Thesis (Ph. D.)--Florida State University, 2005. / Advisor: Teng Ma, Florida State University, College of Engineering, Dept. of Chemical and Biomedical Engineering. Title and description from dissertation home page (viewed Feb. 13, 2006). Document formatted into pages; contains xiv, 164 pages. Includes bibliographical references.

The amniotic membrane is a lining along the inner aspect of the placenta that envelops a developing embryo (then fetus). This component is critical for the adequate growth and nutrition of the fetus and can greatly impact the viability of the fetus. This role in development has led scientists to explore its post-delivery uses in regenerative medicine. Specifically in this paper, current literature was reviewed to determine the applicability of amniotic membranes to neural tissue engineering. The amniotic membrane has been greatly characterized with respect to immune response (including inflammatory effects) and microbial influence. These preliminary characterizations of the amniotic membrane and its components (i.e. stem cells) demonstrated a promising future for clinical implementation. Some fields, such as cardiovascular and orthopedic research, have begun projects using either the amnion-derived stem cells or amniotic membrane as a central element in their research. Both elements have received extensive praise for their versatility and relatively easy implementation into multiple organs and systems in the body. In each of these systems, the amniotic membrane retained its optimal antimicrobial and anti-immunogenic characteristics. After researching the current applications, it was apparent that amniotic membranes could have a place in the future of neural tissue engineering whether it be axillary components (cerebral vessels or surrounding bone) or direct regeneration of nerves. The largest impediment was the lack of basic science understanding in neuroscience. As the specific mechanisms of normal brain behavior and disease states are uncovered amniotic membranes can be added to the pre-clinical testing for the neurological sciences. Similar to the other fields of medicine, amniotic membranes will be specifically useful due to their ability to not evoke an immune response, thereby mitigating the possibility of rejection and infection in the central nervous system. These elements are critical to the research in neurology. Overall, amniotic membrane research would be very valuable to neuroscientists and physicians when exploring the future of neural tissue engineering

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.

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.

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In recent years, significant success has been made in the field of regenerative medicine. Tissue engineering scaffolds have been developed to repair and replace different types of tissues. The overall goal of the current work was to develop scaffolds of native extracellular matrix components for soft tissue regeneration, more specifically, neural tissue engineering. To date, much research has been focused on developing a nerve guidance scaffold for its ability to fill and heal the gap between the damaged nerve ends. Such scaffolds are marked by several intrinsic properties including: (1) a biodegradable scaffold or conduit, consisting of native ECM components, with controlled internal microarchitecture; (2) support cells (such as Schwann cells) embedded in a soft support matrix; and (3) sustained release of bioactive factors. In the current dissertation, we have developed such scaffolds of native biomaterials including hyaluronic acid (HA) and collagen. HA is a nonsulphated, unbranched, high-molecular weight glycosaminoglycan which is ubiquitously secreted by cells in vivo and is a major component of extracellular matrix (ECM). High concentrations of HA are found in cartilage tissue, skin, vitreous humor, synovial fluid of joints and umbilical cord. HA is nonimmunogenic, enzymatically degradable, non-cell adhesive which makes HA an attractive material for biomedical research. Here we developed new photopolymerizable HA based materials for soft tissue repair application. First, we developed interpenetrating polymer networks (IPN) of HA and collagen with controlled structural and mechanical properties. The IPN hydrogels were enzymatically degradable, porous, viscoelastic and cytocompatible. These properties were dependent on the presence of crosslinked networks of collagen and GMHA and can be controlled by fine tuning the polymer ratio. We further developed these hydrogel constructs as three dimensional cellular constructs by encapsulating Schwann cells in IPN hydrogels. The hydrogel constructs supported cell viability, spreading, proliferation, and growth factor release from the encapsulated cells. Finally, we fabricated scaffolds of photopolymerizable HA with controlled microarchitecture and developed designer scaffolds for neural repair using layer-by-layer fabrication technique. Lastly, we developed HA hydrogels with unique anisotropic swelling behavior. We developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked regions. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions. / text

Currently, organ transplant procedures are insufficient to address the needs of the number of patients that suffer of organ failure related disease. In the United States alone, only around 19% of the patients are able to get an organ transplant surgery and 25% die while waiting for a suitable donor. Tissue engineering (TE) has emerged as an alternative to organ transplant; thus, the aim of the present study was to validate a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel system as a model for material scaffolding in TE applications. This work explores the influence of scaffold material properties on cell behavior. Specifically, scaffold modulus, mesh size, and biochemical stimuli were characterized and their influence on cell response was analyzed at the biochemical, histological and microenvironmental levels. Three different TE targets were evaluated: vocal fold restoration, vascular grafts and osteochondral applications. Vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by initial scaffold mesh size and modulus. The results showed increasing levels of SM-α-actin and collagen production with decreasing initial mesh size/increasing initial modulus, which indicated that VFFs were induced to take an undesirable myofibroblast-like phenotype. In addition, it was possible to preserve VFF phenotype in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). On the other hand, regarding vascular graft applications, smooth muscle cell (SMC) phenotype was enhanced by increasing scaffold mesh size and modulus. Finally, the effect of scaffold inorganic content (siloxane) on rat osteoblasts and mouse mesenchymal stem cells was evaluated. Interestingly, the impact of inorganic content on cell differentiation seemed to be highly dependent on the initial cell state. Specifically, mature osteoblasts underwent transdifferentiation into chondrocyte-like cells with increasing inorganic content. However, Mesenchymal stem cells appeared to be preferentially driven toward osteoblast-like cells with an associated increase in osteocalcin and collagen type I production.

Cell-based treatments for insulin-dependent diabetes (IDD) may provide more physiologic regulation of blood glucose levels than daily insulin injections, thereby reducing the occurrence of secondary complication associated with IDD. An autologous cell source is especially attractive for regulatory and ethical reasons and for circumventing the need for immunosuppression, which is currently standard for islet transplantation. Our approach focuses on using adult non-β-cells engineered for physiologic insulin secretion. Specifically, we utilize enteroendocrine L-cells, which naturally exhibit regulated secretion of GLP-1 in response to physiologic stimuli, and upon genetic engineering, co-secrete insulin in a regulated manner. The overall goal of this project is to develop a tissue engineered pancreatic substitute based on a recombinant enteroendocrine cell line and test the efficacy of the pancreatic substitute by implantation into diabetic mice. The specific aims of this thesis were to (1) to modify murine L-cells for regulated insulin secretion and evaluate the insulin secretion properties of the recombinant cells; (2) to incorporate insulin-secreting L-cells into an implantable construct containing small intestinal submucosa (SIS) and to evaluate insulin secretion from the construct in vitro; and (3) to test the efficacy of the tissue engineered pancreatic substitute in vivo by implanting it intraperitoneally in mice made diabetic by streptozotocin. Thus, this proposal takes a tissue engineered pancreatic substitute for IDD from in vitro development to in vivo testing.