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

Severe tissue injuries pose a significant challenge in therapeutic interventions, often resulting in suboptimal functional recovery due to inadequate regenerative signals. Traditional clinical methods, such as autografts, have variable success rates, partly because they fail to provide the necessary cellular and tissue guidance for advanced regeneration. This dissertation investigates the potential of electrospun polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE), a piezoelectric polymer, in creating a conducive environment for tissue regeneration. The inherent piezoelectricity of PVDF-TrFE generates electric charges under mechanical stress, actively engaging tissue cells, including fibroblasts essential for extracellular matrix formation and Schwann cells vital for peripheral nervous system healing, thereby promoting nerve function and broad tissue regeneration. This research focuses on PVDF-TrFE scaffolds, specifically electrospun piezoelectric nanofiber scaffolds, meticulously characterized for their material and electrical attributes. The study explores the incorporation of decellularized extracellular matrix (dECM) to enhance the bioactivity of the scaffolds, introducing bioactive components that provide better functional repair. Additionally, the effects of ultrasonic stimulation on these scaffolds are examined, aiming to induce electrical stimulation through mechanical deformation triggered by sound waves, thereby enhancing cellular activity to aid nerve regeneration. Experiments with NIH-3T3 fibroblast cultures on these scaffolds show increased metabolic activity following ultrasound stimulation, illustrating the scaffold's capability to support cellular processes essential for peripheral nervous system recovery. The dissertation also examines the impact of post-processing annealing on PVDF-TrFE scaffolds, analyzing how different annealing temperatures affect their crystallization behavior, morphology, mechanical properties, and piezoelectric responsivene (open full item for complete abstract)

Committee: Leyla Esfandiari Ph.D. (Committee Chair); Daria Narmoneva Ph.D. (Committee Member); John Martin Ph.D. (Committee Member); Greg Harris Ph.D. (Committee Member) Subjects: Biomedical Engineering

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

Micro- and nanoscale technologies can be engineered to modulate the biochemical cues expressed by adipose tissue, thereby influencing their fate and reprogramming cascade. By leveraging these revolutionary platforms, regenerative therapies can be tailored to the unmet specific needs of patients, amplifying controlled tissue repair, wound healing, and immunomodulation. Adipose tissue-related abnormalities are characteristic of a disease state but engineering of in vitro or in vivo models can be an optimistic avenue to study, explore, and mitigate such irregularity. This could potentially be achieved by mimicking the surface energy and topography of the extracellular matrix (ECM). The presented research highlights promising micro/nanotechnology-based contrivances for augmenting adipose tissue-related cellular and reconstructive therapies. Micro/nanotechnology-based platforms hold immense, yet still untapped potential of adipose tissue-derived stem cells to advance personalized treatments. Thus, the technologies described here in the research and their future derivatives could usher in a new era of regenerative medicine by lending intelligent solutions for more effective patient outcomes. The first chapter therefore provides an overview of adipose tissues intricacies and how micro- and nanotechnology can empower our understanding of the same to perform in-situ cell transformation or develop biomaterials which could bolster cellular microenvironment. The second chapter introduces promising direct reprogramming of one cell to another cell type using implantable micro- and nano-channel based gene reprogramming therapy (TNT) device. Such a device offers cellular reprogramming with precision and can transform a fibroblast into brown adipose tissue. The third chapter focusses on a different route using injectable electrospun biopolymer for tissue reconstructive purposes. The last chapter brings a holistic viewpoint highlighting the potential of micro/nanotechnologies fo (open full item for complete abstract)

Committee: Daniel Gallego Perez (Advisor); Derek Hansford (Committee Member); Natalia Higuita Castro (Committee Member); Kristin Stanford (Committee Member) Subjects: Biomedical Engineering; Genetics; Nanoscience; Nanotechnology; Neurosciences

Master of Science, The Ohio State University, 2023, Biomedical Engineering

Development of Tissue-Engineered Vascular Grafts (TEVG's) for use in treatment of pediatric cardiovascular disease offer a promising alternative to current synthetic graft models, which typically result in the patient needing additional surgeries to correct for complications related to the inability of these grafts to assimilate into the body. Understanding this potential for TEVG performance improvement, we saw opportunity in optimizing the scaffolds design. Herein, we utilized an accelerated degradation mechanism in a basic environment to begin to characterize the degradation space of our six novel scaffold variants: comparing their microstructure, chemical composition, and mechanical integrity at nine different timepoints (Day 0 (control), 1, 3, 5, 7, and 9) to our clinical TEVG scaffold. Current analyses have shown differing chemical compositions of PGA, PLA, and PCL for each scaffold at baseline (day 0), as well as similarities and differences between mechanical and microstructural characteristics of each scaffold variant. While initial degradation of samples is complete, data collection and analysis for this study is ongoing. Once complete, this data will be applied to a multiscale fluid-solid growth (mFSG) computational model that can identify future scaffold designs for optimizing the microstructure, degradation rate, and mechanical profile of the TEVG polymeric scaffold.

Committee: Daniel Gallego-Perez (Committee Member); Christopher Breuer (Advisor) Subjects: Biomedical Engineering

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

A trachea defect from malformation or obstruction can result in significant morbidities and mortalities. Long segment tracheal defects, where the length of the defective tracheal segment is >50% for adults and >30% for the pediatric population, are often fatal due to the lack of autologous tissue suitable for reconstruction. Tissue engineered tracheal grafts (TETG) have the potential to be used to replace diseased tracheal tissue. However, TETG success is often limited by delayed epithelial formation and biocompatibility. The Chiang lab developed a partially decellularized tracheal graft (PDTG) that removes the immunogenic epithelium and lamina propria while preserving donor cartilage that is immune-protected. We have previously shown that PDTG can support host-derived neotissue formation but the composition, long-term outcome, and drivers of PDTG neotissue formation have been largely uncharacterized. The characterization of PDTG neotissue will facilitate the translation of PDTG into large animals as we better understand the regenerated tissue and possible drivers behind neotissue formation. This dissertation aims to examine PDTG neotissue using a combination of immunofluorescent and transcriptomic approaches to characterize PDTG neotissue before uncovering drivers of tracheal graft epithelialization. Functional vasculature in PDTG was established by 2 weeks post-implant. Graft epithelialization was reestablished by 1 month. PDTG neo-epithelialization is driven by basal progenitor cells that were able to proliferate and differentiate into secretory and multiciliated cells. This neo-epithelialization process mirrors the epithelial repair processes seen in epithelial injury models. There was also a macrophage-dominant immune response post-implantation for both PDTG and syngeneic transplants. I then examine the immune response associated with graft implantation to study the impact of macrophages on graft epithelialization. To do that, macrophage phenotypes in the (open full item for complete abstract)

Committee: Tendy Chiang (Advisor); Susan Reynolds (Committee Member); Kaitlin Swindle-Reilly (Committee Member); Christopher Breuer (Committee Co-Chair) Subjects: Biomedical Engineering; Medicine

Doctor of Philosophy, The Ohio State University, 2022, Biomedical Sciences

Regenerative medicine has the potential to revolutionize the field of surgical medicine. More specifically, tissue-engineered vascular grafts (TEVGs) offer a promising solution to current challenges associated with the use of synthetic conduits in cardiac diseases. Since its first use in humans back in 1999 numerous advances have been made describing the remodeling, performance, and outcomes of TEVGs; however, the barriers to its widespread adoption remain largely the same. First, there remains a tendency for the lumen of TEVGs to narrow due to excessive tissue formation. Second, an issue broadly implicated within the field of cardiothoracic surgery, is the development of adhesions following repeat operations thus hindering access and function. This dissertation seeks to overcome both issues through the application of novel therapeutic agents. The findings reported further advance mechanistic knowledge of the regenerative process that may one day improve outcomes of associated with tissue engineering.

Committee: Christopher Breuer (Advisor); Philip Binkley (Committee Member); Jeffrey Parvin (Committee Member) Subjects: Biology; Biomedical Engineering; Biomedical Research; Cellular Biology; Medicine

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Tissue engineered scaffolds and regenerative medicine-based therapeutics hold great potential for a growing patient population in need of alternative tissue replacements. The initial work of this dissertation is on efforts to improve the translational capability of tissue engineered vascular grafts (TEVGs) to the clinic. A challenge in translating our group's TEVGs, as well as is seen with other tissue engineered scaffolds, is balancing the host response where an appropriate amount of healthy neotissue is created and remodeled overtime replacing the biodegradable scaffold and avoiding complications such as graft thrombosis and stenosis. Approaches to optimize tissue engineered scaffolds for use in patients often focuses on material alterations, cell seeding, bioreactor growth, or drug/small molecule co-administration. Seeding our TEVGs with bone-marrow derived nucleated cells has proven to be an effective approach to minimize graft occlusion and alter neotissue development; however, the exact mechanism underlying this remains unclear. The initial focus of this dissertation sought to elucidate what effect interleukin-10, an anti-inflammatory cytokine, had on graft patency and neotissue development from cells seeded onto TEVGs, from the host TEVG recipient, and from a recombinant protein drug delivery. This work demonstrated interleukin-10 from the host was critical in maintaining TEVG patency. Another promising approach optimizing a thrombosis and stenosis resistant TEVG has been our group's investigations into a novel wound healing modulator known as the lysosomal trafficking (LYST) protein. The protein, encoded by the LYST gene, is poorly understood with much of existing information coming from observations into disease states and cellular dysfunction that occurs in presence of a LYST gene mutation. A notable cellular finding is the perniculear clustering of enlarged lysosomes in mutant LYST cells due to defects in lysosomal fusion/fission. We serendipitously (open full item for complete abstract)

Committee: Christopher Breuer (Advisor); Ginny Bumgardner (Committee Member); Ryan Roberts (Committee Member); David Dean (Committee Member) Subjects: Biomedical Engineering; Cellular Biology; Experiments; Histology; Immunology; Medicine

Master of Science, Miami University, 2021, Chemical, Paper and Biomedical Engineering

Newts have exceptional capability of regenerating the lens throughout their lifetime. Since the 1890s, lens regeneration has been documented with ex vivo imaging techniques only. For the first time, we demonstrate that Optical Coherence Tomography (SD-OCT) can capture the in vivo essential morphological characteristics with abundant dynamic features. Monitoring lens regeneration using a single newt is now possible. The results show that the lens originates below the pupillary margin of the middle dorsal region and its early regenerating lens has an irregular elliptical shape. The lens volume expands quadratically, where its regenerating rate is linear from 14 to 60 days post-lentectomy. These findings warrant future research for tailoring OCT to study newt lens regeneration in vivo dynamically.

Committee: Hui Wang Dr (Advisor); Katia Del Rio-Tsonis Dr (Committee Member); Justin Saul Dr (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Engineering

Tissue engineered vascular grafts (TEVGs) have the potential to advance the surgical management of infants and children requiring congenital heart surgery by creating vascular conduits with growth capacity. While TEVG development has progressed much in recent years, many developments have been based on empiric design changes with limited animal numbers, hindering detailed understanding and external validity of the results. Rational design of TEVGs, combining careful experimental design with computational modeling and statistical methods, has the ability to accelerate next generation TEVG development through gaining mechanistic understanding of the mechanisms at play in neotissue formation and remodeling. To this end, this dissertation seeks to individually examine several of the mechanisms at play guiding neovessel formation and remodeling, namely: mechanical properties of implanted scaffolds, biologic properties of the recipient, and the effects of long term implantation in a large animal model on neotissue formation and remodeling.

Committee: Christopher Breuer MD (Advisor); Keith Gooch PhD (Committee Member); Mitchel Stacy PhD (Committee Member); Stuart Cooper PhD (Committee Member) Subjects: Biomedical Engineering; Medicine; Polymers

Doctor of Philosophy, Case Western Reserve University, 2020, Biomedical Engineering

There is a widely known mismatch between the number of donor organs available and patients on waiting lists in need of such organs. Indeed, many patients die while waiting on transplants while others suffer a significant decline in health. Tissue engineering presents an attractive alternative to organ transplantation to treat tissue defects and failing tissues. Many studies have shown the ability to generate constructs with phenotypic properties similar to native tissue. However, a major impediment to the clinical translation of tissue engineering technologies is that the size of these constructs is limited by the diffusion capacity of oxygen and nutrients. Therefore, there is a critical need within the field to develop a microvasculature within these constructs to facilitate oxygen and nutrient delivery and waste removal from constructs of clinically relevant dimensions. This thesis attempts to make progress towards such a microvascularization strategy under the hypothesis that co-culture aggregates may be able to allow self-assemble of a microvasculature within them while the aggregates themselves and be made into controlled geometries for tissue engineering applications. In an attempt to mimic native embryonic vasculogenisis, human endothelial cells and human mesenchymal stem cells were cultured together in a co-culture cell aggregate system that allowed for extensive cell-cell interactions. This is in contrast to biomaterial scaffold or hydrogel based tissue engineering approaches which seed cells onto a matrix or suspend cells uniformly within a matrix, thus interfering with cell-cell interactions that occur in native vasculogenic processes. Prevascular structure formation was analyzed as a function of aggregate size, time, cell ratio and media composition in this high-cell density system to determine the conditions under which self-assembly of the endothelial cells into cord and/or plexus like structures and vascular maturation would occur. These prevasculari (open full item for complete abstract)

Committee: Eben Alsberg (Advisor); Horst von Recum (Committee Chair); Diana Ramirez-Bergeron (Committee Member); Agata Exner (Committee Member); Jeffrey Capadona (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy in Engineering, Cleveland State University, 2020, Washkewicz College of Engineering

Regenerative repair of the elastic matrix is naturally limited due to the intrinsically poor elastogenicity of adult vascular smooth muscle cells. Therefore, when the elastic matrix, which provides tissue stretch and recoil are disrupted in a proteolytic milieu, such as in Abdominal Aortic Aneurysms (AAAs), localized rupture-prone expansions of the aorta, the damage is difficult to reverse. This demands providing an external, pro-elastin regenerative- and anti-proteolytic stimuli to aneurysmal SMCs in the AAA wall towards reinstating matrix structure in the aorta wall. Introducing alternative phenotypes of highly elastogenic and contractile cells into the AAA wall, capable of providing such cues, proffers attractive prospects for AAA treatment. In this regard, our previous studies demonstrated superior elastogenicity of bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs (BM-SMCs) and their ability to provide pro-elastogenic and anti-proteolytic stimuli to aneurysmal SMCs in vitro. However, for cell therapy a large cell inoculate is required for which these derived cells must be cultured extensively as well as retain their superior elastogenicity and anti-proteolytic benefit in long term culture as well as in vivo collagenous environment which is not conducive to elastogenesis. Accordingly, in this study we assessed the proelastogenic and antiproteolytic benefits of the BM-MSC derived cells in vitro and in vivo. The overall goal of this dissertation is to understand the pro-elastogenic and anti-proteolytic behavior of BM-MSCs derived SMCs in vitro and in vivo towards their implication as an alternative cell source for elastin regenerative repair in AAAs. Our results indicate that the stem cell derivatives retain their phenotype and superior elastogenic and anti-proteolytic properties in 2D as well as 3D collagenous culture in vitro. The results of our in vivo studies indicate that the stem cell derivatives (a) possess natural homing abilities similar to the und (open full item for complete abstract)

Committee: Anand Ramamurthi (Advisor); Margot Damaser (Committee Member); Joanne Belovich (Committee Member); Chandrasekhar Kothapalli (Committee Member); Mekki Bayachou (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

Master of Science (MS), Wright State University, 2018, Biological Sciences

Mesenchymal stem cells (MSCs) are adult multipotent stem cell that can differentiate into mesodermal lineages such osteoblast, adipocytes, and chondrocytes, or can be transdifferentiated into clinically relevant lineages such as cardiac or neural cells using in vitro reprogramming techniques. In addition to the multilineage differentiation potential, MSCs from most tissue origins such bone marrow derived mesenchymal stem cells (BM-MSCs) or adipose derived mesenchymal stem cells (AD-MSCs) have immune modulatory functions which indicate their promise in clinical applications for cell-based therapy, tissue engineering, and regenerative medicine. In recent years, three-dimensional (3D) cell culture systems, which utilize 3D scaffolds or hydrogels to provide a physiological 3D microenvironment that mimics the native extracellular matrix, have been largely embraced to study cellular function and cell fate determination using different cell types. Although many investigators have shown that stem cell self-renewal/differentiation is modulated by the 3D scaffold chemistry and/or its surface topology, less attention has been given to undercover how such 3D scaffold affects chromatin organization and gene expression of key developmental regulators or lineage specifiers at the transcription and translation level during the course of stem cell differentiation. In this novel study, we have established a Corning PuraMatrix Peptide Hydrogel based 3D culture system for BM-MSCs and have analyzed key transcription factors regulating osteogenic (RUNX2, OSX, TWIST1) and adipogenic (PPARy, C/EBPa, CHOP10) differentiation of BM-MSCs grown in a 2D culture and a PuraMatrix peptide hydrogel-based 3D culture. BM-MSCs adopt a completely different morphology and metabolism when encapsulated in PuraMatrix peptide hydrogel. Moreover, expression of these key osteogenic (RUNX2, OSX, TWIST1) and adipogenic (PPARy, C/EBPa, CHOP10) transcription factors are differentially expressed between 2D and 3D c (open full item for complete abstract)

Committee: Katherine Excoffon Ph.D. (Committee Co-Chair); Jaime E. Ramirez-Vick Ph.D. (Committee Co-Chair); Barbara Hull Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member) Subjects: Biology; Biomedical Engineering; Biomedical Research; Cellular Biology; Developmental Biology

Master of Sciences, Case Western Reserve University, 2018, Biology

All bone regeneration requires bone forming cells (osteogenic connective tissue progenitors, CTP-Os). This thesis examines a clinically practiced method for selecting and rapidly concentrating CTP-Os for transplantation at point-of-care. Annually in the US alone, more than 1.5M bone regeneration procedures are performed. Autografting is the most common and often involves harvesting bone and bone marrow from the patient's hip and transferring to the site where new bone formation is desired. Autografting is preferred because it provides a graft material that contains viable ostoegenic cells (CTP-Os) that are immune compatible, has correct bone formation inducing proteins, and provides structural scaffold for bone ingrowth. However, due to pain, bleeding and scarring from the additional surgery, less invasive solutions have been demanded. Selective retention provides the properties needed for bone growth without the need for an additional procedure. This thesis describes the life cycle of a product from the lab to the market.

Committee: George Muschler MD (Committee Chair); Christopher Cullis PhD (Committee Member); Elizabeth Sump MS (Committee Member); Stephen Haynesworth PhD (Committee Member) Subjects: Biology; Biomedical Engineering; Entrepreneurship

Doctor of Philosophy, The Ohio State University, 2017, Pharmaceutical Sciences

Messenger RNA (mRNA) as an emerging new drug class is being extensively investigated in the fields of cancer immunotherapy, infectious disease vaccines, protein replacement therapy, and regenerative medicine. The inherent nature of mRNA renders it several advantages as a genetic medicine. For example, mRNA translates into protein in the cytoplasm which makes its expression independent of cell cycle. mRNA does not integrate into host genome and only expresses transiently, which minimizes potential side effects. Recently, mRNA-based therapeutics has become a clinical reality. However, there are two significant obstacles limiting the broad applications of mRNA-based therapeutics in clinic practice: mRNA instability and lack of efficient delivery vehicles.The objective of this dissertation is to improve mRNA translation and develop lipid-like nanoparticles (LLNs) for the safe and efficient delivery of mRNA. In chapter 2, we constructed two sets of chemically modified mRNAs encoding firefly Luciferase (FLuc) or enhanced green fluorescent protein (eGFP) and evaluated the protein expression under various conditions. N1-methylpseudouridine (me1φ), 5-methoxyuridine (5moU), and pseudouridine (φ) which significantly improved protein expression were identified as promising nucleosides for mRNA modifications. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Chapter 3 described the design and synthesis of lipid-like compounds N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT) derivatives and established an effective approach for formulation optimization through orthogonal array experimental design. The lead formulation O-TT3 LLNs fully recovered the human factor IX (hFIX) level to normal physiological values in FIX-knockout mice. From a material optimization perspective, chapter 4 showed that position change of functional groups on lipid-like compounds can dramatically improve delivery efficiency, and chapter 5 demonstrated efficient deliver (open full item for complete abstract)

Committee: Yizhou Dong (Advisor); Robert Lee (Committee Member); Mitch Phelps (Committee Member); Dan Shu (Committee Member) Subjects: Pharmacy Sciences

Master of Engineering, Case Western Reserve University, 2017, Biomedical Engineering

Lower-limb fracture exhibits a limited capacity to heal critically sized defects and biomimetic tissue engineering is a promising approach for addressing this clinical need. Here, scaffold-free tubular mesenchymal condensations featuring temporally controlled TGF-Β1 and BMP-2 morphogen release from incorporated microparticles were engineered to form self-assembled rings and tubes. In vitro culture, subcutaneous, and femoral defect implantation was performed to establish bone forming capacity with the treatment groups: 1) TGF-Β1, 2) BMP-2, or 3) BMP-2+TGF-Β1. Bone formation was evaluated by biochemical, μCT, and histological analyses. Dual-delivery enhanced bone volume versus single morphogens at 6wks, and histology revealed bone within tubular geometries with enhanced cartilage, mimetic of endochondral ossification. Overall: 1) geometry of scaffold-free mesenchymal condensations guided 3D ossification, 2) BMP-2+TGF-Β1 presentation augmented ectopic bone formation, and 3) tubular architecture promoted femoral defect bridging over random organization.

Committee: Eben Alsberg Dr. (Advisor); Steven Eppell Dr. (Committee Member); Ozan Akkus Dr. (Committee Member) Subjects: Biology; Biomedical Engineering; Biomedical Research; Cellular Biology; Engineering; Health Sciences; Histology; Medical Imaging; Microbiology; Morphology

Doctor of Philosophy, The Ohio State University, 2017, Biomedical Engineering

The aim of tissue engineering is to fabricate functional tissue constructs for treating diseases. Contemporary approach for tissue engineering is to embed cells in homogenous three-dimensional (3D) macroscopic scaffolds for mimicking the function of extracellular matrix (ECM) in tissues. However, the native ECM is usually not homogenous and these macroscopic scaffolds are suboptimal due to the limited diffusion length of oxygen and nutrients in cellularized tissues. In this dissertation work, cell microencapsulation and microfluidic technologies are utilized to fabricate multiscale heterogeneous biomaterials for resolving these issues. First, the development of a novel non-planar microfluidic flow-focusing device for high-throughput encapsulation of mouse embryonic stem cells (mESCs) in a liquid core of microcapsules with an alginate hydrogel shell is reported. Using the non-planar microfluidic device, mESCs can be encapsulated in the core with high viability to form uniform sized aggregates, to mimic the growth of totipotent-pluripotent stem cells in early pre-hatching embryos. Further, it is shown that mESCs cultured in the biomimetic microcapsules have higher pluripotency and differentiation potential than the cells cultured under traditional two-dimensional (2D) condition. Utilizing the same microfluidic device, biomimetic ovarian microtissue consisting of an ovarian follicle embedded in microcapsules with a collagen core and alginate hydrogel shell is fabricated. This miniaturized 3D culture of early secondary preantral follicles facilitate their development to the antral stage. The study revealed the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. It is also demonstrated that the proliferation, differentiation, and development of mESCs and preantral follicles can be modulated by changing the composition of ECM in the core of the microcapsules. Next, a bottom-up approach for fabricating 3D (open full item for complete abstract)

Committee: Xiaoming He (Advisor); Gunjan Agarwal (Committee Member); Keith Gooch (Committee Member); Yi Zhao (Committee Member) Subjects: Biomedical Engineering; Medicine

Doctor of Engineering, Cleveland State University, 2016, Washkewicz College of Engineering

Localized host inflammatory microenvironment resulting from several neuropathologies (e.g., trauma, amyotrophic lateral sclerosis (ALS), glioblastomas) leads to progressive degeneration of neuronal tissue and destruction of axonal tracts in the adult central nervous system (CNS). Failure to reinstate healthy cells and axonal connections under these conditions can severely compromise locomotion and cognitive function, resulting in muscle atrophy, paralysis and even death. Annually, thousands of people are diagnosed with various neuropathologies and a majority of them succumb to these conditions soon after. The adult CNS has a limited ability for self-repair, which necessitates repair strategies focused on ameliorating secondary cellular degeneration, promoting endogenous repair mechanisms, and exogenous cell replacement therapy. Currently, pharmacological and surgical treatments options are limited in their outcomes for these types of ailments. Neural stem cells (NSCs) isolated from the embryonic and adult striatum have the capacity to divide and differentiate into various neuronal and glial lineages, thus demonstrating their utility in regenerating lost neuronal populations. To further investigate their clinical potential, in this work, we first developed and tested the utility of uncrosslinked 3D biological hydrogels (compressive strength < 600 Pa) for their ability to promote murine NSC survival, differentiation into desired lineages and neurite outgrowth, in the presence (or absence) of exogenous cues such as retinoic acid. In the second step, the influence of an activated murine microglia in regulating the phenotype and genotype of murine NSCs within a localized 3D coculture microenvironment was investigated, and the key cytokines and chemokines which regulate NSC survival, differentiation and neurite outgrowth were identified. Finally, in the third step, the effects of paracrine-signaling between adult human NSCs and human pediatric glioblastoma cells within a (open full item for complete abstract)

Committee: Chandra Kothapalli Ph.D. (Committee Chair); Nolan Holland Ph.D. (Committee Member); Xue-Long Sun Ph.D. (Committee Member); Joanne Belovich Ph.D. (Committee Member); Moo-Yeal Lee Ph.D. (Committee Member) Subjects: Biomedical Engineering; Medicine; Neurosciences

Doctor of Philosophy, Case Western Reserve University, 2015, Neurosciences

Contusive spinal cord injury (SCI) is a devastating condition that leads to permanent disability due to the lack of neuronal regeneration and functional plasticity. It is well established that an upregulation of glial derived chondroitin sulfate proteoglycans (CSPGs) within the scar creates a barrier to axonal regrowth. Additionally, CSPGs in the perineuronal net (PNN) distal to the injury site prevent remodeling of spared pathways that could provide functional recovery. Protein tyrosine phosphatase-sigma (PTPs), along with its sister phosphatase leukocyte common antigen-related (LAR), and the Nogo receptors 1 and 3 (NgRs) have recently been identified as receptors for the inhibitory glycosylated side chains of CSPGs. We found that PTPs plays a critical role in converting growth cones into a stabilized dystrophic state within CSPG gradients. Furthermore, we identified a critical regulatory wedge domain within the first intracellular phosphatase repeat of PTPs. In vitro, a peptide mimetic of this wedge bound to PTPs and released CSPG inhibition. In vivo, we utilized this peptide as a daily systemic treatment to prevent inhibition and promote axonal growth following SCI. This treatment paradigm induced functional recovery of both locomotor and bladder systems, and restored a large volume of serotonergic innervation to the caudal spinal cord below the level of the lesion. Our results provide strong validation of the critical role of PTPs in mediating the growth-inhibited state of neurons due to CSPGs within the injured adult spinal cord.

Committee: Heather Broihier (Committee Chair); Jerry Silver (Advisor); Lynn Landmesser (Committee Member); Paul Tesar (Committee Member) Subjects: Biology; Biomedical Research; Neurobiology; Neurology; Neurosciences

Master of Science, The Ohio State University, 2014, Comparative and Veterinary Medicine

Objective – To assess the efficiency of gravity filtration to enrich equine bone marrow for blood component recovery, stem cell recovery and replication, and progenitor cell differentiation. Animal – 12 healthy adult horses Procedures – Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to capture, and release, marrow elements. Raw, filtered, and harvested bone marrow were evaluated for white blood cell and platelet counts, automated and cytomorphologic cell differentials, bone marrow-derived mesenchymal stem cell colony forming units, cell viability by flow-cytometry, and differentiation capacity. Isolated marrow mesenchymal stromal cells were analyzed for CD90 and MHC class I and II antigens. Results - Eleven of fourteen marrow aspirates (79%) successfully produced filtered marrow and 100% of filtered marrow produced a harvested marrow product. Mean cell viability of harvested marrow was 95.9%. Total white blood cells and platelets were efficiently captured by the filter (> 95%), but recovery in harvested marrow was a mean of 30%. Cytologic cell differentials indicated that neutrophils (%) were significantly less (P<0.05) and the progenitor cell population 1.56-fold greater in the harvested marrow compared to raw marrow. Flow cytometry and culture characterized harvested marrow cells as CD90+, MHCI-, and MHCII- indicating a stem cell phenotype that were multipotent and differentiated into chondrocytes, osteocytes, adipocytes, and tenocytes. Conclusions and Clinical Relevance – Gravitational filtration of bone marrow efficiently captured platelets and cells and significantly enriched a viable progenitor/stem cell population while decreasing the neutrophil population. Filter modifications are anticipated to improve efficiency of cell harvest.

Committee: Alicia Bertone DVM, PhD, DACVS, DACVSMR (Advisor); Matthew Brokken PhD, DACVS, DACVSMR (Committee Member); Maxey Wellman DVM, MS, PhD, DACVP (Committee Member) Subjects: Veterinary Services

Master of Arts, Case Western Reserve University, 2014, Biology

Sanford "Sandy" Markowitz, MD, PhD, the Markowitz-Ingalls Professor of Cancer at the Case Western Reserve (CWRU) School of Medicine and practicing physician at University Hospitals of Cleveland (UH), is currently developing a first-in-class tissue-regenerating drug (TRD). TRD has the potential to benefit patients across a number of indications including liver surgery, bone marrow transplants, and ulcerative colitis attacks. TRD inhibits 15-prostaglandin dehydrogenase (15-PGDH), a prostaglandin-inhibiting enzyme, and enhances tissue regeneration in various tissue types. TRD is in the preclinical stage of development and the efficacies of each of its indications are being explored in vitro and in vivo by Dr. Markowitz and his team of researchers at CWRU. The purpose of this thesis is to analyze TRD's potential to enhance the standard of care and to navigate the commercial and regulatory landscapes over a variety ofpotential indications.

Committee: Christopher Cullis PhD (Advisor); Glen Gaughan PhD (Committee Member); Arnold Caplan PhD (Committee Member) Subjects: Biology