Coronary artery disease is the leading cause of death in industrialized countries. Strategies to treat atherosclerotic coronary artery disease include coronary artery bypass grafting, which is often complicated by vein graft occlusion or stenosis. Severely occluded vein grafts can completely obstruct blood flow to the myocardium, resulting in a myocardial infarction, and eventually lead to heart failure. Heterotopic heart transplantation remains the treatment of choice for end-stage heart failure, however its widespread applicability is limited by the chronic shortage of donor organs. The therapeutic potential of stem cells in cardiac repair following myocardial infarction has generated a great deal of interest. Many types of stem/progenitor cells including embryonic stem cells and bone marrow-derived mesenchymal stem cells (MSCs) have been used to regenerate the infracted heart with promising results. Adipose tissue is an abundant source of multipotent stem cells that can be easily obtained from liposuction waste tissue. The yield of stem cells per gram of fat is higher when compared with marrow-derived MSCs, making adipose tissue an attractive source of autologous stem cells for cardiovascular cell therapies. The goal of this research effort was to examine the differentiation potential of adipose-derived stem cells (ASCs) along the leiomyogenic and cardiomyogenic lineages. ASCs were extracted from human subcutaneous adipose tissue from female donors during elective abdominoplasty, cultured in the presence of biomolecules responsible for vascular and cardiac development, and subjected to uniaxial cyclic strain in magnitudes comparable to the in vivo conditions. Protein and gene expression of smooth muscle- and cardiomyocyte-specific markers were assessed via immunoctytochemistry, Western blot analysis, and RT-PCR. Our results indicated that uniaxial cyclic strain inhibited cell proliferation, resulted in alignment of ASCs perpendicular to the direction of strain, and down-regulated protein expression of early smooth muscle cell markers A-SMA and h1-calponin. Transforming growth factor B1 significantly up-regulated the expression of A-SMA and h1-calponin in ASCs. Cardiac-specific proteins sarcomeric A-actinin, troponin-I, troponin-T were undetected in ASCs exposed to demethylation agent 5-azacytidine. Expression of cardiac transcription factors Nkx2.5 and GATA4 were also absent. These results suggest that human ASCs may not be capable of cardiomyogenic differentiation via exposure to 5-azacytidine.

Blood-soluble drag reducing polymers (DRPs) have been shown to produce considerable beneficial effects on blood circulation, including an increase in tissue perfusion and tissue oxygenation and a decrease in vascular resistance, when injected in blood at minute concentrations in animal models of normal and especially pathological circulation. DRPs have potential applications in treating tissue hypoperfusion caused by cardiovascular disease, stroke, peripheral vascular disease, diabetes, and other illnesses. To help to translate this novel therapy from the lab bench to the clinic, standard tests need to be developed for characterization and efficacy testing of candidate polymers. Furthermore, elucidation of the mechanisms of the observed DRP effects on blood circulation is extremely important for their future medical applications. Finally, effective, biocompatible and stable polymers which can be easily produced in large quantities must be identified. In this work a sequence of tests was developed to characterize and assess efficacy of DRPs for possible use in treating circulatory disorders. This research study also provided a better understanding of mechanical degradation of DRPs, especially in the presence of blood cells or particles. It was discovered that an increase in particle concentration led to an increase in degradation rate, and that rigid particles caused an even higher degradation rate than deformable red blood cells (RBCs). Microfluidic studies in models of microvessels showed that DRPs prevented RBC movement from the walls of microchannels toward the center and lessened plasma skimming at bifurcations, delivering more RBCs to smaller branches and thus to capillaries. In vivo, this may lead to a reduction of the near-wall plasma layer, which would facilitate gas transport, increase local wall shear stress and promote vasodilation decreasing vascular resistance in microvessels. Three polymers, including an aloe vera derived polysaccharide (AVP), poly(N-vinyl formamide), and hyaluronic acid (HA), were evaluated and characterized as new drag reducers for potential clinical use and found to be very effective. HA and AVP were found to be the most resistant to mechanical degradation of the tested polymers. Finally, relaxation time and gyration radius were found to be the polymers physical properties which best predicted their drag reducing effectiveness.

Isolated medial collateral ligament (MCL) injuries occur frequently (95,000 per year in the US) and heal with conservative treatment. Long-term clinical outcome is generally excellent because the structural properties of the Femur-MCL-Tibia complex (FMTC) naturally return to normal especially the stiffness. However, the quality of the healing ligament, as described by its histomorphological appearance, as well as biochemical, mechanical, and viscoelastic properties, remain poor [37, 70, 108, 109, 116]. Functional tissue engineering techniques such as the use of extracellular matrix (ECM) bioscaffolds have shown promise in improving healing of soft tissues after injury. In particular, small intestine submucosa (SIS) is especially attractive due to its chemoattractant properties, organized fiber alignment, and natural concentration of growth factors [9, 13, 48]. The objective of this thesis is to use SIS to improve MCL healing in a clinically relevant injury model. Sixteen New Zealand white rabbits were subjected to a mop-end tear (Weiss et al. 1991) in order to simulate a clinically relevant injury, which included damage to the ligament insertion sites, over-stretching of collagen fibers, and a frayed appearance of the torn ligament ends. After 12 weeks of healing, seven rabbits per group were euthanized and subjected to a well-established biomechanical testing protocol [111], including a load to failure test. The remaining rabbits (n=2 per group) were evaluated histologically. It was found that SIS treatment resulted in a marked improvement for the tangent modulus of the healing MCL midsubstance over non-treatment (404 ±120 MPa vs. 273 ± 91 MPa, respectively, p<0.05). However, this difference did not translate into a change in the measured structural properties of the FMTC. Nearly half of the specimens in each treatment group failed at the tibial insertion, this indicates asynchronous healing between the ligament insertion and midsubstance. In conclusion, these results confirm SIS enhances the quality of the healing MCL. SIS positively effects the local healing response of an MCL regardless of injury model. This work provides a basis to explore the effect of applying SIS to ligaments which do not heal well naturally, such as the anterior cruciate ligament.

The aortic valve (AV) is essentially a passive organ that permits unidirectional blood flow from the left ventricle to the systemic circulation and prohibits regurgitant flow during diastole. The extracellular matrix (ECM) of the AV leaflet is tri-layered with type I collagen making up the fibrosa layer (aortic side), glycosaminoglycans constituting the middle spongiosa layer, and elastin fibers largely in the ventricularis layer. Each component of the ECM is synthesized, enzymatically degraded, and maintained by the resident population of interstitial cells (AVICs) dispersed throughout the leaflet. The AVICs have been recognized as a heterogeneous mix of cells which include fibroblasts, smooth muscle cells, and myofibroblasts, which have characteristics of both fibroblasts and smooth muscle cells but are unique from each. The hypothesis of this dissertation is that the phenotype and function of the AVIC is predicated on the mechanical environment in which it resides, and during times of activated remodeling (increased myofibroblasts), the mechanobiological response of the AVIC may be contributor to changes in valvular tissue integrity. To test this hypothesis, we examine 1) the mechanical properties of the AVIC and the correlation to biosynthesis, 2) the strong connectivity of the AVIC to the ECM which is demonstrated by the AVICs ability to generate tissue-level forces due to contraction, 3) potential tissue remodeling capabilities of the AVIC via collagen gel contraction, 4) the micromechanics of the AVIC to increasing strain levels, and 5) synergistic response of the in situ AVIC to TGF-â1 and cyclic strain. Results from this work highlight the mechanobiological properties of the AVIC myofibroblast phenotype and its role in valvular tissue homeostasis, remodeling, and dysfunction. Moreover, these results demonstrate the unexamined mechanical properties of the AVIC and the strong correlate with ECM biosynthesis. As the AVIC is situated in a tissue with large strains and varying modes of deformation, the mechanical properties of the cell are likely prominent in their function. We believe that these results will add to the growing body of AVIC literature and further believe that our focus on the AVIC micro-mechanical environment will be very relevant to understanding the mechanobiologic function of the AVIC.

For underground coal mining, over 3,000 musculoskeletal disorder related injuries were reported to the MSHA injury and illness database in 2002, of which 17% were to the knee. When seam heights approach 56 inches or less, these injuries may result from the fact that workers are confined to their knees. Therefore, the industry has attempted to reduce the injury rate by providing equipment such as knee pads that distribute forces and stresses. However, these knee pads are currently designed without knowledge of the forces and stresses in the stabilizing structures within the knee during mining activities. This information is essential in understanding, and ultimately preventing injuries to the knee using interventions such as knee pads. Therefore, this work developed experimental methodologies to collect input and validation data for one subject-specific finite element model of the tibio- and patellofemoral joints consisting of: 1) geometry, 2) joint kinematics, 3) magnitudes of ligament in situ and meniscal resultant forces, and 4) ligament structural properties. Specimen geometry was reconstructed from MR images and verified by comparing to measurements from the actual geometry. The specimen was then mounted within a robotic/UFS testing system that applied external loads at deep knee flexion and recorded resulting kinematics and measured soft tissue forces (to be used for validation). These forces were determined by the principle of superposition as has been done previously; however, a novel surgical technique that removed bone blocks was developed in this work such that the ligaments remained intact. Thus, an innovative approach to clamp bone blocks of the required shape and size for structural testing was also developed. The finite element model was constructed from the experimental data, and displacements and rotations about all axes were applied to the model to verify reasonable motions were achieved. Thus, a finite element model of the knee was developed whereby the properties of only the articular cartilage and meniscus were not subject-specific. Future efforts will include model validation and use of the model for evaluating and designing interventions for the mining community.

A percutaneous respiratory assist catheter is being developed to partially support native lung function in patients with acute respiratory distress syndrome (ARDS) and acute exacerbations of chronic obstructive pulmonary disease (COPD). Current clinical therapies include pharmacotherapy, mechanical ventilation, and ECLS, but are associated with high mortality rates. The catheter is intended for insertion through the femoral vein for placement in the vena cava where it actively processes venous blood. The artificial lung device consists of a hollow fiber membrane (HFM) bundle that supplements oxygenation and carbon dioxide removal through diffusional processes. The catheter is a second generation concept of the Hattler Catheter with a design goal of size reduction to accommodate percutaneous insertion. The tradeoff in available HFM surface area requires the catheter to be more efficient in removing CO2 per unit surface area. Two prototypes utilizing rotational mechanisms to actively mix the blood and reduce mass transfer boundary layers were evaluated. The first prototype consisted of a rotating HFM bundle capable of rates of 10,000 RPM but required a structure for vessel wall protection. The second prototype employed a rotating impeller within a stationary HFM bundle to internalize rotational components within the device. The prototypes were evaluated in vitro and in vivo to assess design and performance. Development of impeller geometries, a saline seal purge, and device flexibility were notable design highlights. Acceptable hemolysis levels were observed in testing the concept of using a high-speed rotational HFM bundle. Standard gas exchange characterization tests in DI water showed over a two-fold increase in CO2 removal efficiency of 450 and 529 ml CO2/min/m2, rotational catheter and impeller catheter respectively, over the Hattler Catheter. The impeller catheter was evaluated in a bovine model and an average efficiency of 513 ± 20 ml CO2/min/m2 was attained at 20,000 RPM. Catheter size reduction and CO2 removal efficiency enhancements were successfully achieved. A separate novel method to augment CO2 concentration gradients is being researched to integrate with HFMs and attain overall gas exchange project goals.

Stress urinary incontinence (SUI) is common in women after vaginal delivery (VD) in childbirth or pelvic trauma, and may be associated with altered biomechanical or functional properties of the urethra. The goal of this dissertation was to identify biomechanical and functional changes in the urethra in a rat model of VD, as well as to understand the role of longitudinal smooth muscle in the healthy urethra. Female rat urethras were isolated in a rat model of SUI induced by VD. Controls were urethras isolated from normal rats. Our established ex vivo urethral testing system was utilized for biomechanical and pharmacological assessments. In this system, outer diameter was measured via a laser micrometer, and recorded along with applied intraluminal pressure to a computer. Urethral thickness was assessed histologically. Biomechancial properties of the urethra were markedly altered by VD for the baseline, passive (via calcium chelation), and active (stimulation via adrenergic and muscarinic receptors) states, most notably in the proximal urethra. Additionally, contractile responses to phenylephrine and bethanechol increased in the proximal urethra in VD rats compared to controls. There were also changes in the VD mid urethral segment. Functional and biomechanical parameters indicated that basal activity was increased for VD compared to controls in the middle segment, as well as adrenergic active biomechanical properties at low strains. VD impaired the basal tone distally compared to controls, but this was the only difference observed. VD urethras had evidence of altered collagen and elastin. Additionally, there was a lack of PGP 9.5, tyrosine hydroxylase, and vesicular acetylcholine transferase in the urethras of the VD group. This suggests that VD has mechanically damaging effects on urethral innervation. Finally, the role of the longitudinal smooth muscle in the urethra was further clarified via a modified urethral testing system. Circumferential and longitudinal testing of baseline, active, and passive urethral properties and function supported the idea that the role of longitudinally-oriented components of the urethra was to lengthen or shorten to enable the circumferential muscle to fully contract and shorten as required. In summary, this dissertation has provided evidence of damaged muscular, neural, and matrix components of the urethra associated with VD. The combination of these changes may contribute to SUI induced by VD.

ABSTRACT DIVALENT CATIONINDUCED REGULATION OF á5â1FIBRONECTIN INTERACTION FORCE ASSESSED USING ATOMIC FORCE MICROSCOPY Nicolas Andres Perrusquia, Ph.D. University of Pittsburgh, 2007 Cellular attachment to the extracellular matrix (ECM) via cell surface receptors is essential for signaling of the most basic of biological function such as differentiation and motility (Hynes, 1992). The integrin á5â1 binds its sole ECM ligand, fibronectin, through recognition of the RGD and (synergy) sequences; establishing a bi-directional signaling pathway between the cytoplasm and the ECM (Leahy, 1996; Redick, 2000; Krammer, 2001). During motility, forward cellular motion results in a rearward pull on the ECM, which physically loads the binding interface between á5â1 and fibronectin, forcing molecular separations at various speeds. Divalent cations play a critical role in the á5â1fibronectin interaction as evidenced by (1) regulation of the affinity of interaction by cations [e.g., Ca2+ down regulates and Mg2+ or Mn2+ up regulates á5â1 binding affinity (Gailit, 1988; Mould, 1995)] and (2) loss of molecular interaction between á5â1 and fibronectin upon chelation using EDTA (Mould, 1995; Li, 2003). The primary goal of the present study was to investigate the mechanisms underlying the cation-induced changes in á5â1fibronectin interaction. We used atomic force microscopy (AFM) to directly examine the á5â1-fibronectin interaction in the presence of affinity regulating divalent cations (i.e. either Ca2+, Mg2+, Mn2+, CaMg or CaMn) and at load rates that encompassed the known range of cellular motility speeds. Complimentary biochemical analyses were performed to examine the competitive binding of various cations to á5â1. The rupture force was linearly proportional to load rate and two distinct patterns for this relationship were observed. There was only one linear region for the down regulated state of á5â1 for all load rates. In contrast, the up regulated state resulted in two piecewise linear segments; one segment was associated with low load rates (&lt ~ 10,000 pN/s) and the other with high load rates (&gt ~10,000 pN/s). Further, the data pattern associated with down regulated á5â1 results from a single (outer) energy barrier, while the up regulated data pattern results from two energy barriers; an outer barrier for low load rates and an inner energy barrier for high load rates. No significant difference in bond rupture force (P = 0.68) existed at low load rates between the down and up regulated forms of á5â1, since each condition encountered the same outer energy barrier. However, the up regulated form of á5â1 encountered an additional energy barrier (i.e., the inner barrier) at high load rates, resulting in a sharp increase in rupture force (i.e. the second piecewise linear segment). Although both Mg2+ and Mn2+ up regulated á5â1 (i.e., both inner and outer barriers present), the addition of Ca2+ down regulated á5â1 (i.e., eliminated the inner barrier) only for Mg2+; it was unable to do so for Mn2+. The complimentary biochemical assays showed that (45Ca2+) preferentially labelled á5â1 in the presence of Mg2+ (but not Mn2+) indicating that Mg2+ is displaced by Ca2+. Overall, these results support the premise that a cation related mechanism is responsible for both down and up regulation of á5â1 binding affinity to fibronectin. Furthermore, this cation-induced regulation is related to the changes in the energy landscape (single vs. double energy barriers).

Electric powered wheelchairs (EPWs) are an important form of mobility for many persons with disabilities. However, little quantitative data exists on how much people use their EPWs in real-world environments. Previous devices have been successful at measuring EPW usage, but they have been limited by the size or the battery life of the device. This study describes the design, development, and validation of caster data logger (CDL) suitable for long-term collection of EPW usage data in real-world environments. Also included in this study is a description of EPW usage data collected during and after the National Veterans Wheelchair Games (NVWG). Several device concepts for logging EPW usage were evaluated. A caster data logger concept was chosen and functional prototypes were fabricated. The prototypes were subjected to a variety of bench tests before being deemed suitable for field use. 10 CDLs were constructed of data collection at the NVWG. At the NVWG, subjects who used an EPW as their primary means of mobility were recruited for this study. In 5 days at the games the participants (n=5) traveled a distance of 7751 ± 3439m per day, while traveling 3397±1300 m (n = 4) per day during 5 days the following week. The results were limited due to small sample size; however, they could provide useful pilot data for future studies. Overall, the CDL showed potential as a useful tool for measuring EPW usage. Future development should focus making the device easier for researchers and clinicians to use.

Selective delivery of drugs to localized regions of tissue within the body is a complex problem, representing one path through which the efficacy of many pharmaceutical compounds can be enhanced. Many pharmaceutical compounds show excellent activity in vitro, but their uses are severely limited in vivo. Unstable active conformations, limited membrane diffusion, rapid metabolism and/or clearance, decreased solubility, and dose-limiting systemic toxicity are just a few areas in which potential problems exist, halting drug development. Compounds exist possessing ideal pharmacologic activity for treating specific disease states, but they are simply unable to be delivered in adequate quantities or in the proper active conformation to the target site in the body. The following dissertation details the synthesis, characterization, and performance of a series of polyurethane drug delivery systems based on amino acids and the simple carbohydrates. The materials were synthesized from lysine diisocyanate (LDI) and glycerol with the aid of various tertiary amine and organometallic urethane catalysts. Candidate drugs were incorporated into the materials by way of labile urethane and urea linkages; subsequent drug release relied on the passive hydrolysis of the tethering bonds. Drug release from the materials correlated to material morphology, urethane catalyst, and chemical functionality of the incorporated drug. A single-phase polyurethane material was designed, synthesized, and shown capable of simultaneously releasing multiple pharmacologic agents at different rates. Finally, naturally occurring ionic ligands were incorporated into the LDI-glycerol polyurethanes to alter their swelling characteristics and release kinetics. This endeavor has resulted in the formulation of a series of polyurethane materials, capable of long-term controlled release of pharmacologic agents within the body. The structure-function relationships elucidated provide key design criteria, which can ultimately be used to develop such advanced degradable polyurethane materials.