Doctor of Philosophy, The Ohio State University, 2020, Biomedical Engineering

The extracellular matrix is extensively reorganized throughout breast cancer progression. This reorganization contributes to cancer cell invasion and intravasation and is an independent prognostic factor for breast cancer patients. Cancer-associated fibroblasts appear to play a major role in this reorganization but the cellular signaling pathways contributing to this reorganization remain unclear. We show here that loss of the tumor suppressor phosphatase and tensin homolog (Pten) in fibroblasts promotes extracellular matrix alignment both in vitro and in vivo through increasing cell traction forces. Furthermore, low stromal PTEN expression correlated with high mammographic density, one of the major risk factors for breast cancer development. Matrix reorganization was concomitant with a marked increase in collagen deposition within the mammary gland. We therefore investigated the mechanism of collagen deposition and showed that loss of PTEN upregulated SPARC, which mediated both collagen and fibronectin assembly without modulating cell traction force. To further determine how Pten loss connected to matrix alignment, we designed a novel screening platform to examine matrix alignment in vitro using fibroblast-derived matrices, automated microscopy, and automated image analysis through MATLAB. We discovered a number of novel matrix alignment modulators, including protein kinase C (PKC), dual specificity tyrosine regulated kinase 1b (DYRK1b), platelet-derived growth factor receptor β (PDGFRβ), and Janus kinase (JAK), among others. The effect of these markers on patient survival was examined using publicly available patient datasets. Finally, we examined the effects of hypoxia and matrix metalloproteinase activity on matrix organization.

Committee: Jennifer Leight (Advisor); Samir Ghadiali (Committee Member); Jonathan Song (Committee Member) Subjects: Biomedical Engineering

MS, University of Cincinnati, 2024, Medicine: Biomedical Research Technology

The ECM plays an important role in the structure, function and signaling within tissues. Changes to the ECM can cause long-lasting alterations that have been seen to influence disease progression. In vivo models currently lack the technological capabilities to clearly visualize the ECM. The inability to study the ECM and monitor changes to the ECM in vivo has created a need to use in vitro models to learn more about the ECM. Using in vitro models in combination with in vivo models' researchers can mimic the in vivo ECM to visualize and study changes that occur within the ECM. Of the current in vitro models, a cell-derived matrix (CDM) assay can be used to study the composition, structure and cell-matrix interactions. Use of this model can show how the ECM changes in response to injury and disease. In this project, we adapted and troubleshooted a CDM assay published by Franco-Barraza et al., in 2017 to work with primary lung fibroblasts. We worked out the best way to allow cells to attach to the plate, grow, and then secrete matrix. Using confocal microscopy we evaluated the deposited ECM protein fibronectin. We worked out the protocol to work that works with murine primary lung fibroblasts. We can now use this adapted protocol for further testing of ECM protein secretion and studying how other cells use the produced matrix.

Committee: Anne-Karina Perl Ph.D. (Committee Chair); Jason Tchieu Ph.D. (Committee Member); Kristin Hudock (Committee Member) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2012, Biomedical Engineering

Cells can sense, signal and organize via mechanical forces. The ability of cells to mechanically sense and respond to the presence of other cells over relatively long distances across extracellular matrix (ECM) has been attributed to the strain-hardening behavior of the ECM. In this study, we explore an alternative hypothesis that the fibrous nature of the ECM makes long-range stress transmission possible, which could provide a mechanism for long-range cell-cell mechanical signaling. To test this hypothesis, we built 2-D and 3-D finite element models of stress transmission within cell-seeded collagen gels. To examine the role of collagen fibers in lateral stress transmission, confocal reflectance microscopy was used to develop 2-D image-based finite-element models. Models that account for the gel's fibrous nature were compared with homogenous linear-elastic and strain-hardening models to investigate the mechanisms of stress propagation. To examine the role of collagen fibers in cell thickness sensing, 3-D finite element models with idealized fiber networks were built, and the stress transmissions in fibrous and homogeneous ECM were compared. Finite-element analysis revealed that stresses generated by cell contraction are concentrated in the relatively stiff ECM fibers and are propagated farther in a fibrous matrix as compared to linear elastic or strain-hardening homogenous materials. These results support the hypothesis that ECM fibers, especially aligned ones, play an important role in long-range stress transmission. Further, fluid-structure interaction models were built to investigate the interplay between collagen fibers and interstitial fluid. The results suggest that in cell culture, cell movement is the key factor in defining fluid-flow development at the microscopic ‘cellular' level, and the cross-links are the key factor that determines the micro-mechanical environment.

Committee: Richard T. Hart (Committee Chair); Keith J. Gooch (Committee Member); Samir N. Ghadiali (Committee Member); Jun Liu (Committee Member) Subjects: Biomechanics; Biomedical Engineering

MS, University of Cincinnati, 2024, Engineering and Applied Science: Biomedical Engineering

Burn injuries are devastating and leave patients without their first line of defense – the skin. Today's advanced therapeutics for major skin loss includes implementation of tissue engineered skin substitutes, but even these are lacking in a few areas including substrate mechanical strength, elasticity, durability, encouraging angiogenesis, and innervation. Here, we aim to address the lack of innervation and mechanical strength of tissue engineered skin substrates by utilizing piezoelectric polyvinylidene-trifluoroethylene (PVDF-TrFE) for its biocompatibility, mechanics, and mechano-electrical properties to promote Schwann cell elongation and sensory neuron extension. In this work, we characterized electrospun PVDF-TrFE scaffolds over a variety of electrospinning parameters, including 1, 2, and 3 h aligned and unaligned fibers, to determine ideal thickness, porosity, and tensile strength to mimic in vivo skin tissue. We then electrically activated PVDF-TrFE through mechanical deformation from low-intensity pulsed ultrasound waves as a non-invasive means to trigger the piezoelectric properties of the scaffold and deliver electric potential to cells. Using this therapeutic modality, neurite integration in these in vitro tissue-engineered skin substitutes was quantified including neurite alignment, elongation, and vertical perforation into PVDF-TrFE scaffolds. Results show that stimulation with LIPUS promoted cellular alignment on the aligned scaffolds. Further, LIPUS stimulation significantly increased Schwann cell elongation and sensory neuron extension separately and in coculture on aligned scaffolds but significantly decreased the elongation and extension on unaligned scaffolds. This was also seen in cell perforation depth analysis into scaffolds which indicated that LIPUS enhanced perforation of Schwann cells, sensory neurons, and cocultures on scaffolds. Taken together, this work demonstrates the immense potential for non-invasive electric stimulation of an in vi (open full item for complete abstract)

Committee: Greg Harris Ph.D. (Committee Chair); Stacey Schutte Ph.D. (Committee Member); Leyla Esfandiari Ph.D. (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, Case Western Reserve University, 2024, Physiology and Biophysics

Functional elastin fibers within extracellular matrix of the arterial wall facilitate passive distensibility and recoil, which is critical to maintaining blood flow. Elastin fragmentation decreases functional elastin level and is associated with aging, contributing to age-related arterial stiffening, an independent risk factor for the development of hypertension. The effects of elastin deficiency in large elastic vessels are well-known, but the impact on resistance vessels, crucial for renal perfusion and blood pressure regulation, is less explored. Furthermore, elastin insufficiency in mice is associated with elevated blood pressure, which is more pronounced in male mice than female mice when compared to their respective wild-type counterparts. However, the mechanisms underlying the sex-related differences in elastin-insufficient mice have not been explored. Using an animal model of elastin haploinsufficiency (Eln+/-), we demonstrated that loss of elastin exacerbates structural and biomechanical properties of intra-renal arteries in young Eln+/- female mice. These changes manifest as increased vascular stiffness and increased fragmentation, leading to blunted responses to increased renal perfusion pressure and impaired renal autoregulation. We further explored whether renal dysfunction contributes to hypertension in these mice and whether this relationship is modulated by ovarian hormones. Our findings establish that ovarian hormones mitigate the hypertensive phenotype in female Eln+/- mice. Though these mice exhibit impaired pressure natriuresis response, the hypertensive phenotype is not sodium-dependent. Instead, the sustained elevation in blood pressure is in part driven by increased activity of the renin-angiotensin system (RAS). Additionally, diuresis and urine concentrating ability were found to be impaired in female Eln+/- mice despite increased aquaporin 2 channel expression. Furthermore, the reduced sensitivity to vasopressin (V2) receptor blockade in Eln (open full item for complete abstract)

Committee: Patrick Osei-Owusu (Advisor); George Dubyak (Committee Chair); Jessica Wagenseil (Committee Member); Julian Stelzer (Committee Member); Jeffery Garvin (Committee Member) Subjects: Physiology

PhD, University of Cincinnati, 2023, Medicine: Molecular and Developmental Biology

As approximately 2.5% of the U.S. population is diagnosed with cardiac valve disease, it is becoming increasingly important to understand the mechanisms governing cardiac valve development and disease. Cardiac valves ensure that blood flows in one direction through the heart. To function properly, the cardiac valve has a distinct structure composed primarily of three layers of concentrated extracellular matrix (ECM) interspersed with valve interstitial cells (VIC) and surrounded in a monolayer of valve endothelial cells (VECs). In valve disease, blood begins to flow in both directions as the ECM becomes disorganized. Thus, understanding the molecular mechanisms governing valve development disease can lead to the identification of novel pharmacological therapies. Recently, localized VEC populations with distinct molecular profiles have been identified. The Coapt-VEC population is localized near the tip of the leaflet on the ventricularis side of the aortic valve (AoV) while the Lymph-VEC population is localized to the fibrosa side of the AoV. Interestingly, the Lymph-VEC population expresses the transcription factor Prox1, a well-known master regulator of lymphatic valve development. In Chapter 2 of this dissertation, we overexpressed Prox1 in VECs on the ventricularis side of the AoV, resulting in an increase in Lymph-VEC and a decrease in Coapt-VEC gene expression. Furthermore, the ectopic expression of Prox1 resulted in an enlarged AoV with a myxomatous ECM phenotype, including increased deposition of collagens and proteoglycans and mis-localized elastin. Strikingly, we also found the Prox1 is naturally mis-expressed on the ventricularis side of the myxomatous AoV of a Marfan syndrome (MFS) mouse model with a mutation in the fibrillin1 gene (Fbn1C1039G/+). Naturally occurring ectopic Prox1+ cells colocalized with Lymph-VEC gene expression and corresponded with a loss of Coapt-VEC gene expression in MFS AoVs. Together, these results suggest that Prox1 is suffic (open full item for complete abstract)

Committee: Katherine Yutzey Ph.D. (Committee Chair); Douglas Millay Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member); Jason Shearn Ph.D. (Committee Member); Phillip Owens Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2023, Medicine: Neuroscience/Medical Science Scholars Interdisciplinary

Psychological loss is something that most people will experience in their lifetime. Losing something of value (e.g., interpersonal relationships, financial stability, secure housing, health) can erode well-being and negatively impact quality of life, generating a unique set of depression-like symptoms. While loss is common, it is difficult to track clinically and has received little attention in preclinical studies. Previously, our lab developed a protocol to study this experience of losing a positive stimulus in rats, called enrichment removal (ER). Environmental enrichment is a well-known positive, rewarding experience, and ER generates behavioral phenotypes that are reminiscent of loss symptoms in humans. Given this face validity, my dissertation work seeks to better understand the molecular mechanisms of loss using enrichment removal and a range of molecular and behavioral techniques. We first identified the basolateral amygdala (BLA) as an important region in ER. We then utilized a comprehensive multi-omics approach to identify novel candidate mechanisms in this region, identifying microglia and the extracellular matrix (ECM) as potential targets driving removal phenotypes. Further investigation into these candidates revealed that ER leads to a loss of function in microglia and an accumulation of ECM. The latter effects were especially intriguing, prompting further study of the matrix itself and the parvalbumin interneurons which it typically surrounds in the form of perineuronal nets. Overall, the present molecular results demonstrate that ER increases ECM in the BLA, decreasing overall plasticity in the region and altering parvalbumin phenotypes to promote inhibition. Next, we expanded our behavioral profiling of ER, focusing on behaviors regulated by the BLA. These results revealed an interesting phenotype of impaired salience evaluation, in which the strength of various stressors and the behavioral responses that they elicited were mismatc (open full item for complete abstract)

Committee: Teresa Reyes Ph.D. (Committee Chair); James Herman Ph.D. (Committee Member); Eric Wohleb (Committee Member); Diego Perez-Tilve Ph.D. (Committee Member); Robert McCullumsmith M.D. (Committee Member) Subjects: Neurosciences

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Engineering

Cancer metastasis is a complex, systemic, and non-random process requiring tumor cells to both adapt to and manipulate a multitude of microenvironments. Given this complexity, traditional 2D cell culture models offer insufficient structural and biological relevance, while animal models face obstacles in real-time analysis, experimental control, and translational success. As an alternative to address these barriers, this dissertation discusses development of tissue engineered microfluidic device-based tumor-on-a-chip platforms to isolate phases of metastatic colonization and study premetastatic microenvironmental changes. In this dissertation, this hydrogel-based technology was applied in three different aspects of metastatic progression. First, a thyroid metastasis-on-a-chip model was developed to study metastasis suppressor gene RCAN1-4 and its impact on downstream lung colonization. Second, 3D hydrogel scaffolds were implemented to investigate colorectal cancer-induced collagen remodeling by stromal fibroblasts and pericytes during premetastatic niche development. Third, observations of cancer-induced collagen remodeling were used to inform design of a liver premetastatic niche-on-a-chip model to further interrogate immune-myofibroblast crosstalk in response to colorectal cancer signaling and establish the relationship between this crosstalk and metastatic colonization.

Committee: Aleksander Skardal (Advisor); Daniel Gallego-Perez (Committee Member); Jennifer Leight (Committee Member); Jonathan Song (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Oncology

Bachelor of Science (BS), Ohio University, 2023, Biological Sciences

Fibrosis, a pathological process characterized by excess extracellular matrix (ECM) deposition, can occur in many internal organs and tissues in response to various stimuli. As fibrosis progresses, scarring occurs, which ultimately leads to tissue dysfunction and organ failure. Patients with acromegaly, a rare disease usually caused by a benign, GH-producing pituitary tumor, have been reported to have prominent ECM deposition and scarring in certain tissues, which is indicative of fibrosis. In bGH transgenic mice, which express high levels of bovine growth hormone, several tissues [white adipose tissue (WAT), heart, intestine, and kidney] demonstrate a fibrotic phenotype. However, there is no previous research that investigates various bGH tissues – particularly from mice derived from a single cohort – for fibrosis. Additionally, WAT fibrosis is associated with obesity and lipodystrophy, and seems to be particularly associated with excess GH. This study aims to investigate the role of different cell types and genes involved in the development and progression of WAT fibrosis and determine if fibrosis is increased in BAT, liver, quad, kidney, lung, and spleen of aged bGH mice. Results of this thesis included a striking observation of increased fibrosis in all bGH tissues examined. For WAT, decreases in fibrosis-associated RNA expression in 3-month-old bGH mice via qPCR analysis was only observed in the perigonadal depot and not the subcutaneous depot that has more prominent collagen deposition. Interestingly, we observed an intriguing increase in fibrosis-associated RNA expression in a population of adipose stem and progenitor cells in 6-month-old mice within subcutaneous bGH WAT. These results indicate a potential common GH-induced mechanism of fibrosis across bGH tissues and pave the way for future research into WAT fibrosis.

Committee: Darlene Berryman (Advisor) Subjects: Biology; Biomedical Research

PhD, University of Cincinnati, 2022, Engineering and Applied Science: Chemical Engineering

The use of biomaterials for tissue repair applications presents immense potential for regenerative medicine. Soft tissue such as skin, muscle, and nerves share a similar overarching molecular paradigm for initiating the process of repair after injury. Namely, the interaction between individual cells and the surrounding microenvironment is a clear predictor of regenerative success. Phenotypic changes in cells such as stem cells, glial cells, or other progenitor cells are, largely, determined by changes in the deposited extracellular matrix. However, as cells often play some role (whether direct or indirect) in secreting, assembling, and modifying the connective matrix, this interaction is dynamic and reflects the litany of considerations for human induced changes to the microenvironment. While there is great variance in the repair success of individual damage, the microenvironment of cells is typically a useful target for improving regenerative outcomes. This work models the interdisciplinary reach of biomaterial design by exploring the use of numerous engineered substrates to direct the behavior of cell line models for soft tissue applications. A piezoelectric, electrospun polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) was specifically identified as a model substrate for nerve repair. With extensive materials, chemical, and electrical characterizations, it was emphatically determined that PVDF-TrFE fibrous scaffolds can be used to precisely influence the microenvironment of 3T3 Fibroblasts and RT4- D6P2T Schwann Cells to promote a prolonged regenerative phenotype. Additional modifications in scaffold fabrication produced numerous methods for functionalization with bioactive, decellularized extracellular matrix (dECM) proteins that are known to contribute significantly to repair. Current clinical solutions for nerve damage consist of nerve grafts, nerve conduits, and neurorrhaphies. (open full item for complete abstract)

Committee: Greg Harris Ph.D. (Committee Member); Aashish Priye Ph.D. (Committee Member); Jonathan Nickels Ph.D. (Committee Member); Leyla Esfandiari Ph.D. (Committee Member) Subjects: Chemical Engineering

PhD, University of Cincinnati, 2021, Engineering and Applied Science: Chemical Engineering

Peripheral nervous system (PNS) injuries currently lack effective treatments for regaining full functional recovery, and thus remain a major challenge in healthcare. Schwann cells (SCs) as the principal glial cells within the PNS, play a vital role in peripheral nerve regeneration due to their inherent capacity for altering phenotype to enhance the regenerative capacity of the PNS post injury. However, the regenerative phenotype of SCs is challenging to maintain through the time-period needed for regeneration and can be impacted by the properties of the surrounding extracellular matrix (ECM) such as protein composition and stiffness. Furthermore, the properties of the ECM also regulate cell morphology including spreading area and cellular elongation, which directly impact SC regenerative capacity. Therefore, deciphering the complex interplay between SCs and the ECM to provide alternative therapeutic solutions for enhancement of regenerative potential in SCs leader to functional recovery in traumatic nerve injury is essential. To address the role the ECM plays in SC phenotype, this research mechanistically analyzes how matrix stiffness, cell morphology, and protein composition cooperatively control regenerative phenotype. In this work, ECM proteins were adsorbed onto mechanically tunable polydimethylsiloxane (PDMS) substrates, which provided a cell culture platform where stiffness and protein composition can be modulated. SCs were cultured on tunable substrates as critical cellular functions and transcriptional factors that represent the dynamics of SC phenotypes were assessed at differing time points. To illustrate the dynamic reciprocity between a regenerative phenotype and cell morphology, single-cell microcontact-printed cell adhesive patterns and line-patterned substrates were utilized, and SC regenerative phenotypes were characterized by immunofluorescence (IF) staining, western blot, and microarray assay. Lastly, SCs were grown on tunable or patterned sub (open full item for complete abstract)

Committee: Greg Harris Ph.D. (Committee Chair); Yoonjee Park Ph.D. (Committee Member); Aashish Priye (Committee Member); Jason Shearn Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, Case Western Reserve University, 2020, Pharmacology

Despite extensive advances in early breast cancer detection, diagnosis and treatment, effective cancer therapies to prevent tumor cell dissemination and overt metastasis remain an area of unmet clinical need. Many factors underlying metastatic processes have been identified; however, the central nodes that govern these elements are poorly understood. Our studies elucidate two nodal pathways that drive metastatic phenotypes in cancer cells: the activin/follistatin signaling pathway and the BCL11A-governed transcriptome. We found that breast cancers display decreased follistatin expression, a selective inhibitor of activin with high-affinity binding. Simultaneously, breast cancers also have increased expression of the INHBA subunit of activin, suggesting unabated activin signaling in this disease. Restoration of follistatin expression in mammary tumors arising in HER2/Neu transgenic mice completely abrogated metastasis to the lungs, indicating that follistatin is capable of suppressing metastatic progression. Follistatin is also prognostic of extended recurrence-free and overall patient survival. These studies suggest that therapeutics that inhibit activin signaling may be useful for the prevention of metastatic disease. We also present data demonstrating that the transcription factor, BCL11A drives optimal invasion and metastatic outgrowth in cell line and mouse models of triple-negative breast cancer (TNBC). We identified two pathways governed by BCL11A that facilitate its metastasis promoting properties. First, we found that BCL11A sustained the invasive capacity of TNBC cells by suppressing the expression of muscleblind-like splicing regulator 1, a splicing regulator that suppresses metastasis. This ultimately increased the levels of an alternatively spliced isoform of integrin-α6 that is associated with worse patient outcomes. We also uncovered that the BCL11A-controlled transcriptome was enriched in genes encoding matrisome proteins. Notably, matrix m (open full item for complete abstract)

Committee: Ruth Keri Ph.D. (Advisor); Marvin Nieman Ph.D. (Committee Chair); Donny Licatalosi Ph.D. (Committee Member); Mark Jackson Ph.D. (Committee Chair); Hung-Ying Kao Ph.D. (Committee Chair) Subjects: Molecular Biology; Pharmacology

Doctor of Philosophy, The Ohio State University, 2020, Biomedical Sciences

Mitral valve prolapse (MVP) affects 3-6% of the total population including those with connective tissue disorders. Current treatment options are limited, and patients commonly require surgery which can be impermanent and insuperable. MVP is diagnosed as increased length and thickness of the mitral leaflets with abnormal prolapse of the leaflets into the left atria, often accompanied by mitral regurgitation due to the inability of the leaflets to close and coapt. This is due to compromised biomechanics of the leaflet, caused by disturbances to the composition and organization of the extracellular matrix (ECM), that weaken mechanical function. This process, known as myxomatous degeneration, is characterized by an abnormal accumulation of proteoglycans, in addition to collagen fiber disruption and elastic fiber fragmentation. The underlying mechanisms that promote myxomatous degeneration to the point of biomechanical failure are unknown. However, previous histological studies of end-stage diseased leaflet tissue have reported abnormal alpha-smooth muscle actin (SMA) expression in a subset of heart valve interstitial cells (VICs) characteristic of activated cells, and these cells are localized within regions of myxomatous ECM changes. As the contribution of activated, SMA-positive VICs to valve disease has not been explored, the goal of this study is to explore the regulation, and contribution of these abnormal cells in the progression of MVP pathogenesis. To do this, we utilized mice harboring a Fbn1C1039G mutation that mimic human features of Marfan Syndrome and develop MVP, to examine temporal and spatial changes in SMA expression relative to myxomatous degeneration via histological techniques. We showed that SMA-positive VICs are present prior to myxomatous degeneration and increase in number throughout disease progression. In vitro, we further showed that SMA is required for expression of Collagen I and proteoglycans via direct and indirect knockdown of SMA in (open full item for complete abstract)

Committee: Joy Lincoln (Advisor); Brenda Lilly (Committee Chair); Timothy Plageman (Committee Member); Noah Weisleder (Committee Member) Subjects: Biomedical Research

Master of Sciences (Engineering), Case Western Reserve University, 2020, Biomedical Engineering

The project goal is to dissect the mechanical feedback of cells onto the extracellular matrix (ECM) using De Novo ECM synthesis and traction force microscopy (TFM). In this thesis, we devised a method to apply traction force microscopy to De Novo ECM. We first stimulated 3T3s to synthesize ECM (>=20μm in 10days) using ascorbic acid with experimental conditions of hyperglycemia. After cell removal by lysis, the ECM was measured with AFM and fresh cells were replated for TFM. To achieve TFM on de novo ECM, we developed a serial labeling approach to deposit carboxylated fluorescent beads during multi-day ECM synthesis. We observe that ECM stiffness is increased in high vs low glucose. We also observe a unimodal response of cell traction to increasing stiffness in polyacrylamide substitutes. With the generated TFM bead displacement data, we plan to analyze 3t3s replated onto bead-labeled de novo ECM to better understand the mechanical response of cells.

Committee: Samuel Senyo PhD (Committee Chair); Zheng-Rong Lu PhD (Committee Member); Ozan Akkus PhD (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, The Ohio State University, 2020, Biomedical Engineering

The extracellular matrix (ECM) occupies the interstitial space of tissue and provide a scaffold for cell attachment. The ECM composition and structure, regulated mainly by fibroblasts, confer tissue with mechanical stiffness and transport regulation functions. These functions become altered in disease such as cancer, where activated fibroblasts continuously alter ECM by excess deposition of ECM molecules such as collagen and hyaluronan leading to increased levels of stiffness and therapeutic resistance due to decreased transport across the interstitial space. Quantification of these ECM features in vivo has been limited due to challenges in experimentally manipulating ECM composition and estimating stiffness and transport parameters with controlled fashion. Furthermore, current in vitro techniques for quantifying matrix parameters have lacked a framework for characterization of ECM biophysical features as function of composition and fibroblast mediated remodeling. This dissertation describes a framework for the experimental characterization scheme of the biophysical properties of reconstituted acellular and fibroblast seeded hydrogels matrices based on indentation testing, quantification of transport via microfluidics, and confocal reflectance microscopy analysis. While methods for characterizing hydrogels exist and are widely used, they often do not measure diffusive and convective transport concurrently, determine the relationship between microstructure and transport efficiency, and decouple matrix mechanics and transport properties. The integrated approach enabled independent and quantitative measurements of the structural, mechanical, and transport properties of hydrogels as a function of ECM components and alterations to fibroblasts. I used fibrillar type I collagen as the base matrix and investigated the effects of 1) matrix supplementation with exogenous non fibrillar components and 2) fibroblast mediated regulation of hydraulic permeability in v (open full item for complete abstract)

Committee: Jonathan Song Ph.D. (Advisor); Gina Sizemore Ph.D. (Committee Member); Daniel Gallego-Perez Ph.D. (Committee Member) Subjects: Biomedical Engineering

Master of Science, The Ohio State University, 2019, Mechanical Engineering

Tumor growth and metastasis are believed to be promoted by alterations to the mechanical properties of the extracellular matrix (ECM). These mechanical alterations in tumors are also highly dynamic, but our understanding of the exact causes and consequences of tumor ECM remodeling is constrained by the current methodologies used to measure them. Therefore, the goal of this study was to better understand stability and matrix properties by developing a new sensor capable of detecting for alterations in the mechanical properties of the ECM with improved temporal and spatial resolution compared to current methodologies. To achieve this goal, we used the NanoDyn, a DNA origami nanostructure. Our study aimed to adapt the NanoDyn for measuring stability and behavior within biological material found in the ECM. The NanoDyn detection scheme consists of a double barrel structure which can be either in an open or closed state depending on the environment in which it is placed. The state of the device is found by fluorescence resonance energy transfer (FRET) due to the changing proximity of two fluorophores. By measuring stability and overlap concentration of well-characterized polymer solutions, the NanoDyn was initially characterized. The main objective of this thesis was to conduct stability testing for the NanoDyn in collagen and hyaluronan, which are two of the main constituents of the ECM using various measurement techniques. The NanoDyn has been found to be stable in hyaluronan, collagen, and solutions of combinations of the two for extended periods of time. In future investigation, the NanoDyn will be analyzed using single molecule experiments, flow experiments, and further stability studies in cell culture media.

Committee: Jonathan Song PhD (Advisor); Carlos Castro PhD (Committee Member) Subjects: Mechanical Engineering

Doctor of Philosophy in Engineering, Cleveland State University, 2019, Washkewicz College of Engineering

Controlled inflammation is crucial for normal wound healing. Our main aim in this study was to investigate the effect of loss of tumor necrosis factor-stimulated gene-6 (TSG-6) in cutaneous wound closure and inflammation. TSG-6 by its enzymatic action modifies the extracellular matrix molecule, hyaluronan (HA), through the transfer of heavy chain (HC) proteins from inter-α-trypsin inhibitor to form HC-HA complexes. Both TSG-6 and HC-HA have been associated with inflammation. Here, we showed that loss of endogenous TSG-6 and HC-HA in TSG-6 null mice results in significantly delayed wound closure and differential neutrophil recruitment compared to wildtype mice. Both of these phenotypes were successfully rescued by reintroduction of TSG-6 into null wounds. We also observed leukocyte recruitment behavior upon chemical injury and propose interesting differences between wildtype and TSG-6 null animals. Further, we showed that levels of the pro-inflammatory cytokine TNFα, and the presence of M1 proinflammatory macrophages, were elevated in TSG-6 null wounds compared to wildtype wounds. To facilitate the analysis of wound macrophages, we have described a detailed protocol to isolate single cells from cutaneous wounds. In a nutshell, our study indicates that TSG-6 is required for normal wound closure and plays an important role in regulating inflammation during wound repair.

Committee: Edward V. Maytin MD, PhD (Committee Chair); Nolan B. Holland PhD (Committee Member); Mark Aronica MD (Committee Member); Margot Damaser PhD (Committee Member); Carol de la Motte PhD (Committee Member) Subjects: Biology; Biomedical Research; Immunology; Molecular Biology

PhD, University of Cincinnati, 2019, Medicine: Molecular and Developmental Biology

Mitral valve insufficiency is a major source of morbidity and mortality worldwide and a common cause of heart failure, ventricular dysfunction, arrhythmias, and sudden cardiac death. This loss of unidirectional blood flow is often due to myxomatous valve degeneration (MVD), a progressive deterioration of the heart valves characterized by leaflet thickening, excessive accumulation of glycosaminoglycans, and disruption of elastin and collagen fibers. Insight into early disease progression is lacking because valves are generally examined once patients are symptomatic and undergo surgery or autopsy, thereby representing advanced disease. As a result, the molecular and cellular mechanisms underlying MVD pathogenesis and disease progression continue to remain elusive and no pharmacological therapies exist to prevent or reverse this condition. Instead, surgical valve repair or replacement remains the current standard of care, despite the need for re-operation as a result of limited durability and/or growth potential of the transplanted valve, particularly for pediatric patients. In recent years, there have been significant advances in our understanding of cardiac valve disease and a greater realization that acquired valve disease is not simply a passive degenerative process. A prevailing theory is that valvular endothelial cells (VECs) lining the surface of the heart undergo an endothelial-to-mesenchymal transition (EndMT) event that converts them into highly invasive and synthetic mesenchymal valvular interstitial cells (VICs). This view, however, has been controversial because it has been largely based on large surgical animal models that lack the capability to temporally track the fate of VECs during disease. In Chapter 2, we test this hypothesis by genetically labeling VECs and tracking them over time in a mouse model of Marfan syndrome (MFS) that exhibits MVD. We find no evidence of an EndMT event and instead discover hematopoietic cells within the diseased (open full item for complete abstract)

Committee: Katherine Yutzey Ph.D. (Committee Chair); James Cnota M.D. (Committee Member); Tony De Falco Ph.D. (Committee Member); Vladimir Kalinichenko M.D. (Committee Member); Jason Shearn Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, University of Akron, 2019, Biomedical Engineering

Decellularized myocardial tissue has shown great promise as a biological scaffold for cardiac repair and regeneration. Compared with other natural or synthetic biomaterials, decellularized myocardial tissue provides many benefits including the preservation of cardiac-specific microstructure, ECM composition, and perfusable hierarchical vascular bed, which supports the cell attachment, growth, migration, and differentiation. Because of these advantages, decellularized myocardium has been used to construct engineered myocardial tissue (EMT) at various complexities. However, current EMTs fabricated using decellularized myocardium have not been able to precisely mimic native cardiac tissue in terms of cell density, cell distribution, vasculature, and communications among cells. One of the main technical barriers that researchers are facing is the lack of the effective approaches to recellularize acellular myocardium. To address this issue, we aim to develop an innovative recellularization strategy to facilitate the fabrication of EMT using decellularized porcine myocardial slice (dPMS). In this dissertation, we examined the key variables affecting the interaction between dPMS and reseeded stem cells. We focused on investigating three major recellularization parameters during our strategy development: optimal scaffold thickness, cell source, and seeding method. Based on our results, human mesenchymal stem cells (hMSCs) were chosen to be used as one of the cell sources to promote vascularization. Cardiomyocyte sheet derived from the human induced pluripotent stem cells (hiPSCs) was used to improve cell density and cell-to-cell communications. Bilateral cell seeding method was used to enhance uniform cell distribution throughout the dPMS. The influence of types of stem cells, seeding strategy, and scaffold thickness on cell attachment, infiltration, proliferation, cardiovascular differentiation, and scaffold degradation were thoroughly investigated. Additionally, two innov (open full item for complete abstract)

Committee: Ge Zhang (Advisor); Jiang Zhe (Committee Member); Rebecca Willits (Committee Member); Rouzbeh Amini (Committee Member); Yi Pang (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, The Ohio State University, 2018, Comparative and Veterinary Medicine

Osteoarthritis (OA) is a progressive disease associated with cartilage injury and is the most common form of arthritis, affecting millions of people worldwide. Most common cartilage healing and treatments have unsatisfactory outcomes due to the inherently limited repair capability of cartilage. The goal here was to produce a sConstruct from decellularized synovial-derived extracellular matrix (sECM) seeded with synovial-derived mesenchymal stem cells (sMSCs) that could house normal or engineered sMSC with little immune reaction while improving cartilage healing. The first part of this work investigates the sMSC migration, differentiation, and distribution into the sECMs as determined by CD90, viability, histologic morphology, expression of GFP, BMP-2, hyaluronic acid (HA), and proteoglycan (PG). At day 14, sMSCs were viable, had multiplied 2.5-fold in the sECMs, had a significant decrease in CD90 expression and significant increases in HA and PG expression. Seeding with BMP-2-sMSCs enhanced the expression of BMP-2, and increased soluble HA and PG. These results indicate sMSC produce anabolic agents and differentiate in the sECM. The second portion of the thesis has two parts; 1) an in vitro model where the sConstructs were co-cultured with chondrocytes, and 2) in vivo, placing sConstructs adjacent to a cartilage lesion in a rat knee. The in vitro study showed increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte co-cultures increased the sConstruct sMSC production of HA, PG, and BMP-2 in a positive feedback loop. 2) In the in vivo study, sECM alone, GFP- or BMP-2-sConstructs were implanted adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area was resected and gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct im (open full item for complete abstract)

Committee: Alicia Bertone (Advisor) Subjects: Animal Sciences; Veterinary Services