One third of adults from ages 20 to 44 have untreated dental caries, typically treated through irreversible fillings and root canals (Centers for Disease Control and Prevention, 2012). Current technologies require professional consultation for caries detection – an inconvenient and costly process. Our objective is creating an optical system of detecting early caries formation to be performed at home on a weekly basis via spectral imaging of the plaque on teeth. The bacterial growth in plaque and demineralization of underlying enamel is detected by optically monitoring the fluorescent byproduct of bacterial metabolism, porphyrin. By imaging teeth in blue reflectance light and red fluorescence signal, concentration and growth of plaque deposits are measured using target versus background (T/B) pixel peak values (PPV) and T/B pixel count (PC) of PPV. Repeated measurements are plotted for plaque deposits and trends. Statistical analysis and comparison of T/B ratios and PCs trending can identify the most efficient measurement for locating plaque deposits most susceptible to caries development, prompting more efficient preventative care and triggering a dental examination. This new concept was simulated in vitro using a dental training model of teeth, measured applications of red stain, image stitching software (123D, Autodesk), image analysis software (ImageJ), and a mobile phone. Red stain was applied in increasing concentration and volume to both occlusal and interproximal crevasses that would most likely be missed by brushing and flossing. At each time period, a series of 2D images were acquired to reconstruct 3D models of simulated bacterial growth and were processed using ImageJ for T/B ratios of PPVs and PCs, which were plotted against each time period. T/B ratios of PPVs produced the most consistent measure of trending of the simulated growth, R^2 values 0.77, with statistically significant differences in variance with PC ratios (f-test).

Thrombotic microangiopathy is characterized by endothelial activation and microvascular thrombosis, and is associated with a group of life-threatening disorders, such arteriothrombosis, sepsis, and other small vascular diseases. With systemic inflammation, the arteriolar and capillary vessels are often found to be obstructed by platelet rich thrombi, leading to tissue ischemia and organ failure. Understanding the mechanism behind endothelial activation and induced thrombi formation during inflammation and vessel injury has important clinical implications that can lead to therapeutic development. Here, I developed a novel in vitro microvascular platform that mimics the in vivo arteriole architecture and function. This platform is generated based on a biocompatible polymer, polydimethylsiloxane, using injection molding. The device is lined with an extracellular matrix (ECM) using type I collagen for structural support. Human coronary artery smooth muscle cells (hCASMCs) are seeded within the bulk collagen to allow spontaneous reorganization and migration. A channel is created within the ECM using needle-based subtraction molding and is then seeded with human umbilical vein endothelial cells (HUVECs) to form an endothelium. Optimization was performed to provide consistent cell seeding of HUVECs within the channel, to allow cellular reorganization and alignment, and to form a functional small-diameter arteriole. We observed reorganization and alignment of both HUVECs and hCASMCs after 3 days of culture under continual flow. In addition, luminal patency was maintained with proper hCASMC contractile function. On-going studies include development of functional assays to recapitulate the inflammatory response and thrombi formation in arterioles. The study is expected to not only further elucidate the fundamental mechanism of arteriothrombosis, but to also provide a base platform to develop preventative or therapeutic medicine for those affected by these pathological phenomena.

The primary function of the kidneys is to filter waste from blood, and the kidney glomeruli are the major site for filtration. The glomerulus is a blood filtration barrier with low resistance to small molecules, but is relatively impermeable to macromolecules. Each glomerulus consists of a blood capillaries network with endothelial cells on a basement membrane, the other side of which is lined with specialized epithelial cells known as podocytes. The integrity and function of the glomerular filtration barrier is critical for kidney function, and disruption of this barrier correlates with kidney disease progression, and eventually kidney failure. Current research models for kidney disease include animal models, 2D, and 3D kidney epithelial cell cultures. However, animal models suffer from high cost and are too complex for studying detailed cellular interactions, while existing in vitro models lack the architecture and components to fully reproduce in vivo conditions. We propose a 3D microphysiological system that recapitulates the endothelial-epithelial interface found in the kidney glomerulus. We have developed a method to form independent microscale channels within a collagen I substrate, separated by a very thin layer of collagen. Collagen I is a native matrix protein that supports cell growth, invasion, and exchange of cellular signals. Primary endothelial and epithelial cells has been seeded into the channels to form closed lumina, whose overlaps form the endothelial-epithelial interface. However, culture conditions remain to be optimized to support both types of cells. Structure, morphology, and function of the interface are monitored by confocal microscopy, biochemical assays, and electron microscopy. When fully developed and validated, this culture system can be used to study the transport properties and injury responses of glomerular filtration. It can also provide a platform for drug screening to identify compounds toxic to the kidney, or therapeutic compounds for kidney diseases.

In the United States, kidney cancer has consistently been in the top 10 most prevalent cancers with 62,700 new cases and 14,240 fatalities each year. Renal cell carcinoma (RCC) derives from the epithelium of the kidney’s proximal convoluted tubule, and comprises the overwhelming majority of all kidney cancers. A critical step in the formation and spread of all cancers, including RCC, is angiogenesis: the formation of new blood vessels. Cancerous cells secrete numerous signals to recruit endothelial cells to form new blood vessels; these vessels provide primary tumors with oxygen, nutrients, and a path to metastasize throughout the body. While cell and animal models have been utilized to study angiogenesis and test anti-angiogenesis drugs in RCC, successes fail to translate in clinical human studies. Because of the added complexity of the human body, a better model to represent human RCC is required to study angiogenesis in vitro. In order to address this need, we are developing a tissue engineered vascularized RCC microphysiological 3D system. Microfluidic devices will be utilized to recapitulate the angiogenic interaction between blood vessels and RCC cells. Sprouting from the endothelial-lined channel, simulating kidney microvasculature, toward collagen suspended RCC cells will be analyzed to determine the efficacy of our device. In addition, we have unprecedented access to normal and cancerous tissue sample pairs from the same kidney cancer patient at the UW Medical Center. Cultures from the tissue sample pairs will be utilized to validate the device and display differences in angiogenesis between RCC and normal kidney tissue. Tissue pairs were also utilized in qRT-PCR to determine up regulated pro-angiogenesis genes in RCC. Commercial drugs along with those currently in development or clinical trials will be administered to our device to determine their efficacy in preventing angiogenesis in RCC.

Vessels of different organs have heterogeneous permeability to serve distinct physiological functions. Differences in basal vascular permeability may result from heterogeneity in the endothelial cells that line the vessel walls, which are influenced by many factors—including differences in the complexity of cell-cell junctions and the expression of various diaphragms that span the cell body. In addition, many diseased states are often associated with elevated levels of permeability, including viral hemorrhagic fever and tumor-induced angiogenesis. To understand such fundamental heterogeneity of the blood vessels and further investigate the change of vascular structure and function in disease scenarios, we characterized the permeability of endothelial cells harvested from different areas of the body. Human primary cells—including endothelial cells from various organs and epithelial cells from the proximal tubules of the kidney (PTECs)—were seeded on gelatin-coated transwells. We measured transendothelial electrical resistance (TEER) using a Millicell ERS Voltohmmeter, and determined solute permeability by tracking the perfusion of fluorescent dextran across the cell monolayer over time. Immunostaining was performed to observe monolayer formation, while transmission electron microscopy (TEM) was utilized to observe ultrastructure. Preliminary TEM images demonstrate distinct morphological differences between endothelial cell types, specifically in the complexity of cell-cell junctions. Investigations into the impact of these morphological differences on vascular permeability are ongoing, and a definitive conclusion will be made upon the study’s completion.

In the aging community, our ultimate goal is to lifespan and healthspan, the period of time that an individual lives healthily. Previous studies have found that aging results in decline of heart functions and increases prevalence of cardiovascular disease. Characteristics of cardiac aging include enlargement of the left ventricle, impaired diastolic functions, and reduced myocardial performance. Oxidative stress has been implicated as one of the mechanisms underlying cardiac aging. SS-31 peptide, a tetrapeptide that targets the inner membrane of the mitochondria, has been shown to interact with cardiolipin and enhance the electron carrying function of cytochrome c, an essential component of the electron transport chain. SS-31 peptide also limits oxidative damage to cardiolipin, a phospholipid that is almost exclusively located in the inner mitochondrial membrane, by reducing peroxidase activity of cytochrome C. SS-31 treatment has been shown to protect mitochondria in pressure-overload induced heart failure and oxidative injury in other tissues. In this study, we performed echocardiography and treadmill running and showed that 8 weeks of the SS-31 treatment improved diastolic function, myocardial performance, and enhanced exercise endurance in old mice. Using quantitative real-time PCR, I am assessing the gene expression changes regulated by the SS-31 treatment. Understanding the mechanism of how SS-31 improves cardiac aging will provide insight on potential therapies to improve cardiac health in the elderly populations.

Hydrostatic pressure is important to many vascular processes, including endothelial activation (inflammation), vasodilation, vasoconstriction and angiogenesis (branching of new blood vessels from existing ones). The problem of pressure regulation in in vitro studies is usually solved by syringe pump based systems. Harvard Apparatus provides a catalogue of “Constant Pressure Syringe Pumps,” the cheapest of which costs $4500. This is prohibitively expensive for many applications. Thus, there is a need to create an affordable device that can reliably regulate hydrostatic pressure. I present a syringe pump based system that maintains liquid pressure within a flow chamber at a fraction of the cost of commercially available devices. My pump was designed from open-source and off the shelf components, including a pressure transducer and Arduino Microcontroller, at a total cost of ~$350. I have tested the pump’s ability to maintain constant liquid pressure across a high-impedance microfluidic system and have shown that it maintains pressure at a user-defined set point between 0 and 70 cmH2O of gauge pressure. This device illustrates the power of open-source software and hardware, 3D-printing and rapid prototyping to allow labs to design customized hardware to meet specific research needs at a reasonable price. Once completed, I will use it to study the pressure dependence of angiogenesis and vasculogenesis (formation of new blood vessels by endothelial cells embedded in interstitium). We will vary hydrostatic pressure to optimize and document blood vessel network formation. This is relevant to studying embryonic development and tumor vascularization and to engineering grafts to repair the damage from myocardial infarction (heart attack). Other possible applications include eliminating the confounding variable of pressure variation in Doppler echocardiography or in studies of endothelial cell response to variation in microvessel geometries.

Extracellular matrices are more than structural scaffolds used merely to provide mechanical support to cell and tissue organization—rather, they provide physical and biochemical signals to change the cell structure and function. Here, my project is to investigate on the diffusion properties of human kidney extracellular matrix (kECM) and their impact on proximal tubular epithelial cells (PTECs) in an engineered 3D physiological system. Outcomes of the project include: 1) diffusion coefficients of dextran of different sizes in the matrices made from pure type I collagen, pure kECM, and mixtures of type I collagen and kECM (3:1, 1:1, and 1:3). 2) A standard protocol and an assistive Matlab program for diffusion measurement and vessel perfusion experiments. 3) characterization of structure, morphology and transporters (Immunostaining) of proximal tubular epithelial cells inside the flow-derived microphysiological system. I expect the study to provide supportive understanding of cell-matrix interactions and facilitate the further development of the bioengineered human kidney microphysiological system to study kidney function and response to drugs and toxins.