This thesis investigates modifications to an injectable cell-scaffold construct and evaluates the constructs ability to induce chondrogenesis of encapsulated marrow stromal cells for cartilage tissue engineering applications. The scaffold was formed from an unsaturated macromer, oligo(poly(ethylene glycol) fumarate), developed in our laboratory. A matrix metalloproteinase sensitive peptide was used to crosslink the macromer, allowing for enzymatic degradation of the hydrogel and providing more void space for the production and deposition of extracellular matrix. Various formulations of OPF and crosslinker concentration were used to form the crosslinked hydrogels employed in the studies, and degradation and swelling of the hydrogels were analyzed. The differentiation stage of and the extracellular matrix production by marrow stromal cells embedded within the hydrogels were evaluated. This thesis provides information concerning the effect of the hydrogel macromolecular structure on its degradation and chondrogenesis of encapsulated cells.

This thesis details the development of a targeted nanoshell therapy for cancer specific photothermal ablation. By attaching targeting antibodies or ligands to the nanoshell surface, these targeted nanoshells preferentially bind to tumor sites. When NIR light is applied over the tumor region containing nanoshells, the nanoshells heat, thus destroying the tumor. The targeted nanoshells therapy is demonstrated here in vitro and in vivo, targeting both the cancer cells and the angiogenic vasculature. In vitro, anti-HER2 antibody was used to bind nanoshells directly to the cancer cells, which express HER2. The cancer vasculature was targeted in vitro and in vivo by vascular endothelial growth factor (VEGF), which binds to the VEGF receptor on endothelial cells. Nanoshells targeted against cancer cells were conjugated with anti-HER2 antibodies to facilitate the binding on nanoshells to SKBR-3 breast cancer cells. Upon NIR excitation, the nanoshell-laden cells were thermally ablated. Both membrane-bound nanoshells and NIR laser irradiation are required simultaneously to destroy the cancer cells. Cells incubated with targeted nanoshells without laser irradiation continued to be viable. When healthy cells and cancerous cells were co-cultured, cancer cells could still be targeted and ablated without damaging the adjacent healthy cells. Similar to anti-HER2 nanoshells binding cancer cells, nanoshells conjugated with the soluble VEGF bound vascular endothelial growth factor receptors on endothelial cells. Selectively killing endothelial cells removes the blood supply sustaining the tumor and demonstrates the feasibility of targeted nanoshells as an anti-angiogenic strategy. VEGF nanoshells incubated with endothelial cells in vitro produced a circular area of cell death after laser irradiation. A tumor-bearing mouse model further validated the vascular targeting when VEGF nanoshells induced tumor regression after systemic nanoshell delivery and laser irradiation. Both in vivo and in vitro studies confirmed the ability to selectively induce cell death with the photothermal interaction of immunonanoshells and NIR light. Immunonanoshells exposed to laser irradiation produced targeted cell death of cancer cells even when cancer cells were in close proximity to normal healthy cells. Immunonanoshells are a promising minimally invasive cancer therapy due to their biocompatibility, selective cell specific binding, and NIR-assisted photothermal destruction of tumor tissue.

Trace gas sensing is a rapidly developing field, with many applications in breath diagnostics and therapeutic monitoring. For this thesis, laser spectroscopic techniques were employed to measure exhaled nitric oxide (NO), carbonyl sulfide (OCS) and carbon dioxide (CO2). A pulsed quantum cascade laser operating at 4.86 microns was coupled to a 36-m optical path length Herriott cell for simultaneous measurement of exhaled OCS and CO2. Exhaled OCS was evaluated as a marker of acute rejection in lung transplant recipients. A continuous wave thermoelectrically cooled quantum cascade laser at 5.47 microns coupled with off-axis integrated cavity output spectroscopy (ICOS) was developed to simultaneously measure exhaled NO and CO2. Using the Bland-Altman method, the ICOS platform was found to be in good agreement with two commercial gas sensors---a chemiluminescent analyzer for NO and a non-dispersive infrared capnograph for CO2. The ICOS NO sensor was used to measure exhaled NO in patients with moderate to severe COPD. Four flow-independent NO exchange parameters were determined by measuring exhaled NO at six exhalation flow rates. Large airway NO flux (J'awNO) was reduced in current smokers compared to former smokers (p < 0.05). Alveolar NO (CANO) was correlated with declining functional capacity (r = -0.63, p < 0.05) and was unaffected by smoking status, indicating its potential use as a marker specific to peripheral lung inflammation in COPD. This thesis demonstrates the merits of mid-infrared laser-based trace gas sensing of multiple target gas species. This technology can be used for rapid human breath analysis and has diagnostic and therapeutic applications.

Optical techniques based on spectroscopic analysis have the potential for in vivo detection of early malignancies in tissue. Many optical modalities, including reflectance spectroscopy, use fiber optics as the means of light delivery and detection. However, the influence of fiber geometry on sampled optical spectra is not well understood. Since various configurations of fiber-optic probes may produce disparate optical spectra that are unique to individual optical systems, direct comparison among spectroscopic measurements using different optical systems may be difficult. Despite the various configurations of fiber-optic probes and optical modalities, light undergoes an identical sequence in all optical diagnostic techniques, including photon delivery, light-tissue interaction, and photon detection. Therefore, design and optimization of fiber-optic geometry must be combined with a strong understanding of tissue-photon interaction. To meet this requirement, computational models have been constructed and experiments conducted to investigate the influence of fiber geometry on the reflectance spectra. Monte Carlo simulations of photon propagation in stratified tissue models show that the spatial distribution of reflected photons varies as a function of the angles with respect to the tissue surface. More specifically, the spatial distribution of the reflectance favors superficially scattered photons when the exit trajectories of the reflected light become increasingly oblique. Therefore, it is possible to vary the collection angles of fiber probes to achieve spatially-selective reflectance from the epithelial layers, which is particularly pertinent to the diagnosis of early dysplastic transformation occurring at superficial depths. By testing angularly-variable fiber geometry on layered tissue phantoms, we confirm the theoretical predictions of the computational models. Furthermore, the angled fiber geometry may be coupled with gold nanoshells to provide significantly enhanced scattering contrast when nanoshells are selectively conjugated with dysplastic epithelial tissue. An equal magnitude of scattering contrast can be induced with markedly less gold nanoshell dosage when the angled fiber geometry is used in place of the conventional orthogonal fiber geometry. Combining reflectance-based diagnostic modalities with enhanced scattering contrast offers greater diagnostic sensitivity for clinical practitioners and greater safety for patients.

Due to their invasiveness as well as the complex properties of articular cartilage current treatments often fail to restore cartilage damage. Thus, tissue engineering approaches have received attention as a promising alternative to treat cartilage disease. A variety of natural and synthetic materials have been developed as potential carriers for cells or therapeutic agents for cartilage repair. In particular, injectable and biodegradable materials hold promise for cartilage tissue engineering due to the ease of administration and their biomimetic properties. In this thesis, an injectable hydrogel based on oligo(poly(ethylene glycol)) in conjunction with cells and gelatin microparticles loaded with growth factors is examined. First, bovine chondrocytes were encapsulated in hydrogel composites based on the biodegradable polymer, oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MP) loaded with transforming growth factor-beta1 (TGF-beta1). The results demonstrated that the presence of loaded gelatin microparticles promoted cell proliferation while maintaining a chondrocytic phenotype. A following study investigated OPF hydrogel composites encapsulating gelatin microparticles as the delivery vehicle for rabbit marrow mesenchymal stem cells (MSCs). Gene expression results showed that chondrocyte-specific gene expression of type II collagen and aggrecan were only evident in groups containing TGF-beta1-loaded MPs and varied with TGF-beta1 concentration in a dose dependent manner. OPF with two different repeating units (PEG 10K and PEG 3K) were then prepared for encapsulation of rabbit MSCs and TGF-beta1-loaded MPs to examine the effect of swelling ratio on chondrogenic differentiation of encapsulated rabbit MSCs both with and without TGF-beta1. The result demonstrated OPF hydrogel composites with the higher swelling ratio resulted in the higher level of chondrocyte-specific gene expressions. Finally, OPF hydrogel composites were evaluated as the delivery matrix for dual growth factors (TGF-beta1 and IGF-1) for in vitro chondrogenic differentiation of encapsulated MSCs. The presence of IGF-1-loaded MPs promoted the aggregation of rabbit MSCs encapsulated in hydrogels. Additionally, rabbit MSCs had the upregulation of the chondrocyte-specific genes such as collagen type II and aggrecan only in the presence of TGF-beta1-loaded MPs.

This study is concerned with the relationship between the intra- and extracellular potentials associated with the His bundle of the rabbit heart. A Hodgkin-Huxley type model of the Purkinje cell is developed and subsequently used to simulate conduction in a cable-model of the His bundle. The simulation results indicate that the field potential waveform can be decomposed and analyzed in terms of the component currents of the His bundle model. A field-theoretic model is also developed to analyze the relationship between the intra- and extracellular potentials of the His bundle. This relationship is treated as an equivalent filtering problem, wherein the extracellular field potential is predicted by specifying the action potential waveform and the equivalent filter characteristic.

Mathematical modeling of the electrical activity of a single cardiac cell and of strands of cardiac cells are problems of fundamental interest in the area of electrophysiology. New and increasingly comprehensive data on the electrophysiological behavior of single, isolated cardiac myocytes have facilitated the development of more 'complete' models and are used here for the development of mathematical characterizations of cells exhibiting 'normal' electrophysiologic behavior as well as those exhibiting 'depressed' activity. Simulation and parameter estimation techniques are utilized to investigate model-generated single cell electrical behavior and to adjust this behavior to best fit observed responses. Suitably 'identified' models may be used in further simulations of electrical activity in linear strands of resistively coupled cardiac cells. It is hoped that together with advances in experimental methods, these methods for analysis and adjustment of model behavior will provide new and meaningful insight into the mechanisms of cardiac electrical activity.

The effect of flow on the adhesion of neutrophils (PMNL) to vascular endothelium was investigated using a parallel plate flow chamber. Human umbilical vein endothelial cells (HUVEC) were cultured on a glass slide which was fitted onto the flow cell. Formyl-methionyl-leucyl-phenyl-alanine (FMLP), a chemotactic tripeptide, interleukin-1 (IL1), lipopolysaccharide (LPS), and thrombin were used to stimulate the PMNL and HUVEC in order to model the effect of inflammatory mediators on leukocyte adhesion under controlled flow conditions. PMNL adhesion to HUVEC monolayers was measured over a range of wall shear stresses estimated to be representative of flow conditions in the microcirculation. HUVEC monolayers were treated with interleukin-1 (IL1, 2U/ml, 4 hours) preceding the experiment. At 2.0 dynes/cm$\sp2$ wall shear stress, 371 $\pm$ 25.8 PMNL/mm$\sp2$ (mean $\pm$ SEM) adhered to IL1-treated HUVEC and 28 $\pm$ 2.9 PMNL/mm$\sp2$ adhered to control HUVEC after ten minutes of flow (p $<$ 0.01 on the adhesion ratio, n = 5). At 3.0 dynes/cm$\sp2$ wall shear stress, 10.2 $\pm$ 3.8 PMNL/mm$\sp2$ adhered to IL1-treated HUVEC and 6.8 $\pm$ 3.5 PMNL/mm$\sp2$ adhered to control HUVEC (n = 5). PMNL adherence to IL1-treated HUVEC decreased significantly at 3.0 dynes/cm$\sp2$ compared to adherence at 2.0 dynes/cm$\sp2$ (p $<$ 0.005). The CD18 family of leukocyte glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to the inflammatory response. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to IL1-treated HUVEC at 2.0 dynes/cm$\sp2$. TS1/18 inhibited migration of PMNL beneath IL1-treated HUVEC monolayers by 82 $\pm$ 6.8%. In flow experiments with CD18-deficient PMNL, no transendothelial migration was observed. The effect of FMLP (10$\sp{-8}$ M) on PMNL adhesion to untreated HUVEC was investigated at wall shear stresses ranging from 0.25 to 2.0 dynes/cm$\sp2$. FMLP stimulation did not significantly increase PMNL adherence at shear stresses above 0.5 dynes/cm$\sp2$. It was possible using a flow system to demonstrate that the initial attachment (or margination) of PMNL does not involve the same membrane associated adhesive proteins as does subsequent migration beneath HUVEC monolayers. Additionally, by controlling the level of shear force, it was possible to distinguish CD18/ICAM-1 mediated attachments to HUVEC from CD18-independent ones. The CD18/ICAM-1 dependent component appears to contribute to attachment at wall shear stresses below 1.0 dyne/cm$\sp2$, while the CD18/ICAM-1 independent component appears to form stronger or more numerous bonds that mediate adhesion at higher wall shear stresses. These observations indicate that local blood flow rates in the vasculature can play an important role in regulating the margination and attachment of leukocytes to the blood vessel wall.

Endothelial cell biology has become a major area of research during the last decade. An increasing body of evidence suggests that the local mechanical forces arising from blood flow play a key role in the normal physiology and pathobiology of the endothelium. In this study, monolayers of human endothelial cells were subjected to hydrodynamically-induced shear stress using a parallel plate flow chamber. This allows for an in vitro simulation of hemodynamic shear stress effects on the endothelium without pressure-induced mechanical strain, as would occur in an arterial vessel. The molecules chosen for this study were thrombomodulin and c-Fos. Thrombomodulin is a plasma membrane protein which binds thrombin and then converts protein C to activated protein C which has several anticoagulant properties. Thus, thrombomodulin is able to control the extent of thrombosis by indirectly blocking the coagulation cascade. c-Fos is a transcription factor which has been implicated in the regulation of several biomolecules synthesized by endothelial cells including tPA, PAI-1 and PDGF. A four-fold increase in thrombomodulin activity was observed in endothelial cells subjected to arterial levels of shear stress for twenty-four hours with respect to stationary controls. This is the first report of a membrane bound protein whose activity is modulated by mechanical forces. Northern blot analysis of total cellular RNA isolated from endothelial cells exposed to arterial levels of shear stress for twenty-four hours for thrombomodulin mRNA yielded no conclusive results due to weak signals. Attempts to measure c-fos mRNA in shear stress-stimulated endothelial cells yielded negative results. To provide a framework for future experiments, a nuclear transduction model concerning differential gene regulation was developed to explain the published in vitro transcription and secretion data for peptides and proteins by mechanically-stimulated endothelial cells based on the AP-1 complex. A reporter gene construct consisting of bacterial CAT under the control of the human tPA promoter was successfully introduced in to endothelial cells in vitro via an adenovirus vector. Results show a four-fold increase in CAT activity in shear stress-stimulated endothelial cells with respect to controls. This result mimics the tPA secretion and mRNA results observed in vitro.

This study demonstrates that the phenotype of zonal articular chondrocytes can be dramatically affected by monolayer passaging, three-dimensional culture, substrate modification, and growth factor stimulation. Retention of the chondrocytic phenotype is important for tissue engineering applications and cell transplantation procedures in which large numbers of cells are required. Defining a culture environment that is conducive to chondrocytic growth and development could help promote the formation of a functional tissue in a short amount of time. This work focuses on the phenotypic changes that occur in zonal chondrocytes cultured in vitro. Initial differences in gene expression are found to exist between superficial and growth zone articular chondrocytes. Results indicate that monolayer expansion of these cells causes a rapid loss in chondrocytic phenotype, especially for zone-related genes. By using novel growth environments, this work shows that there is a possibility for retaining the phenotype of cultured cells. In particular, dedifferentiated zonal chondrocytes seeded onto aggrecan-coated polystyrene show a limited ability to re-express the chondrocytic phenotype in monolayer. The presence of growth factors can also stimulate matrix synthesis and improve gene expression. This work shows that significant changes occur under a variety of treatments, but in general, biosynthesis is localized to growth zone chondrocytes. As shown throughout the combined studies, the zonal populations respond differently to stimuli, a finding that requires consideration when replicating the native organization of articular cartilage.