Diffusion-weighted Magnetic Resonance Imaging (DWI) has become a useful tool in medicine for the purpose of diagnosis, tracking disease progression, and monitoring response to therapy. The current techniques used for DWI suffer from artifacts due to magnetic field inhomogeneities, image distortion, and low spatial resolution. The aim of the presented work is to advance DWI by improving upon and developing novel high-resolution acquisition techniques. The approach taken for this purpose was to utilize radial fast spin-echo data acquisitions, which have been shown to produce high-resolution DWI without artifacts due to magnetic field inhomogeneities. In addition, there is little image distortion in radial fast spin-echo DWI, which allows for direct overlay onto anatomical MRI. However, a draw back is that radial methods require longer scan times. By increasing the imaging speed of existing radial fast spin-echo acquisitions, it may become a more practical clinical tool. In addition, novel acquisition techniques are developed that push high-resolution to all three dimensions. By employing a three-dimensional radial fast spin-echo acquisition, voxels in an image have equal size in each dimension and can be on the order of 1mm3. By decreasing the voxel size, the tissue contained within a voxel is more homogeneous. This is important for DWI applications that aim to measure the microscopic integrity of the tissue. The development and analysis of the novel radial fast spin-echo techniques are presented in this work along with several clinical applications. The remaining issues to be addressed for application to quantitative DWI measures are also presented, along with possible solutions.

Laparoscopic liver resection is increasing accepted as a standard of care with results comparable to open cases while incurring less trauma and reducing recovery time. The tradeoff is increased difficulty due to limited visibility and restricted freedom of movement. Image-guided surgical navigation systems can help localize anatomical features to improve patient safety and achieve negative surgical margins. Previous research has demonstrated that intraoperative surface data can be used to drive a finite element tissue mechanics organ model such that high resolution preoperative scans are registered and visualized in the context of the current surgical pose. In this paper we present an investigation of using sparse data as imposed by laparoscopic limitations to drive a registration model. Surface swabs and subsurface data were used in tandem to reconstruct a displacement field on the posterior of the organ to optimize the fit between the intraoperative data and the preoperative liver model. Tests based on laboratory phantoms were used to validate the potential of this approach. Experimental results based on a liver phantom demonstrate that Target Registration Errors (TRE) on the order of 5mm were achieving using only surface swab data, while use of only subsurface data yielded errors of about 6mm. Registrations using a combination of both datasets achieved TRE on the order or 2.4mm and represent a sizeable improvement over either dataset alone.

Biodegradable tissue engineering scaffolds are commonly fabricated from poly(lactide-co-glycolide) (PLGA) or similar polyesters that degrade by hydrolysis. PLGA hydrolysis generates acidic byproducts that trigger an accelerated, autocatalytic degradation mechanism that can create mismatched rates of biomaterial breakdown and tissue formation. Reactive oxygen species (ROS) are naturally produced by cells, and induction of inflammation and ROS is an inevitable in vivo response to biomaterial implantation. Thus, polymeric biomaterials that are selectively degraded by cell-generated ROS may have potential for creating scaffolds with better-matched rates of tissue in-growth and cell-mediated scaffold biodegradation. To explore this approach, a series of novel poly(thioketal) (PTK) urethane (PTK-UR) biomaterial scaffolds that degrade specifically by an ROS-dependent mechanism were synthesized. Unlike poly(ester-urethane) (PEUR) scaffolds, the PTK-UR scaffolds were stable under aqueous conditions out to 25 weeks but were selectively degraded by ROS. The in vitro oxidative degradation rates of the PTK-URs followed first-order degradation kinetics, were significantly dependent on PTK composition (p<0.05), and displayed dose-dependence with respect to ROS levels. In subcutaneous rat wounds, PTK-UR scaffolds supported cellular infiltration and granulation tissue formation, followed first-order degradation kinetics over 7 weeks, and produced significantly greater stenting of subcutaneous wounds compared to PEUR scaffolds. These combined results indicate that PTK-UR tissue engineering scaffolds have significant advantages over analogous polyester-based biomaterials and provide a robust, cell-degradable substrate for guiding new tissue formation.

Tuberculosis infects one out of three individuals worldwide. In order to control tuberculosis infection, accurate diagnostics are needed at the point-of-care. An ideal point-of-care diagnostic for tuberculosis would include an integrated system for lysis of the bacteria, extraction of the DNA from the bacterial lysate and the sputum, and detection of a specific biomarker. We compared the use of two different isothermal amplification methods, cross priming amplification (CPA) and loop mediated amplification (LAMP), for the detection of tuberculosis within a previously developed extraction cassette designed for low resource areas. Under ideal laboratory conditions, CPA and LAMP had a limit of detection of 500 copies and 50 copies respectively. As part of an integrated system, CPA and LAMP detected a concentration of bacteria at 1X103 cells/mL at 46 ± 5.8 minutes and 57 ± 4.6 minutes. For the integrated system of tuberculosis detection, CPA generates faster results. However, LAMP was shown to have a lower limit of detection and more specificity under ideal conditions. Overall, this study supports the continued investigation of using isothermal amplification methods combined with a low resource extraction cassette as a point-of-care diagnostic test.

RNA interference (RNAi) by small interfering RNA (siRNA) possesses great promise as a therapeutic for pathologies whose etiology is related to gene overexpression. However, due to the poor pharmacokinetic properties of siRNA, it requires a carrier for in vivo intravenous delivery. Historically, nucleic acid delivery systems have utilized cationic lipids or polymers as carriers, but such agents are poorly translatable in vivo, as they have inadequate hemo-stability, a short blood circulation half-life, and can lead to unexpected toxicity. Here, we introduce a series of novel mixed micelles that modulate the molar concentration and lengths of poly(ethylene glycol) (PEG) on the corona of the micelles to achieve charge shielding that improves the pharmacokinetic properties of the siRNA-micelle complex, while maintain significant levels of gene knockdown. Hemocompatibility and in vitro stability is increased for micelles with greater PEG surface concentration and for micelles with higher molecular weight PEG in the corona. When delivered intravenously in vivo, micelles with a higher molecular weight PEG in the corona demonstrate a significantly improved blood circulation half-life (17.8 minutes for micelles with a 20 kDa PEG vs. 4.6 minutes for micelles with a 5 kDa PEG) and a 4-fold decrease in lung accumulation. These improved in vivo pharmacokinetics have the potential to be applied to leverage the enhanced permeation and retention (EPR) effect for biodistribution to and gene silencing in vascularized tumors.

Gelatin is a hydrolyzed and denatured form of collagen, which comprises the majority of extracellular matrix. Despite its numerous advantages for tissue engineering, its use as a thermostable hydrogel has been achieved only recently by conjugating hydroxyphenyl propionic acid to the gelatin backbone, resulting in injectable in situ crosslinking hydrogels through a H2O2- and peroxidase-mediated reaction. Gelatin hydrogels gelled rapidly, and exhibited excellent biocompatibility in vitro where mesenchymal stem cells (MSCs) were encapsulated over 15 days. Furthermore, MSCs organized into tubular network and differentiated into an endothelial lineage purely by material effects. Finally, 2-week long subcutaneous implantation of gelatin hydrogels delivering MSCs in vivo confirmed the vasculogenic and angiogenic effect where implanted MSCs expressed Flk-1, an endothelial cell marker, and increased blood vessel formation was observed via microangiography. Histology showed no fibrous capsule formation around the gelatin hydrogel, and qPCR also confirmed favorable host macrophage responses by up-regulating MRC1, a reparative/regenerative macrophage marker, and down-regulating iNOS, an inflammatory macrophage marker. Based on these observations, we conclude that injectable in situ crosslinking gelatin hydrogel is a promising biomaterial for tissue engineering/regenerative medicine where robust angiogenesis and favorable host immune response are required.

Calorimetry has many applications in the physical and life sciences including measuring phase changes, determining reaction equilibria, detecting protein binding events, and quantifying enzyme kinetics. Towards the goal of creating more sensitive calorimeters, we examined nanowatt scale reactions utilizing commercial IR sensors. With this information, we created heat flow models to aid in the optimization of future device designs. From there, best in class nano-calorimeters with 375 pW/Hz1/2 resolution were fabricated and applied to the need for better point of care (POC) assays in the medical field. We developed nanoliter scale thermometric enzyme-linked immunosorbent assay (TELISA) for use in measuring the anti-cancer monoclonal antibody trastuzumab. We measured therapeutic levels of trastuzumab (10-100 µg/ml) in human serum to help enhance clinical outcomes and aid in further drug development. By utilizing standard ELISA reagents this assay can be applied to a broad range of analytes, bringing with it cost, sample, and time savings. In order to better manage metabolic diseases related to the loss of function of key enzymes and transporters in the metabolic pathway, we demonstrated POC detection of phenylalanine down to 5 mM. The incorporation of capillary microfluidic channels into our calorimeter allowed for automatic sample delivery from a finger prick blood draw. With improvement, this could lead to the first POC device for management of Phenylketonuria.

Calcific aortic valve disease (CAVD) is the predominant valvular disease in the developed world, affecting over five million individuals in the United States alone and manifests itself as a progressive disease resulting in the obstruction of left ventricular outflow, decreased cardiac output, and eventual heart failure. Presently, treatment for CAVD is limited to surgical aortic valve replacement, a high risk procedure especially for the population affected, and although significant advances have been made to reduce the associated risk with this procedure, a non-surgical treatment option is preferred. Unfortunately, current efforts to develop pharmacological treatments have been largely unsuccessful at preventing or slowing down the progression of CAVD; this lack of efficacy can be attributed to the incomplete understanding of the etiology of CAVD. Thus, focused attention must be placed on elucidating the underlying mechanisms of CAVD initiation and evolution in order to develop novel and effective pharmacological drugs. At the tissue level, normal supple leaflets are transformed into thickened, stiff, and calcified leaflets; these striking changes are attributed to the aberrant behavior of the resident cell population, the aortic valve interstitial cells (AVICs), which are believed to play significant roles in leaflet thickening and calcification. Investigating what factors contribute to AVIC differentiation into pathological phenotypes and further how they generate valvular calcification is essential to the understanding of CAVD etiology. It has been demonstrated that CAVD development is tightly regulated by biomolecular, mechanobiological, and genetic factors. Previous studies have focused on describing the effect of biomolecular cues on CN development; however, many mechanobiological and genetic factors that have significant in vivo relevance have not been thoroughly assessed. In an effort to gather insight towards CAVD processes in vivo, we believe that investigations into the role of mechanical strain and the effect of Notch1 mutation on AVIC biology and calcification would provide novel insights towards CAVD etiology that can contribute to the development of effect therapeutics.

The age-related risk of skeletal fracture is a significant problem in modern medicine, such that both hip fracture and osteoporotic fracture are listed in the World Health Organization top 12 sources of disease burden, with nearly 9 million osteoporotic fractures in the year 2000 alone. Dual energy X-ray absorptiometry or (DXA) is the clinical gold standard in the diagnosis of fracture risk for disease like osteoporosis. However, DXA based assessment of areal bone mineral density does not explain the age related increase in fracture risk. Despite the excellence in advances of X-ray based technologies and complementary etiological factors (FRAX), the DXA method is fundamentally limited to the assessment of the mineral phase of bone. Therefore growing efforts to improve fracture risk assessment have led to the development of several complementary tools to analyze bone quality beyond its mineral density. At the core of this interchange between laboratory science and clinical diagnosis, Raman Spectroscopy (RS) has become a critical tool for measuring bone composition. The stable chemical composition and crystalline nature of bone tissue makes RS a powerful tool suited for the needs of bone assessment. Despite significant work in identifying the RS features of bone tissue, little evidence correlates RS to fracture resistance of bone. This thesis focuses on the evaluation of RS as a clinically relevant tool for bone fracture resistance assessment. I will detail how I established the technique of manipulating light polarization to concurrently measure both the organization and composition of bone according to optical theory, despite the complications of biological tissue. Application of the methods will show how RS measures of organization explain bone brittleness when measures of composition do not. Finally, I will detail how multivariate expressions of RS describing the interplay between organization and composition led to the first nondestructive explanation of fracture toughness in the largest human sample study to date. The analysis reveals that fracture toughness is driven by microstructural heterogeneity and not bulk composition, with significant implications for our understanding of clinical fracture resistance and future designs applications of RS instruments to material quality.

Clinical translation of siRNA therapeutics has been limited by the inability to effectively overcome the rigorous delivery barriers associated with intracellular-acting biologics. Here, in order to address both potency and longevity of siRNA gene silencing, siRNA conjugated to palmitic acid (siRNA-PA) was paired with pH-responsive micellar nanoparticle (NP) carriers in order to improve siRNA stability and endosomal escape, respectively. Conjugation to hydrophobic PA improved NP loading efficiency relative to unmodified siRNA, enabling complete packaging of siRNA-PA at a lower polymer:siRNA ratio. PA conjugation also increased intracellular uptake of the nucleic acid cargo by 35-fold and produced a 3.1-fold increase in intracellular half-life. The higher uptake and improved retention of siRNA-PA NPs correlated to a 2- to 3-fold decrease in gene silencing IC50 in comparison to siRNA NPs in both mouse fibroblasts and mesenchymal stem cells for both the model gene luciferase and the therapeutically relevant gene PHD2. PA conjugation also increased longevity of silencing activity, as indicated by an increase in silencing half-life from 24 hours to 186 hours. Thus, conjugation of PA to siRNA paired with endosomolytic NPs is a promising approach to enhance the functional efficacy of siRNA in tissue regenerative and other applications.