we have shown that the deformable registration pipeline and the template selection strategies lead to improved accuracy in the brain MR image registration and segmentation, and the automated WMH segmentation and localization method provides more specific and more accurate information about volume and spatial distribution of WMH than traditional visual grading methods. Using the developed methods, our clinical study provides evidence for altered DMN connectivity in LLD. The correlation between WMH volume and DMN connectivity emphasizes the role of vascular changes in LLDs etiopathogenesis.

Complete transection of peripheral nerves can result from trauma, tumor removal, infection, or as adverse consequences of various surgeries. Current commercially available nerve guides cannot repair large nerve defects because these guides are engineered to provide mechanical support for the developing axon and do not actively promote axonal growth. For large nerve gaps, targeting axonal growth is particularly important because the length of the nerve that must be regrown is the distance from the lesion to the innervated muscle. Therefore, there is enormous clinical potential for a nerve guide capable of improving axonal outgrowth across large nerve defects. Our underlying hypothesis is that delivery of Glial Cell Line-Derived Neurotrophic Factor (GDNF) from a nerve guide will improve peripheral nerve regeneration across large defects. To test this hypothesis, biodegradable poly(caprolactone) (PCL) nerve guides were prepared with manufacturing parameters optimized for protein delivery and retention at the injury site. Quantitative changes in the diffusion of small molecular weight proteins and glucose through PCL conduit walls were measured to determine the independent and combinatorial effects of three fabrication variables: wall thickness, pore size and porosity percentage. Double-walled microspheres were then fabricated as a method of sustained protein delivery, and were incorporated within the luminal wall of PCL nerve guides using a novel solvent specific embedding technique. The overall efficacy of our nerve guide design was confirmed by encapsulating and delivering GDNF in the rat sciatic nerve injury model. Evaluation of sensory reinnervation following a long gap, 1.5cm nerve injury at 16 weeks showed a significant increase in animal response time to stimuli from animals treated with GDNF as opposed to negative control PCL guides. Furthermore, the measured gastrocnemius contraction force in animals treated with GDNF was significantly higher than negative controls and was not significantly different from the isograft positive control group. Histological assessment of explanted conduits after 16 weeks showed improved tissue integration within GDNF releasing nerve guides compared to negative controls. Nerve fibers were present across the entire length of GDNF releasing guides, while nerve fibers were not detectable beyond the middle region of negative control guides. Therefore, the results reported within this dissertation support our original hypothesis that; the long-term delivery of a neurotrophic factor from nerve guides results in improved functional recovery above negative controls following large axonal defects in the peripheral nervous system.

The autogenous saphenous vein remains the graft of choice for both coronary (500,000 annually in the US) and peripheral (80,000 annually) arterial bypass procedures. Failure of arterial vein grafts (AVGs) remains a major problem, and patients with failed grafts will die or require re-operation. Intimal hyperplasia (IH) accounts for 20% to 40% of all AVG failures. It is believed that this adverse pathological response by AVGs is largely due to their abrupt exposure to the significantly elevated circumferential wall stress (CWS) associated with the arterial system. We believe that if an AVG is given an ample opportunity to adapt and remodel to the stresses of its new environment, cellular injury may be reduced, thus limiting the initiating mechanisms of IH. The goal of this work was to develop a new mechanical conditioning paradigm, in the form of a peri-adventitially placed, biodegradable polymer wrap, to safely and functionally arterialize AVGs in situ. The polymer wrap was tuned so that as it degraded over a desired period of time, the mechanical support offered by it was reduced and the vein was exposed to gradually increasing levels of CWS in situ. To investigate the effects of mechanical conditioning on AVGs, we utilized both our well established, validated ex vivo vascular perfusion system (EVPS) as well as an appropriate preclinical animal model. The engineering component of this bioengineering study was to enhance our EVPS capabilities. Enhancements were made in the form of rigorous mathematical modeling, via subspace system identification, and automatic feedback control, via proportional integral and derivative control, of the arterial CWS and shear stress waveform generation capabilities of the EVPS. Pairs of freshly harvested porcine internal jugular veins (PIJVs) were perfused ex vivo under several biomechanical conditions. The acute hyperplastic response of PIJVs abruptly exposed to arterial hemodynamic conditions was compared to PIJVs perfused under normal venous conditions. In an attempt to attenuate this acute hyperplastic response, an ex vivo mechanical conditioning paradigm was imposed onto the PIJVs both via manual adjustment of EVPS parameters and via an adventitially placed tuned electrospun biodegradable polymer wrap. Early markers of IH were evaluated post-perfusion, and they included vascular smooth muscle cell apoptosis, proliferation, and phenotypic modulation. Quantification of these markers via immunohistochemical techniques provided the foundation for the final stage of this work. To assess the efficacy of the tuned electrospun biodegradable polymer wrap in attenuating the development of intimal hyperplasia in AVGs, a series of preclinical studies was performed in a pig model. PIJVs abruptly exposed to arterial levels of CWS showed a significant increase in apoptosis and in the number of synthetic smooth muscle cells, as well as a decrease in proliferation. Mechanical conditioning, via both manual adjustment of the EVPS parameters and placement of the biodegradable adventitial wrap, appeared to have beneficial effects on the acute hyperplastic response of PIJVs perfused ex vivo. The beneficial effects of the adventitially placed polymer wrap was also observed in vivo, however the results did not achieve significance over unwrapped controls. Future work should be aimed at enhancing the beneficial effects of the electrospun biodegradable polymer wrap by incorporating the delivery of drugs and/or stem cells in addition to the delivery of structural support to AVGs.

The field of neurobiology focuses on the development, maintenance, and function of the nervous system. Of particular interest is the formation of synapses, the junctions which allow for transmission and control of information between neurons. Synapse formation can be broken into two general processes: structural formation and activity-dependent validation. Structural formation requires transmembrane adhesion proteins that connect the two sides of the synapse. This newly-formed connection is then validated through neurotransmitter-mediated activity, which is deciphered by receptors on the postsynaptic side. In order to compare the role of two adhesion molecules (NL1 and SynCAM) and two glutamate receptors (NMDAR and AMPAR) on synaptogenesis, heterologous synapse systems were created between neurons and HEK cells expressing various combinations of these proteins (NL1 alone; SynCAM alone; NL1/NMDAR; NL1/AMPAR; SynCAM/NMDAR; SynCAM/AMPAR). These heterologous synapses were then stained for synapsin, and the size of the presynaptic contact (determined by the area of synapsin staining) was compared between the experimental groups. Results show that receptor expression causes the formation of smaller contacts than when the adhesion molecule is expressed on its own. These results suggest a role for the glutamate receptors in refining synaptic contacts during the process of synaptic validation.

Conducting polymers are synthesized on electrode surfaces, conduct electricity, and can incorporate different molecules. These properties make them ideal for biocompatible application to interface with the nervous system, particularly for drug release. This thesis describes the development of system based on nanoporous conducting polymers for the controlled release of dopamine. Polypyrrole, a conducting polymer, was demonstrated to release the neurotransmitter dopamine when electrically stimulated. Dopamine release from nanoporous and non-nanoporous polypyrrole films was characterized. Diffusion from unstimulated polypyrrole accounts for much of the dopamine release, while a fraction of the dopamine was released in a controllable fashion when the polypyrrole film was stimulated. Dopamine was retained by holding the releasing electrode at a negative potential. Dopamine release was quantified by fast-scan cyclic voltammetry using carbon-fiber microelectrodes. Successful controlled release of dopamine from conducting polymer films is promising for treatment of neurological conditions characterized by low dopamine levels, neuroscience research investigating the effects of neurotransmitters on network activity, and it also serves as a model system for controlled release of other similar molecules of pharmaceutical interest.

Embryonic development involves a fundamental biomechanical process that constructs complicated three-dimensional tissue structures through massive cellular movements. During early gastrulation stages, polarized cell intercalation movements drive the dramatic extension of the Xenopus laevis frog embryo in the anterior-posterior direction. How those individual cellular protrusive forces integrate to produce the bulk force at the tissue level remains unknown. Furthermore, the embryo is shaped not only by active forces, but also by the mechanical properties such as the viscoelastic properties of the constituent tissues. Although rapid progresses have been made to identify the genes or proteins involved in this process, there is much less known about mechanical roles of the genes and proteins in the process. By investigating the contribution of subcellular-, cellular-, and tissue-level structures to the tissue mechanical properties, we found that, on the tissue-level, there were large temporal and spatial variation in tissue stiffness of dorsal isolates and the stiffness was largely dependent on paraxial mesoderm tissues, while notochord tissue, which has been proposed to support the early embryos, was not a major contributor to the tissue mechanics. On the cellular-level, the mechanical properties of dorsal isolates were mainly dependent on cells, but not their ECM. On the subcellular-level, the mechanical properties of the embryonic cells were determined by actin and myosin II contractility, while microtubules indirectly controlled the tissue stiffness by regulating actomyosin network through a Rho-GEF mediated signaling pathway. In order to measure the tissue extension forces, we developed a high throughput technique combining imaging techniques and finite element models. Using this technique, we identified two cases of mechanical adaptation. In the first case we found that dorsal axial tissues generated less force to compensate for their own lower mechanical resistance. In the second we found that dorsal axial tissues encountering a stiffer environment were capable of generating nearly 2-fold greater force. These cases of adaptation demonstrate that force production is quantitatively balanced during CE and that the mechanisms responsible for this adaptation are able to ensure robust morphogenesis against environmental and genetic variation in physical force production and tissue stiffness.

The human learning and rewards system is comprised of a number of cortical and subcortical neural regions, including the orbitofrontal cortex, striatum, and anterior cingulate. While modern neural imaging methods such as functional magnetic resonance imaging (fMRI) and functional positron emission tomography (PET) can successfully detect the activity of these regions, they cannot discern temporal activation patterns, due to the slow onset of the blood oxygen level dependent (BOLD) effect. Magnetoencephalographic imaging (MEG) is able to capture these temporal patterns but traditionally has been unable to detect activity originating from the deeper regions of the brain due to signal attenuation and high noise levels. The recently published exSSS method has shown significant promise extracting deep signals from MEG data. To elicit appropriate subcortical activity we utilized a previously published gambling task. This paradigm has been shown to differentially activate a number of subcortical regions within the rewards system, including the orbitofrontal cortex (OFC), striatum, and anterior cingulate cortex (ACC), based on reward-related feedback. MEG analysis using source localization methods in conjunction with source signal reconstruction techniques yielded neural activation time courses for each of the regions of interest. Granger causality was used to identify the temporal relationships between each of these regions, and a possible functional connectivity map is presented. The behavioral paradigm was replicated using functional magnetic resonance imaging. fMRI activity patterns were similar to those previously reported in the literature using this paradigm. Additionally, the fMRI activation patterns were similar to those obtained via MEG source reconstruction of the exSSS-processed data. Our results support the literature finding that the rewards network is differentially activated based on feedback. Additionally, these results demonstrate the efficacy of the exSSS signal processing method for extracting deep activity, and suggest a possible use for MEG in the imaging of deep activity using other behavioral paradigms.