Theses and Dissertations from UMD
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Item A TISSUE-ENGINEERED PLACENTAL BARRIER MODEL FOR TOXICOLOGY AND PHARMACOLOGY APPLICATIONS(2019) Arumugasaamy, Navein; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Throughout history, there have been two major instances where a substance caused thousands of birth defects, yet it took a few years for the causation to be noted: thalidomide, in the late 1950s and early 1960s, and Zika Virus, just recently in 2014 to 2016. In both instances, the developing fetus was indirectly exposed to the substance through the placental barrier. Pregnant women took thalidomide as a medication or were stung by mosquitos and exposed to Zika Virus. These examples clearly show why models of the placental barrier and downstream fetal tissues are critically needed. Herein, I present our work on the development and utilization of a biomimetic placenta-fetus model. The three objectives in this work were to: (1) develop and validate the tissue-engineered BPB model through study of biologically relevant substances; (2) assess the effects of SSRIs on the BPB’s cells and evaluate the drugs’ transport profile across the barrier; and, (3) assess how SSRIs influence cardiomyocyte signaling and injury biomarker release following passage through the BPB. We suggest that this work provides a critically needed and biologically relevant placenta-fetus model, useful as a method to assess pharmacology and toxicology properties of medications and other substances. Moreover, the knowledge gained through the studies performed may hopefully improve clinical care of pregnant women through enhanced understanding of how a medication impacts both the pregnant mother-to-be and her developing fetus.Item USE OF 3D PRINTED POLY(PROPYLENE FUMARATE) SCAFFOLDS FOR THE DELIVERY OF DYNAMICALLY CULTURED HUMAN MESENCHYMAL STEM CELLS AS A MODEL METHOD TO TREAT BONE DEFECTS(2014) Wang, Martha Elizabeth Ottenberg; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This project investigates the use of a tissue engineering approach of an absorbable polymer, poly(propylene fumarate) (PPF) to provide long term mechanical stability while delivering a bioactive material, precultured human mesenchymal stem cells (hMSC) encapsulated in hydrogel, to repair bone defects. Annually over 2.2 million bone grafting procedures are performed worldwide; however, current treatment options are limited for critically sized and load bearing bone defects. Much progress has been made in development of bone tissue replacements within the field of bone tissue engineering. The combination of a polymer scaffold seeded with cells for the eventual replacement by host tissue has shown significant promise. One such polymer is PPF, a synthetic linear polyester. PPF has been shown to be biocompatible, biodegradable and provide sufficient mechanical strength for bone tissue engineering applications. Additionally PPF is able to be photocrosslinked and therefore can be fabricated into specific geometries using advanced three-dimensional (3-D) rapid prototyping. Current technology to culture and differentiate hMSCs into osteoblasts has been enhanced with the development of the tubular perfusion system (TPS). The TPS bioreactor has been shown to enhance osteoblastic differentiation in hMSCs when encapsulated in alginate beads. Although this system is effective in differentiating hMSCs it lacks the sufficient mechanical strength for the treatment of bone defects. Therefore this work suggests a combination strategy of harnessing the ability of the TPS bioreactor to enhance osteoblastic differentiation with the mechanical properties of poly(propylene fumarate) to develop a porous PPF sleeve scaffold for the treatment of bone defects. This is accomplished through four steps. The first step investigates the cytotoxicity of the polymer PPF. Concurrently the second step focuses on designing, fabricating and characterizing PPF scaffolds. The third step investigates the degradation properties of 3D printed porous PPF scaffolds. The fourth step characterizes alginate bead size and composition for use within the PPF sleeve scaffolds. The successful completion of these aims will develop a functional biodegradable bone tissue engineering strategy that utilizes PPF fabricated scaffolds for use with the TPS bioreactor.Item Engineering Zonal Cartilage Through Utilization of a Mesenchymal Stem Cell Population(2012) Coates, Emily Elizabeth; Fisher, John; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Articular cartilage has a limited ability to repair itself after damage due to injury or disease. Regenerative therapies using chondrocytes, the primary cartilage cell population, result in poor quality repair tissue and often cause further damage at the donor site. Furthermore, there are no current therapies which aim to regenerate the zonal organization and function of the tissue. In an effort to address both cell source limitations and zonal tissue regeneration the goal of the presented work was to utilize a mesenchymal stem cell (MSC) population to generate abundant numbers of chondrocytes with zonal phenotypes. To this end, zonal subpopulations of articular chondrocytes were isolated, characterized for differences in gene and protein expression, and exposed to scaffold environments designed to aid in phenotype retention. From these results, and reports in the literature, it was clear a major functional difference between zones was the production of a lubricating protein, proteoglycan 4 (PRG4), in the superficial zone only. Middle and deep zone cells were found to be phenotypically similar and distinct from superficial zone cells. It was further found that gene expression of PRG4 by superficial zone cells in alginate culture can be significantly enhanced by incorporation of matrix molecules hyaluronic acid (HA) and chondroitin sulfate (CS) to the scaffold environment. HA and CS also had favorable effects on MSC chondrogenesis by upregulating chondrogenic transcription factor Sox9 gene expression, and downregulating type I collagen (fibroblastic marker) gene expression. The potential of soluble signals derived from zonal (superficial or middle/deep) cartilage explants to drive MSC chondrogenesis was also investigated. Results show that signals derived from cartilage explants can induce chondrogenesis to varying degrees, with superficial zone explants inducing robust and sustained differentiation. This differentiation was found to be dependent on the proximity of the MSCs and tissue explants, implying that communication between MSCs and chondrocytes is necessary for chondrogenic induction. Coculture with superficial zone explants also upregulated MSC gene expression of PRG4. This research highlights the important functional differences between zonal chondrocyte populations and identifies MSCs as a progenitor population capable of differentiating into zone-specific chondrocytes.Item TUBULAR PERFUSION SYSTEM BIOREACTOR FOR THE DYNAMIC CULTURE OF HUMAN MESENCHYMAL STEM CELLS(2012) Yeatts, Andrew Bryan; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In vitro culture techniques must be improved in order to increase the feasibility of cell based tissue engineering strategies. Limitations of current techniques are largely a result of the slow diffusion of molecules such as oxygen into the interior of three dimensional scaffolds in static culture. In order to enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three dimensional scaffolds. In our design, hMSCs are cultured on scaffolds tightly packed in a tubular growth chamber. Media is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds. In the first part of the work hMSCs are encapsulated in alginate scaffolds and results demonstrate bioreactor culture enhances late osteoblastic differentiation of hMSCs. An investigation into shear stress in the system revealed that osteogenic markers increase with increasing shear stress and that the differentiation of hMSCs is dependent on cell radial position within scaffolds. In order to enhance the ability to implant these constructs in vivo, a method to create an aggregated cell containing construct in vitro in a bioreactor system was developed. In this part of the work hMSCs are cultured in individual alginate beads in the TPS bioreactor and the beads are aggregated to form one large construct. Following this the TPS bioreactor was investigated to culture synthetic poly-L-lactic acid scaffolds which were fabricated using supercritical carbon dioxide gel drying. In addition to investigating the effects of perfusion on hMSC growth in these scaffolds, the effect of microporosity was investigated. In the final part of the work, a study was completed to determine how TPS culture influenced in vivo bone regeneration. Here alginate beads as well as synthetic PLGA/PCL constructs were used as scaffolds. Results revealed the efficacy of using the tubular perfusion system for bone tissue engineering and demonstrated increased bone formation as a result of hMSC implantation in both alginate and PLGA/PCL scaffolds. These studies highlighted the need for bioreactor culture in vitro as well as scaffolds to support in vivo tissue interaction.Item The Delivery of IGF-1 for Skeletal Muscle Regeneration Within Abdominal Wall Hernia Repair(2009) Falco, Erin Elizabeth; Fisher, John P.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)At an ever increasing pace, synthetic biomaterials are being developed with specific functionalities for tissue engineering applications. These biomaterials possess properties including biocompatibility, mechanical strength, and degradation as well as functionalities such as specific cell adhesion and directed cell migration. However, synthetic polymers are often not completely biologically inert and may non-specifically react with the surrounding in vivo environment. An example of this reactivity is the release of acidic degradation products from hydrolytically degradable polymers based upon an ester moiety. These degradation products can lower the local pH and incite an inflammatory response as well as increase scaffold degradation rate. Therefore there has been a concerted effort in the research community to develop alternatives. In order to address this concern, a novel class of biomaterials based upon a cyclic acetal unit has been developed and investigated for both soft and hard tissue repair. This work specifically looks at a cyclic acetal biomaterial based on a 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD) monomer as a scaffold for abdominal wall hernia repair. Abdominal wall hernias are a growing concern for clinicians today as they occur in approximately 10% of all patients that undergo an abdominal procedure. Despite many advances in repair techniques, both wound healing and skeletal muscle regeneration is limited in many cases. This results in both a decrease in abdominal wall tissue function as well as a hernia recurrence rate of up to 50%. To address this high recurrence rate this project aims to create a functional gene delivery scaffold from the EH monomer. Scaffolds with different architectures were fabricated and skeletal muscle myoblast cell compatibility, material properties and protein and gene delivery rates were all investigated.Item Tissue and Metabolic Engineering of Biohybrid Artificial Organs(2005-12-07) Yung, Chong Wing; Barbari, Timothy A; Bentley, William E; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In an effort to develop a biohybrid artificial organ, mammalian cells were engineered with several properties to make them adept in secreting therapeutic proteins and surviving low oxygen levels in an encapsulated environment. Three cell lines, C2C12, Jurkat, and HEK293, were investigated for their ability to secrete human interleukin 2 (hIL2), which can serve as an anti-cancer agent. An intracellular fluorescent protein marker was independently expressed using an internal ribosome entry site sequence so that hIL2 remained active and free for secretion. DsRed fluorescent protein markers were used since red light is known to be transmissible through mammalian tissue. Transient transfection of all three cell lines proved that internal red fluorescence measurements were indeed linearly correlated with the concentration of hIL2 secreted. To increase the survivability of encapsulated cultures, cells were engineered with an anti-apoptotic gene, bcl-2Delta, placed under the control of a hypoxia sensitive promoter. This protective system was found to lessen both hypoxia induced necrosis and apoptosis. To complete the biohybrid system, a novel hydrogel (mTG-Gel), utilizing microbial transglutaminase (mTG) to enyzmatically crosslink gelatin, was developed as a biocompatible cellular scaffold for encapsulating the engineered cells. These gels were stable at physiological temperature (37 oC) and could be tailored for enzymatic stability. Specifically, HEK293 cells that were metabolically engineered with all the previous characteristics were encapsulated in 4% mTG-Gels. In situ analysis of DsRed fluorescence showed that cells overlayed with mTG-Gel exhibited reductions in fluorescence with increasing height of gel. Human IL2 diffusion through the hydrogel into overlaying media was found to exhibit the expected dependence on the square of the gel thickness. Diffusion cells were used to determine an effective diffusion coefficient for hIL2, which compared well to that obtained from the gel-overlay cell culture experiments.