Over 38 million Americans have diabetes, with 90% of diabetic Americans having Type 2 Diabetes. Diabetes causes an increased risk of complications, including diabetic kidney disease (DKD), a disease that affects kidney filtration. This occurs in the glomerulus, a specialized capillary network of single-layered endothelial cells on one side and podocytes on the other, and their extracellular matrix (ECM). Injury, reduced function, or changes in the ECM of either of these cells cause abnormal filtration and kidney disease. Preliminary data from our group suggests that ECM remodeling driven by endothelial cells is a key feature in DKD in both our mouse model and in humans. Additional preliminary data from our lab indicates that two metalloproteases involved in ECM remodeling, ADAMTS6 and ADAMTS9, are increased in endothelial cells in diabetes via elevated lipids. My hypothesis is that very-low-density lipoprotein (VLDL), a lipid often increased in diabetes, induces increased endothelial cell ADAMTS6 and ADAMTS9 expression, contributing to remodeling the ECM and altering the filtration capacity of the glomeruli. To investigate this, isolated endothelial cells from non-diabetic mice were stimulated with varying VLDL concentrations, alone or with elevated glucose, to simulate diabetes. Following the stimulation, I isolated the RNA from these cells and measured the mRNA expression of Adamts9 and Adamts6 using real-time PCR. To test if endothelial cells isolated from mice with diabetes would respond differently, similar experiments are being carried out in cells isolated from diabetic mice. Western blots are used to verify the altered protein expression I observe. I am utilizing already acquired kidney sections from mice with diabetes with different lipid levels and determining the glomerular endothelial cell ADAMTS9 and ADAMTS6 expression by immunohistochemistry. Results from these experiments will help us understand the mechanisms through which endothelial cells respond to diabetes and thus contribute to DKD.

People with diabetes have an increased risk of developing cardiovascular disease (CVD). Hyperglycemia is the hallmark of diabetes, but diabetic dyslipidemia with increased circulating lipid levels is also present, which is believed to contribute to the augmented CVD seen in diabetes. The Cluster of Differentiation 36 (Cd36) receptor mediates fatty acid and lipoprotein uptake in macrophages. Lipid-loaded macrophages are a key feature of atherosclerosis, the underlying CVD pathology. Preliminary data suggest that monocytes (macrophage precursors) are lipid-loaded via increased Cd36. However, it is unclear what drives the increased Cd36 expression in diabetes. Reduction in blood glucose, but not lipid levels, in diabetic mice reduced monocyte cell surface Cd36 expression. Based on these preliminary data, I hypothesize that hyperglycemia induces increased Cd36 mRna expression in monocytes in diabetes. To address whether glucose or lipids increase monocyte Cd36 mRna expression, I will isolate monocytes from the bone marrow of non-diabetic mice and stimulate them ex vivo. To address if hyperglycemia alters Cd36 expression, I will stimulate monocytes with 4 glucose conditions: 5.6 mM, 15 mM, 30.6 mM D-glucose, and an osmotic control of 5.6 mM D-glucose and 25 mM L-glucose. The 5.6 mM represents non-diabetic blood glucose conditions. To address if dyslipidemia alters monocyte Cd36 expression, I will use the same 4 glucose conditions in conjunction with 50 µg/mL of VLDL, a triglyceride-rich lipoprotein that is elevated in diabetic dyslipidemia. Following a 24-hour stimulation, I will isolate monocyte RNA and use qPCR to determine the amount of Cd36 mRna. If elevated glucose induces an increase in Cd36 expression, this suggests that hyperglycemia stimulates increased Cd36 expression in monocytes in diabetes. However, if the presence of VLDL is required to observe an increase in Cd36 mRna, this indicates that dyslipidemia is needed for increased monocyte Cd36 mRna expression. Results from this study will help us understand the relationship between lipids and hyperglycemia in the context of diabetes-induced monocyte lipid loading.

Birth asphyxia is the inability of a newborn to begin and maintain breathing. Twenty-three percent of neonatal deaths globally are caused by birth asphyxia. Birth asphyxia results in a neurological injury called hypoxic ischemic encephalopathy (HIE). Rapid HIE screening within six hours after birth is crucial to identify neonates at risk. Unfortunately, the diagnostic equipment is impractical for low resource settings because it is costly ($20/test and $5,000 for equipment) and requires technical staff, that are in short supply, to operate. We hypothesize that a cost-effective device can be developed for HIE analysis. pHast Cam quickly screens for birth asphyxia and HIE in infants via a paper-based blood pH sensor. The device combines an inexpensive pH sensitive dye, a smartphone camera, and a fixture that controls the imaging environment to quickly identify acidosis from samples. A low-cost paper-based strip is made with a water-soluble resin doped with a pH-sensitive dye, bromothymol blue (BTB), and a membrane to filter out red blood cells. The fixture removes lighting variation. The smartphone camera records the pH indicator image, and an algorithm captures, reduces noise, and accesses color change. pHast Cam incorporates four features: 1) accurate assessment of acidity within 0.05 pH units, 2) require only a few microliters of sample, 3) use electrical hardware and software only from the smartphone, and 4) affordability. At this stage, we have achieved a regressive linear model that predicts buffered solution acidity (y=-589.32x+4684.05 R2=0.9857), with 95% confidence interval of 0.04 pH units. In the future, we will transition from measuring buffered solutions to blood-plasma. Ultimately, we expect pHastCam to screen for birth asphyxia, and other acid-base disorders, by quantifying plasma pH in neonates so that timely therapeutic interventions and plans to address long-term complications may occur.

Capillary microfluidics capitalize on surface tension effects encoded in microchannel geometry and chemistry to transfer liquids without external instruments, making them a user-friendly technology for point-of-care tests. For most applications, hydrophilic surfaces (contact angle < 90Ëš) are necessary to induce surface tension driven flow. Currently, this is achieved with vacuum plasma chambers that alter surface chemistry. Unfortunately, the hydrophilic properties made with plasma processing are temporary and unstable. Alternatively, an inherently stable hydrophilic 3D-printing resin containing polyethylene glycol diacrylate (PEGDA) and acrylic acid (AA) was recently developed for capillary microfluidics. However, this hydrophilic resin has not been thoroughly validated for inexpensive (<$300) liquid crystal display (LCD) printers. Our objective is to optimize and validate 3D-printing parameters including exposure time, UV power, layer thickness, and lift/retract speed using this hydrophilic PEGDA-AA resin with three LCD 3D printers (AnyCubic Photon Mono X 6K, AnyCubic Photon Mono M5s Pro, and Phrozen Sonic Mini 8K). Validation includes measuring hydrophilic properties as well as the dimensional fidelity of the printed channels compared to the design specifications. Our proof-of-concept prints on the Mono X 6K printer had average contact angle measurements of 42.8° ± 8.77. The percent differences between designed and printed channel lengths, widths, and depths were 31.5 ± 0.23%, 28.9 ± 3.41%, and 2.40 ± 13.9% respectively. By optimizing the print parameters of cost-effective 3D printers with the inherently stable hydrophilic resin, we enable capillary microfluidic technologies for users in low income/resource settings who may not have access to vacuum plasma chambers. Future work will explore additional resin modifications to encourage applications like spatial patterning of hydrophilicity and protein immobilization in microchips. [1]V. Karamzadeh, A. S. Kashani, M. Shen, and D. Juncker, “Digital Manufacturing of Functional Ready‐to‐Use Microfluidic Systems,” Advanced Materials, vol. 35, no. 47

Tissues like the meniscus, a wedge-shaped pad of connective tissue found in the knee, are fibrous and have complex architecture that regenerates poorly and undergoes active mechanical stimulation which modifies cell signaling and tissue health. In vitro models are beneficial for characterizing these interactions as they create a controlled environment where single variables can be altered. We previously used the J1 Mechanoculture bioreactor to apply strain on a fibrous polymer scaffold laden with primary meniscal cells and observed nonsignificant variances between testing groups with mock injury vs. no injury. Applied strain was modeled after physiological strain levels, ~10%. Based on the minimal changes in cell behavior observed in mock injury samples, it is likely that the mock injuries in conjunction with the applied strain did not induce comparable plastic deformation to that experienced post injury within the native meniscus. We hypothesize that increasing strain and applied force to achieve plastic deformation within the electrospun samples will create a fibrotic and apoptotic response like that in vivo. Ongoing work is analyzing how the bioreactor will interact with unaligned electrospun polymer samples with no cells present. This will demonstrate the optimal parameters to instigate a significant material response. By inducing significant changes to scaffold material properties and underlying structure, it is more likely cells with demonstrate fibrotic and apoptotic responses in vitro mimicking immediate cell reactions to meniscal injuries in vivo. This response will be assessed by assaying for fibrosis through αSMA activation and apoptosis by caspase-3 activation. On the conclusion of this study, we expect that greater applied stress and associated strain will cause more plastic deformation within the polymer scaffold. This can be applied to an in vitro meniscus injury model to better understand the response of primary meniscal cells to stress in an environment with disrupted mechanics.

It is well documented that women are predisposed to various musculoskeletal injuries, including hip labrum, Anterior Cruciate Ligament (ACL), and meniscus tears. Literature has detailed the regenerative potential of human mesenchymal stromal cells (hMSCs), and clinical trials have confirmed their medicinal application within tissue repair and healing, particularly in the musculoskeletal system. hMSCs are commonly used in the field of tissue engineering due to their immunomodulatory capabilities, differentiation capacity, and autograft availability. Across the lifespan, cells in male and female bodies experience varying levels of estrogen exposure, which elicits different responses. During in vitro cell culture, cells are typically exposed to sources of exogenous estrogens, including phenol red and fetal bovine serum (FBS). Although these additives are commonplace in the practice of cell culture, little research has been done to understand the impact of these additives on hMSCs in in vitro cultures, especially among female hMSCs. To investigate these cellular responses, we studied the impact of these estrogen-mimetic media components on cell proliferation and metabolism in vitro for hMSCs derived from male and female donors. Specifically, we investigated phenol red, which is shown to behave as an estrogen, and FBS, which naturally contains 17β-estradiol (E2). We hypothesized that estrogen-mimetic compounds would be associated with an increase in cellular proliferation and metabolism in a sex-dependent manner. These results clarify the response patterns of male and female hMSCs due to exogenous estrogen exposure, improving our sex-specific understanding of their potency for in vitro studies and regenerative medicine applications.