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Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content. / Ph. D.

Bioreactor landfills offer a sustainable way to achieve increased waste degradation along with benefits such as enhanced landfill gas (LFG) recovery, reduction in leachate pollution potential and rapid increase in landfill volumetric capacity. It also offers significant reduction in post closure management activities as leachate treatment, LFG impact on the environment and improves the potential for land reuse. The regulatory 30 year post-closure period is believed to account for attenuation of organics, metals and trace pollutants of adverse environmental consequences. Methodologies to improve the degradation rate and process are refuse shredding, nutrient addition, pH buffering, and temperature control along with moisture enhancement. Municipal Solid Waste (MSW) settlement and field capacity are of significant beneficial interest to achieve maximum utility of landfill volume and compute water requirements for rapid degradation using bioreactor concepts. Physical and biochemical Municipal Solid Waste (MSW) characteristics were investigated with specific emphasis on the Bio-Chemical methane potential (BMP) test. The impact of waste characteristics on its compressibility and moisture retention capacity was evaluated on a laboratory scale. Traditional in-situ waste compression models from literature were used to compare with the obtained laboratory data. / Master of Science

Regenerative medicine involves formation of tissue or organ for replacement of a wounded or dysfunctional tissue. Healthy cells extracted from the patient are expanded and are seeded on a three-dimensional biodegradable scaffold. The structure is then placed in a bioreactor and is provided with nutrients for the cells, which proliferate and migrate throughout the scaffold to eventually form a desired to tissue that can be transplanted into the patient's body.  Inability to monitor this complex process of regeneration in real-time makes control and optimization of this process extremely difficult. Histology, the gold standard used for tissue structural assessment, is a static technique that only provides "snapshots" of the progress and requires the specimen to be sacrificed. This inefficiency severely limits our understanding of the biological processes associated with tissue growth during the in vitro pre-conditioning phase. Optical Coherence Tomography (OCT) enables imaging of cross sectional structure in biological tissues by measuring the echo time delay of backreflected light. OCT has recently emerged as an important method to assess the structures of physiological, pathological as well as tissue engineered blood vessels. The goal of the present study is to develop an imaging system for non-destructive monitoring of blood vessels maturing within a bioreactor. Non-destructive structural imaging of tissue-engineered blood vessels cultured in a novel bioreactor was performed using free-space and catheter-based OCT imaging, while monitoring of the endothelium development was performed using a fluorescence imaging system that utilizes a commercial OCT catheter. The project included execution of three specific aims. Firstly, we developed OCT instrumentation to determine geometrical and optical properties of porcine and human skin in real-time. The purpose of the second aim was to assess structural development of tissue-engineered blood vessels maturing in a bioreactor. We constructed a novel quartz-based bioreactor that will permit free space and catheter-based OCT imaging of vascular grafts. The grafts were made of biodegradable PCL-collagen and seeded with multipotent mesenchymal cells. We imaged the maturing grafts over 30 days to assess changes in graft wall thickness. We also monitored change in optical properties of the grafts based on free-space OCT scanning.   Finally, in order to visualize the proliferation of endothelial cells and development of the endothelium, we developed an imaging system that utilizes a commercial OCT catheter for single-cell-level imaging of the growing endothelium of a tissue-engineered blood vessel. We have developed two modules of an imaging system for non-destructive monitoring of maturing bioengineered vascular grafts. The first module provides the ability to non-destructively examine the structure of the grafts while the second module can track the progress of endothelialization. As both modules use the same endoscope for imaging, when operated in sequence, they will produce high-resolution, three-dimensional, structural details of the graft and two-dimensional spatial distribution of ECs on the lumen. This non-destructive, multi-modality imaging can be potentially used to monitor and assess the development of luminal bioengineered constructs such as colon or trachea. / Ph. D.

The overall goal of this research project is to develop methods for producing a tissue engineered ligament. The envisioned tissue engineering strategy involves three steps: seeding bone marrow stromal cells (BMSCs) onto electrospun scaffolds, processing them into cords that allow cell infiltration, and conditioning them with uniaxial cyclic stretch. These steps were addressed in three complimentary studies to establish new methods to engineer a tissue with ligament-like cells depositing organized extracellular matrix (ECM). In the first study scaffold topographies were systematically varied to determine topographies that induce cells to orient and differentiate into ligament-like cells in static culture. Scaffolds — electrospun from poly (ester-urethane urea) (PEUUR) with different fiber diameters degrees of fiber alignments — were biocompatible and supported cell growth. Topographic cues guided cell alignment, and cell elongation increased with increasing fiber alignment. Finally, expression of the ligament-like markers collagen type I and decorin were enhanced on the smallest fiber diameters compared to larger diameters. In the second study BMSCs — seeded onto aligned electrospun PEUUR scaffolds — were cyclically stretched to determine the effect of dynamic mechanical stimulation on BMSC alignment and differentiation. BMSCs remained aligned parallel to the direction of fiber alignment and expressed ligament markers (e.g. collagen type I, decorin, scleraxis, and tenomodulin) on electrospun scaffolds after the application of stretch. However, the cyclic stretch regimen was not able to enhance expression of ECM components. In the third study techniques were developed to produce more clinically relevant constructs with improved cell infiltration. Specifically, a co-electrospun scaffold composed of two well integrated components was developed to create larger pores. The scaffold was also embedding in a photo-crosslinkable hydrogel to prevent the fibers from collapsing. These results demonstrate the feasibility of making a tissue engineered ligament by seeding BMSCs on an aligned, co-electrospun scaffold with submicron diameter fibers and then applying cyclic mechanical stretch. Future work will involve combining these three steps to achieve materials suitable for in vivo testing. / Ph. D.

Drainage ditches, or grassed waterways, collect nutrient-laden runoff from agricultural fields and transport it to nearby waterbodies. The high nitrogen and phosphorus content in this water leads to negative effects, such as eutrophication in the receiving waters. In-ditch bioreactors are a simple, inexpensive treatment technology that could potentially remove nitrogen and phosphorus from agricultural runoff. In-ditch bioreactors are intended to reduce flow rate and stimulate denitrification and sedimentation. Using experimental ditch segments and simulated runoff, this study evaluated nutrient removal in 1) vegetated ditches, 2) vegetated ditches with woodchip bioreactors and 3) vegetated ditches with combination woodchip and biochar bioreactors. Biochar was added in an effort to increase phosphorus removal. Inlet and outlet concentrations of nitrate, ammonium and phosphate were measured for each of the three treatments in triplicate. There were no statistically significant differences between treatments on load removed for any of the three nutrients of interest. Issues in measuring outlet flow rate made drawing definitive conclusions on nutrient load reductions difficult. Further experimentation using adjusted outlet flow measuring methods and bioreactor design would help establish whether in-ditch bioreactors are suitable for use as a nutrient removal technology in agricultural grassed waterways. / Master of Science / Drainage ditches, or grassed waterways, are located at the edge of agricultural fields where runoff migrates naturally. These ditches help to direct runoff from the field to receiving waterbodies while reducing erosion. Agricultural runoff often contains high levels of nitrogen and phosphorus from fertilizer added to promote crop growth. When runoff with a high nutrient content reaches a waterbody, it reduces the quality of the water for the plants and animals that live in it and for human recreation or consumption. In-ditch bioreactors are a simple, inexpensive treatment technology that could potentially remove nitrogen and phosphorus from agricultural runoff. In-ditch bioreactors have the potential to remove nitrogen from the water by creating optimal conditions for the microorganisms that transform nitrogen in the water to nitrogen in the air. Phosphorus removal has the potential to be enhanced by in-ditch bioreactors that reduce flow and allow for phosphorus to settle out of the water. In addition, settling of phosphorus may be increased by adding a material, such as biochar, that phosphorus can attach to. Using experimental ditch segments and simulated runoff, this study looked at nutrient removal in 1) vegetated ditches, 2) vegetated ditches with woodchip bioreactors installed and 3) a vegetated ditch with combination woodchip and biochar bioreactors installed. Concentrations of two nitrogen compounds and one phosphorus compound were measured before and after passing through each ditch. There were no significant differences between any of the three ditch types on how much of each compound they could remove. These results are inconclusive due to inaccuracies in measuring flow rate at the outlet of the ditches. Further experimentation using improved flow measuring techniques and bioreactor designs would likely help establish whether in-ditch bioreactors are suitable for use as a nutrient removal technology.

The objective of this study was to determine how the key features of bioreactor landfills; increased temperature, moisture and microbial activity, affect the biological stability of the landfill material. In the first part of the study the solubilization and degradation of lignin in paper exposed to these bioreactor landfill conditions are explored. The solubility of the lignin in paper was observed at different temperatures and over 27 weeks at 55°C and the anaerobic bioconversion of office paper, cardboard and Kraft lignin was observed in bench-scale reactors over 8 weeks. As the temperature rose, lignin solubility increased exponentially. With extended thermal treatment, the dissolution of lignin continues at a constant rate. This rate increases 15 times for paper and 1.5 times for cardboard in the presence of rumen inoculum compared to un-inoculated systems. At around 6 weeks the inter-monomeric linkages between the solubilized lignin molecules began breaking down, releasing monomers. In cardboard and Kraft lignin, a significant amount of the monomers mineralize to CO₂ and CH₄ during this time period. The results indicate that small, but significant rates of lignin solubilization and anaerobic lignin degradation are likely to occur in bioreactor landfills due to both higher temperature and microbial activity. In the second part of the study, field data from the Outer Loop Recycling and Disposal Facility in Louisville, Kentucky was evaluated to determine the effectiveness of an anaerobic-aerobic landfill bioreactor (AALB) vs. the control landfill that is managed as a traditional landfill. Moisture, temperature, elevation and the amount of time the MSW has spent in the landfills (age) were measured and compared to determine the factors that affect the biological stability of the landfill. The results showed that the MSW in the AALB is more biologically stable than the MSW in the control landfill, indicating that they are more degraded. Additionally, elevation or location of the MSW was the key factor in determining the extent of MSW stability within the AALB and temperature is the key factor in determining the biological stability of the MSW in the control landfill. Higher temperatures correlated with a more biologically stable waste. The cellulose to lignin ratio (C/L ratio) and biochemical methane potential (BMP) were the main biological stability parameters used. / Master of Science

There are more than three thousand landfills in the United States, in which approximately 55% (1998, U. S. EPA 1999) of the MSW generated in the US is buried. The majority of the landfills are conventional, but in the last two decades new types of landfills, called leachate recycle and bioreactor landfills, have been designed and tested as an enhanced environment for biochemical degradation of municipal solid waste. All the landfills are regulated under Subtitle D of the Resource Conservation and Recovery Act (RCRA). The shortage of time and money has limited the amount of research done on waste stability analysis. The purpose of this study was to evaluate the importance of lignocelluloses in biodegradation and the secondary settlement based on dry density and typical landfill evaluating parameters. Both parts of the study samples were collected and analyzed from eleven landfills. In the first part of the study, bioreactor landfills were found more effective, faster in the degradation of VS and cellulose as compared to conventional landfills. The time required for stabilization is reduced to about 1/3 that of conventional landfills. The lignocelluloses degradation that occurs in these landfills is happening in two phases. In the initial, rapid degradation phase, the primary degradation substrate is cellulose. In the second phase, after cellulose degraded to 15-20% of the waste, degradation of the remaining cellulose along with lignin and the hemicelluloses takes place. The start of lignin and hemicellulose degradation results in an increase in the biochemical methane potential (BMP). In the second part of the study, the addition of moisture to the landfills presented a contentious issue. Moisture is encouraged for MSW refuse degradation, but for settlement it reduces compressibility. In leachate recycle landfills, the dry density is higher than in conventional landfills; therefore there is more available room for further MSW load. The increase can reach up to 40 percent in total volume. / Master of Science