Children with congenital heart defects and patients with faulty or failing valves have the need for a suitable aortic heart valve replacement. Current treatment options have several downfalls and heavy investigation is being done into the design of an engineered valve to find an alternative that would alleviate many of these issues. Understanding the physiology of how cells interact in vivo is crucial to the construction of such valve. This study investigates the effect of cyclic strain in aortic valve endothelial cells on the adhesion molecules, PECAM-1, ƒÒ1-Integrin, VE-Cadherin and Vinculin. Experiments found that cyclic strain plays a role in the development of cell/cell and cell/extracellular matrix adhesions and junctions and is extremely important in the pre-conditioning of a tissue engineered construct. Without this strain the new valve would be more susceptible to inflammation, injury or possible failure after being implanted into the patient.

Myocardial infarction (MI) and heart failure are leading causes of mortality globally. Recently, cardiac tissue engineering has become an attractive option for MI treatment due to the following advantages: it might provide optimal tissue performance maintained by viable transplanted cells, and might also stimulate the formation of vasculature supplying oxygen and nutrients in the patched region. However, fabrication of a thick viable cardiac patch with 3D scaffolds that are thoroughly recellularized with desired cells remains a challenge. We hypothesize that the decellularized porcine myocardium scaffold can preserve natural extracellular matrix (ECM) structure, cardiomyocyte lacunae, mechanical properties, and vasculature templates that are able to facilitate stem cell reseeding, proliferation, cardiomyocyte differentiation, and angiogenesis. In this dissertation, we have (i) assessed the potential of the decellularized porcine myocardium as a scaffold for thick cardiac patch tissue engineering; (ii) thoroughly characterized the structural and biomechanical properties of the myocardial ECM; (iii) designed and built a novel bioreactor that could provide coordinated mechanical and electrical stimulations, and (iv) evaluated the efficiency of the multi-stimulations on the development of a cardiac tissue construct. An optimized decellularization protocol has been identified to obtain the acellular myocardial scaffold that preserves subtle ECM composition and ultrastructure. We recellularized the acellular scaffold with bone marrow mononuclear cells using a rotating bioreactor and observed successful recellularization with good cell viability, proliferation, and differentiation in a 2-week culture time. Furthermore, we have successfully built a novel bioreactor that is able to provide coordinated mechanical and electrical stimulations for facilitating stem cell differentiation and tissue construct development. We found that cardiomyocyte differentiation and tissue remodeling were more effectively and efficiently promoted with the coordinated simulations, evidenced by good cell viability, proliferation, differentiation, positive tissue remodeling, and a trend of angiogenesis in a short period of time (2 - 4 days). The clinical product that we envision will benefit from the natural architecture of myocardial ECM, which has the potential to promote stem cell differentiation, cardiac regeneration, and angiogenesis. The hopes are that our novel approach will ultimately impact thousands of patients who have suffered significant damage from a prior myocardial infarction.

There is intense interest in developing novel approaches to enhance immune-mediated cancer therapies with cells such as Natural Killer (NK) cells. Previously, a particle-based method was developed for in vitro expansion of highly cytotoxic human NK cells (PM21-NK cells). Here, we tested two approaches to further enhance the antitumor activity of PM21-NK cells. First, we tested the hypothesis that oncolytic Parainfluenza virus 5 (P/V virus) would combine with PM21-NK cells for enhanced killing of lung cancer cells in vitro. Flow cytometry, luminescence and real-time imaging-based methods were used to assay PM21-NK cell-mediated killing of P/V virus-infected lung cancer cells in 2-dimensional (2D) and 3-dimensional (3D) spheroid cultures. In 2D cultures, lung cancer cells were efficiently infected by the P/V virus and PM21-NK cell killing activity was enhanced against P/V virus-infected cancer cells compared to non-infected cells. By contrast, P/V virus infection of 3D lung cancer cell spheroids was restricted to only the outer-most layer of cells. Nevertheless, PM21-NK cells showed enhanced killing in both infected and non-infected spheroid cells. Antibody neutralization assays showed enhanced NK cell killing was due to both type I and III interferon signaling in lung cancer target cells, which increased their killing by PM21-NK cells. In a second approach to enhance NK cell anti-tumor activity, we designed a novel chimeric protein (NA-Fc) which positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. Real time viability assays revealed that stable expression or lentiviral delivery of NA-Fc to A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells increased their killing in vitro by PM21-NK cells, and this depended on CD16-Fc interactions. Our results lay the foundation for further development of oncolytic viruses and/or novel Fc-based molecules to deliver signals for enhanced NK cell mediated anti-cancer therapies.

Pertussis is a highly contagious acute respiratory disease of humans which is mainly caused by the gram-negative obligate human pathogen Bordetella pertussis. Despite the availability and extensive use of vaccines, the disease persists and has shown periodic re-emergence resulting in an estimated 640,000 deaths worldwide in 2014. The pathogen expresses various virulence factors that enable it to modulate the host immune response, allowing it to colonise the ciliated airway mucosa. Many of these factors also directly interfere with host signal transduction systems, causing damage to the ciliated airway mucosa and increase mucous production. Of the many virulence factors of B. pertussis, only the tracheal cytotoxin (TCT) is able to recapitulate the pathophysiology of ciliated cell extrusion and blebbing in animal models and in human nasal biopsies. Furthermore, due to the lack of appropriate human models and donor materials, the role of bacterial virulence factors has been extrapolated from studies using animal models infected with either B. pertussis or with the closely related species B. bronchiseptica which naturally causes respiratory infections in these animals and produces many similar virulence factors. Thus, in the present work, in vitro airway mucosa models developed by co-culturing human airway epithelia cells and fibroblasts from the conduction zone of the respiratory tract on a decellularized porcine small intestine submucosa scaffold (SISser®) were used, since these models have a high correlation to native human conducting zone respiratory epithelia. The major aim was to use the engineered airway mucosa models to elucidate the contribution of B. pertussis TCT in the pathophysiology of the disease as well as the virulence mechanism of B. pertussis in general. TCT and lipopolysaccharide (LPS) either alone or in combination were observed to induce epithelial cell blebbing and necrosis in the in vitro airway mucosa model. Additionally, the toxins induced viscous hyper-mucous secretion and significantly disrupted barrier properties of the in vitro airway mucosa models. This work also sought to assess the invasion and intracellular survival of B. pertussis in the polarised epithelia, which has been critically discussed for many years in the literature. Infection of the models with B. pertussis showed that the bacteria can adhere to the models and invade the epithelial cells as early as 6 hours post inoculation. Invasion and intracellular survival assays indicated the bacteria could invade and persist intracellularly in the epithelial cells for up to 3 days. Due to the novelty of the in vitro airway mucosa models, this work also intended to establish a method for isolating individual cells for scRNA-seq after infection with B. pertussis. Cold dissociation with Bacillus licheniformis subtilisin A was found to be capable of dissociating the cells without inducing a strong fragmentation, a problem which occurs when collagenase and trypsin/EDTA are used. In summary, the present work showed that TCT acts possibly in conjunction with LPS to disrupt the human airway mucosa much like previously shown in the hamster tracheal ring models and thus appears to play an important role during the natural B. pertussis infection. Furthermore, we established a method for infecting and isolating infected cells from the airway mucosa models in order to further investigate the effect of B. pertussis infection on the different cell populations in the airway by single cell analytics in the future. / Pertussis ist eine hoch ansteckende akute Atemwegserkrankung des Menschen, die durch das gramnegative obligat humanpathogene Bakterium Bordetella pertussis verursacht wird. Obwohl seit langer Zeit effektive Impfstoffe verfügbar sind und weltweit eingesetzt werden, stellt die Krankheit nach wie vor ein großes Problem dar und tritt seit einiger Zeit auch in Ländern mit guten Impfraten wieder vermehrt auf. Allein in den letzten 10 Jahren wurden weltweit etwa 24 Millionen Neuinfektionen mit 640,000 Todesfällen pro Jahr gezählt. Die Bakterien exprimieren verschiedene Virulenzfaktoren, die es ihnen ermöglichen, die Immunantwort des Wirts zu modulieren, wodurch sie die Schleimhaut der oberen Atemwege besiedeln können. Viele dieser Faktoren stören auch direkt die Signaltransduktionssysteme des Zellen der oberen Atemwege, was zu einer Schädigung des Flimmerepithels der Atemwege und zu einer starken Erhöhung der Schleimproduktion führt. Von den vielen bekannten Virulenzfaktoren von B. pertussis kann soweit bekannt nur das Tracheale Cytotoxin (TCT) die typische Pathophysiologie des Flimmerepithels verursachen, die durch massive Gewebszerstörung gekennzeichnet ist und z.B. das Herauslösen von Epithelzellen aus der Epithelschicht oder die Ausbildung von bläschenförmigen Epithelzellen beinhaltet. Aufgrund des Mangels an geeigneten menschlichen Modellsystemen bzw. an Spendermaterialien wurden die Virulenzeigenschaften des Erregers entweder mit Hilfe von einfachen Zellkultursystemen oder in Tiermodellen untersucht, die keine natürlichen Wirte für B. pertussis darstellen. Alternativ hierzu wurden auch Daten, die mit dem eng verwandten tierpathogenen Bakterium B. bronchiseptica, das viele aus B. pertussis bekannte Virulenzfaktoren produziert, in entsprechenden Tiermodellen erhoben wurden, genutzt, um auf die Virulenzeigenschaften von B. pertussis zu schließen. Die vorliegende Arbeit verwendet In-vitro-Atemwegsschleimhautmodelle, die durch Co-Kultivierung von menschlichen Atemwegsepithelzellen und Fibroblasten auf einem dezellularisierten Schweine-Dünndarm-Submukosa-Gerüst (SISser®) entwickelt wurden. Die in-vitro-Atemwegsschleimhautmodelle weisen eine hohe Korrelation mit nativen menschlichen Epithelien der oberen Atemwege auf. Mithilfe dieser neuartigen Atemwegsschleimhautmodelle sollte der Beitrag von B. pertussis TCT zur Pathophysiologie der Krankheit und die Bedeutung von TCT als relevanter Virulenzfaktor aufgeklärt werden. Es wurde beobachtet, dass TCT und das bakterielle Lipopolysaccharid (LPS) entweder alleine oder in Kombination die Bildung von Epithelzellbläschen und Nekrose in diesen in-vitro-Atemwegsschleimhautmodellen induzieren. Zusätzlich induzierten diese Toxine eine viskose Hyperschleimsekretion und störten die Barriereeigenschaften der in-vitro-Atemwegsschleimhautmodelle signifikant. Zudem wurde in dieser Arbeit versucht, die Invasion und das intrazelluläre Überleben von B. pertussis in den polarisierten Epithelien zu bewerten, das in der einschlägigen Fachliteratur kritisch diskutiert wird. Die Infektion der Modelle mit B. pertussis zeigte, dass die Bakterien bereits 6 Stunden nach der Inokulation an den Modellen adhärieren und in diese eindringen können. Invasions- und intrazelluläre Überlebenstests zeigten, dass die Bakterien bis zu 3 Tage intrazellulär in die Epithelzellen überleben können. Aufgrund der Neuheit der in dieser Arbeit entwickelten in-vitro-Atemwegsschleimhautmodelle sollte auch eine Methode zur Isolierung einzelner Zellen für scRNA-seq Analysen nach Infektion mit B. pertussis etabliert werden. Dabei wurde festgestellt, dass die Inkubation der Modelle mit Subtilisin A von Bacillus licheniformis in der Kälte eine sehr gute Methode darstellt, um die Zellen zu dissoziieren, ohne eine starke Fragmentierung zu induzieren, wie sie unter Verwendung von Kollagenase und Trypsin / EDTA auftritt. Zusammenfassend wird in der vorliegenden Arbeit gezeigt, dass TCT gemeinsam mit LPS eine extrem destruktive Wirkung auf die menschliche Atemwegsschleimhaut besitzt, die der früher gezeigten Wirkung in Tiermodellen stark ähnelt. TCT sollte deshalb tatsächlich als ein wichtiger Virulenzfaktor von B. pertussis eingeschätzt werden. Darüber hinaus wurden Methoden zur Infektion und Isolierung von infizierten Zellen aus den Atemwegsschleimhautmodellen entwickelt, um künftig die Auswirkung einer B. pertussis Infektion auf die verschiedenen Zellpopulationen in den Atemwegen durch Einzelzellanalytik noch besser erforschen zu können.

Soft tissue engineering has become increasingly relevant in efforts to create complex, functional tissues for tissue replacement in tissue engineering applications or for the development of more complex tissue models for drug screening or fundamental research. Tissue engineering of micro- and nano-scale structures has been explored through a number of biofabrication techniques but most successfully on the nano-scale through electrospinning. Electrospun nanofibers represent one of the most similar structures to natural extracellular matrix (ECM), while electrospinning of hydrogel nanofibers is particularly relevant given that such nanofibers support the high water content environment required by cells to survive. Herein, a reactive cell electrospinning process is demonstrated based on dynamic hydrazone-crosslinked poly(oligoethylene glycol methacrylate (POEGMA) hydrogel nanofibers that can be electrospun from an aqueous solution, allowing for the generation of cell-loaded hydrogel nanofibers in a single fabrication/cell-seeding step. Using the proper collectors, the fabrication of aligned and/or multi-layered scaffolds is demonstrated without the risk of layer delamination due to the dynamic crosslinking of POEGMA hydrogels. Co-electrospun NIH 3T3 fibroblasts and Psi2 12S6 epithelial cells were found to proliferate over 14 days within the networks, while electrospun C2C12 myoblasts were found to align along the direction of aligned fibers. POEGMA hydrogels provide a suitable environment for cells and can be expanded to multi-layer, multi-cellular networks with tunable micro-architectures to better mimic more complex aligned (e.g. muscle) and/or multi-layer (e.g. smooth muscle vasculature, esophageal) tissues. / Thesis / Master of Applied Science (MASc) / Successful regeneration of diseased tissues relies first on understanding the healthy tissue structures and functions that currently exist within the body, and second, how to synthetically replicate those structures using biomaterials. Re-creating the natural networks that cells use to attach and grow has many challenges, including the challenge of creating nano-scale structures, controlling any immune response to the biomaterial(s) used, and ensuring the correct response of cells to the fabricated structures. One method of generating suitable nano-scale structures is through a process called electrospinning, specifically when it is used to produce hydrogel-based nanofibers which can bind large amounts of water. When implanted, hydrogels swell to form a hydrated environment suitable for cells. These nanofibers are generated on a scale that is smaller than the encapsulated cells to allow for guided cell responses to the material. Furthermore, the use of cell-friendly polymer solutions allows for cells to be in contact with the biomaterial without resulting in high cell death. In this thesis, aligned and/or multi-layered nanofiber structures are generated to replicate the naturally existing support structures seen in the body. These fibers are also loaded with cells to create semi-functional body tissues that in the future can serve to replace non-functional tissues or used to better understand cell interactions with their environment.

Articular cartilage (AC) provides an interface between bones within joints that serves to minimize friction, absorb and distribute load along the joint, and facilitate movement. Cartilage has poor self-repair abilities following injury, in part due to the limited migration of chondrocytes and the avascular nature of the tissue which impedes the ability of progenitor cells to reach the site of injury. As a result, cartilage defects are at risk of progressing into osteoarthritis (OA), a degenerative disease of the whole joint. OA affects almost 5 million Canadians and can cause pain, severely reduce joint mobility, and negatively impact quality of life. Cartilage tissue engineering is a field that aims to develop strategies to repair cartilage defects with a combination of cells, biomaterials, and/or cues, either biochemical or biophysical in nature, to guide tissue formation. Tissue engineering strategies can include an in vitro maturation phase to create cartilage constructs with sufficient mechanical properties to withstand the cyclic loads present in the joint upon implantation. Identifying optimal culture conditions during this in vitro maturation phase is key for the generation of functional cartilage constructs. Typical tissue engineering strategies use supraphysiological glucose concentrations to ensure there is sufficient glucose in the media for energy production and proteoglycan synthesis between media changes; however, studies have found that these elevated glucose concentrations may elicit catabolic processes in the chondrocytes. We hypothesized that culturing constructs at physiological glucose concentrations in larger media volumes, to prevent glucose depletion, would generate cartilage constructs with superior biochemical properties. To test this hypothesis, primary chondrocytes were cultured in physiological (5 mM) and supraphysiologic (25 mM) glucose concentrations at low (2 mL) and high (11 mL) media volumes. The composition of the tissue and the different metabolic pathways conducted by chondrocytes were then evaluated. Our results indicated that high media volumes generate constructs with significantly higher proteoglycan and collagen content, the two major components of the extracellular matrix. Physiological glucose concentrations had no apparent effect on matrix accumulation; however, histological sections suggest that this culture condition may provide improved cell morphology. The glucose consumption rate was comparable for all four media conditions which suggests that the constructs may have similar matrix synthesis rates. Lactate concentration was significantly higher in low media volumes which may lead to a more acidic environment. The levels of both bioenergetic molecules quantified in the constructs, adenosine triphosphate (ATP) and inorganic polyphosphate (polyP), follow similar trends as the levels of matrix components; however, the relationship between ATP and polyP remains poorly understood. This thesis provides insight into the optimal culture conditions for engineered cartilage by demonstrating that media volume is an important culture parameter for matrix accumulation. Future work is required to understand the mechanisms behind this effect of media volume, to characterize effects of glucose concentration at the cellular level, and to identify key nutrients that will form functional cartilage constructs.

Cellular senescence, a hallmark of aging, has been implicated in the pathogenesis of many major age-related disorders, including atherosclerosis, metabolic disease, and neurodegenerative disorders such as Alzheimer's disease (AD). AD is characterized by increased cognitive impairment and treatment options available provide minimal disease attenuation. Additionally, diagnostic methods for AD are not conclusive with definitive diagnoses requiring postmortem brain evaluations. Therefore, miRNAs, a class of small, non-coding RNAs, have garnered attention for their ability to regulate a variety of mRNAs and their potential to serve as both therapeutic targets and biomarkers of disease. Several miRNAs have already been implicated with AD and cellular senescence and have been found to directly target genes associated with their pathology. The APP/PS1 mice is an AD model that expresses the human mutated form of the amyloid precursor protein (APP) and presenilin-1 (PS1) genes. In a previous study, crossing long-living growth hormone (GH)-deficient Ames dwarf (df/df) mice with APP/PS1 mice provided protection from AD through a reduction in IGF-1, amyloid-ß (Aß) deposition, and gliosis. Hence, we hypothesized that changes in the expression of miRNAs associated with AD mediated such benefits. To test this hypothesis, we sequenced miRNAs in hippocampi of df/df, wild type (+/+), df/+/APP/PS1 (phenotypically normal APP/PS1), and df/df/APP/PS1 mice. Results of this study demonstrated significantly upregulated and downregulated miRNAs between df/df/APP/PS1 and df/+/APP/PS1 mice that suggest the df/df mutation provides protection from AD progression. Furthermore, we identified a pro-longevity miRNA, miR-449a-5p, downregulated with age in normal mice but maintained in long-living df/df mice. Gene target analysis and our functional study with miR-449a has revealed its potential as an anti-senescence therapeutic. We tested the hypothesis that miR-449a reduces cellular senescence by targeting senescence-associated genes induced in response to strong mitogenic signals and other damaging stimuli and found miR-449a upregulation reduces senescence, primarily through targeted reduction of p16Ink4a, p21Cip1, and the PI3K-mTOR signaling pathway. Our results demonstrate that miR-449a is important in modulating key signaling pathways that control cellular senescence and age-related pathologies and that miRNAs hold great potential as therapeutics and/or biomarkers for disease, namely in Alzheimer's disease.

Background Fetal Alcohol Spectrum Disorders (FASDs) refer to the range of developmental abnormalities that occur in a fetus following prenatal alcohol exposure (PAE). It is unclear how PAE affects the development of the embryonic heart. Recent data indicates that the Wnt-signaling pathway may be implicated in congenital heart defects caused by PAE. In previous RNA-Sequencing (RNA-Seq) studies, Wnt7a, Wnt7b, and Wnt11 showed significantly changed expression in embryonic mouse hearts after a single maternal binge ethanol dose at embryonic day 9.5 (E9.5). Hypothesis We hypothesize that there will be significant change in expression of Wnt7a, Wnt7b, and Wnt11 following maternal ethanol binge at E9.5. We also hypothesize a significant decrease in expression of Wnt7a in C2C12 cells following ethanol exposure. Experimental Methods In-vivo, timed pregnant mice were given a single oral gavage of 0.9% saline or 2.5g/kg ethanol at E9.5. RNA from the embryonic heart was quantified and analyzed after 24 hours. Invitro, C2C12 murine myoblasts were cultured and incubated with ethanol or water for 2-24 hours. Cells at 4 different differentiation stages were also exposed to ethanol or water for 24 hours before expression quantification. Results Out of our 3 genes, only Wnt7a showed sustained depressed expression after 24 hours. We also concluded there is no significant impact of alcohol on Wnt7a expression in DM6 C2C12 cells exposed to different doses of ethanol from 2 to 24 hours following exposure. There was a significant change between Wnt7a expression in DM0 controls vs. UD, DM3, and DM6 controls. Conclusion These results suggest that the stage of differentiation plays a large role in Wnt7a activity and its sensitivity to ethanol. This study creates a greater understanding of the Wnt-signaling pathway's response to alcohol in-vivo and Wnt7a's vulnerability to alcohol at various stages of muscle differentiation.

Children born with congenital heart valve defects require open-heart surgery to implant an artificial replacement valve. These valves are unable to grow with the developing child and need replacing every 5 years. Tissue engineered heart valves, capable of growing with the patient, would alleviate the need for repeat surgery. I hypothesize chitosan and collagen possess advantageous qualities as scaffolding for a tissue engineered heart valve. This study evaluated chitosan and collagen hydrogels as potential scaffold materials. Chitosan scaffolds had suitable pore size/distribution and scaffold strength; however, they were unable to sustain cell attachment or viability. Collagen gels were assessed for compaction, mechanical properties and expression of matrix metalloproteases in the presence or absence of biochemical and mechanical stimuli. Pressure increased the remodeling potential. This was augmented further in the presence of TGF-β. In conclusion, both materials have potential as scaffolding substrate in a tissue engineered heart valve.

Yes