Knee meniscus tears are common musculoskeletal injuries that have difficulty healing on their own. Required surgical interventions often fail to restore the function of damaged tissue resulting in the need to develop more effective therapies. To do so, it is necessary to develop in vitro models for further investigation of mechanisms of regeneration in the meniscus. To model the mechanical environment of the meniscus, our lab developed a pentenoate-functionalized hyaluronic acid (PHA) hydrogel system with tunable elastic, compressive moduli. However, viscoelastic properties, assessed via stress relaxation time, also dictate cellular behaviors that are favorable for regeneration. The objective of this study is to tune the viscous response within the PHA hydrogel system without significantly impacting the compressive modulus. I hypothesize that viscoelastic properties can be modulated by tuning non-covalent chain entanglement by increasing the amount of un-crosslinked (non-functionalized) HA (UHA) in the PHA hydrogel system. Specifically, I expect that increased entanglement will increase the viscous response without significantly altering the compressive modulus. I performed multiple iterations of stress relaxation and uniaxial compression tests on 0, 0.5, 1, and 2% UHA hydrogels on a dynamic analysis system. I found that increasing the amount of UHA in the hydrogel system had no statistically significant effect on the compressive moduli or stress relaxation half-times, suggesting that modulating the UHA in the hydrogel system allowed me to maintain elastic properties but does not yet allow fine control over the viscous properties. Limitations of the DMA indicate the need to also perform rheological assessments to analyze stress relaxation using constant shear strain. Future directions also include seeding meniscal cells on the hydrogels to assess the impact of viscoelastic properties on the regenerative behavior of meniscal cells. Ultimately, results from these studies will contribute to the development of regenerative therapies for meniscal tears.

The meniscus is a fibrocartilaginous tissue that sits in the knee and acts as a stabilizer and shock absorber. Meniscus tears are a prevalent injury among athletes in contact sports. Severe tears often do not heal, even with surgical intervention and synthetic replacements. Our lab seeks to develop treatments that encourage functional regeneration of the original cartilage-like tissue. A critical initial step toward this goal is understanding the specific cell types within the meniscus and the specific extracellular matrix (ECM) proteins that these cells produce.  Our ultimate goal is to find the cells that can best regenerate the damaged matrix after injury. My project entails comparing NIH/3T3 cells, a well researched immortalized mouse fibroblast cell line, to the cells of interest: human meniscal fibrochondrocytes (MFCs), the predominant ECM-synthesizing cells within the meniscus. I am comparing the expression of known and newly-identified ECM protein markers at different stages of in vitro cellular growth via fluorescent antibody staining and confocal microscopy. These images can be compared using analysis tools like ImageJ to quantify the amount and localization of matrix protein expression. I will use relevant quantifications, including  the percentage of total cells expressing each protein and the percentage of surface area covered by each protein. 3T3 cells are far more robust and resilient than MFCs, they replicate more readily, and I hypothesize that they will produce a greater quantity of ECM in a given timeframe. However, as both cell lines are fibroblasts, I expect the localization and percentage of cells expressing each protein to be similar. Comparing the expression and localization of ECM proteins across these two different cell types allows us to better characterize MFCs in a robust way, contributing a novel functional characterization to the literature.

The meniscus is a crescent-shaped fibrocartilaginous structure in the knee joint that plays a crucial role in weight distribution, shock absorption, and joint stability. Women experience higher rates of meniscal tears when controlled for sport and tend to have worse clinical outcomes following treatment. While surgery remains the standard treatment, regenerative therapies using human meniscal fibrochondrocytes (MFCs) have shown promise in repairing damaged tissue and improving joint stability. However, repeated culture of primary MFCs on tissue culture polystyrene (TCPS) is known to alter cell phenotype, leading to loss of native function. These phenotypic changes remove our ability to accurately model differences that are seen in vivo, such as sex differences. One approach to mitigate phenotypic change is culturing MFCs in a 3D environment, which more closely mimics the native extracellular matrix (ECM) and helps maintain cell phenotype. Little research has been done to assess whether 3D cell culture systems preserve sex-based differences in meniscal tissue. Sodium alginate beads offer a well-characterized, accessible, and cost-effective 3D tissue culture system designed for fibrochondrocytes. These beads are formed via ionic cross-linking between sodium alginate and calcium chloride solution. Studies have demonstrated that sodium alginate can maintain cell phenotype in chondrocytes, making it a promising alternative to TCPS for MFC culture. To address the issue of phenotypic changes, we cultured MFCs in sodium alginate beads and examined their ability to preserve sex differences in vitro. Previous data from our lab indicates that female MFCs express higher levels of decorin (DCN), a key ECM regulator protein, compared to male MFCs. Therefore, to determine whether the 3D structure of sodium alginate beads better supports the native phenotype of MFCs by maintaining sex differences, we analyzed DCN immunostaining. These findings establish an in vitro system that preserves and facilitates the study of sex differences observed in vivo.

Diabetes results in hyperglycemia and elevated lipid levels (diabetic dyslipidemia), both of which contribute to complications such as atherosclerotic cardiovascular disease. Preliminary data from our laboratory suggest that monocytes are lipid-loaded in diabetes, and the Cluster of Differentiation 36 (CD36) receptor mediates monocyte lipid-uptake. My preliminary data indicate that Cd36 mRNA expression increases in monocytes treated with high glucose. Thus, I hypothesize that hyperglycemia augments uptake of the Very-low density lipoprotein (VLDL), a triglyceride-rich lipoprotein (TRL) elevated by diabetes. To address this, bone marrow monocytes will be cultured in low and high glucose, ex vivo. An osmotic control will be included. Following the glucose stimulation, monocytes will be challenged with fluorescently labeled VLDL (Dil-VLDL), and the uptake will be measured by fluorescent microscopy. Furthermore, to verify that CD36 is critical for monocyte VLDL-uptake, bone marrow monocytes from control and CD36-deficient mice will be used. This study will help us clarify the relationship between lipid metabolism and hyperglycemia in diabetes-induced monocyte lipid loading.

Over 38 million Americans have diabetes, and over 90% of people with diabetes have Type 2 Diabetes. Diabetes increases the risk for complications, including Diabetic Kidney Disease (DKD), a disease which impacts filtration of the kidneys. This filtration occurs in the glomerulus, a specialized capillary network lined with a single layer of endothelial cells. The glycocalyx, an extracellular matrix (ECM), produced by the endothelial cells, plays a crucial role in regulating filtration. Injury, reduced function, or changes in the ECM of endothelial cells cause abnormal filtration and kidney disease. Previously generated single-cell RNA sequencing data from our lab and the Kidney Precision Medicine Project indicated an upregulation of genes involved in ECM remodeling, such as Adamts6, in diabetes, both in mice and humans. My hypothesis is diabetes induces degradation of the endothelial cell glycocalyx through increased expression of ECM degrading enzymes, contributing to glomerular endothelial cell dysfunction. To test if diabetes induces ECM degradation, I examined the abundance of glomerular glycocalyx in a mouse model of DKD in Type 2 Diabetes and in nondiabetic mice. Glycocalyx levels were assessed using wheat germ agglutinin staining and quantified through immunohistochemistry and flow cytometry. Diabetes significantly reduced glomerular glycocalyx in diabetic versus nondiabetic mice. Additionally, I investigated the role of hyperglycemia and dyslipidemia individually on ECM degradation in cultured glomerular endothelial cells. Further investigation will focus on the role of ADAMTS6 on ECM remodeling in diabetes by using a siRNA to block ADAMTS6 expression in cultured endothelial cells. Additionally, to further test the role of hyperglycemia in glycocalyx degradation, an SGLT2 inhibitor was given to diabetic mice to reduce blood glucose levels and examine the impact on endothelial cell glycocalyx. ECM remodeling may be induced by increased expression of ECM-degrading enzymes in diabetes, contributing to the glomerular filtration barrier breakdown seen in DKD.