Intervertebral disc (IVD) degeneration is a prevalent health problem that is highly linked to back pain. To understand the disease and tissue response to therapies, ex-vivo whole IVD organ culture systems have recently been introduced. The goal of this study was to develop and validate a whole spinal segment culturing system that loads the disc in complex loading similar to the in-vivo condition, while preserving the adjacent endplates and vertebral bodies. The complex loading applied to the spinal segment was achieved with three pneumatic cylinders. The pneumatic cylinders were rigidly attached to two triangular alumni plates at each corner, comprising the loading mechanism. By extending or compressing the pneumatic cylinders, three modes of loading were achieved: flexion-extension, bi-lateral bending, and cyclic compression. The cylinders were controlled via microcontroller, and the entire system was fully automated. The culture container, which housed the spinal segment during culturing, was a flexible silicone container with an aluminum base and lid. The culture container attached to the loading mechanism allows for loading of the spinal segment. It had a vent attached to the aluminum lid that allowed for gas exchange in the system. The dynamic bioreactor was able to achieve physiologic loading conditions with 100 N of applied compression and approximately 2-4 N-m of applied torque. The function of the bioreactor was validated through testing of bovine caudal IVDs with intact endplates and vertebral bodies that were isolated within 2 hours of death and cultured for 14 days under a diurnal cycle. The resulting IVD cell viability following 14 days of loading was approximately 43% and 20% for the nucleus pulposus and annulus fibrosus respectively, which was significantly higher than the unloaded controls. The loading system accurately mimicked flexion-extension, bi-lateral bending, and compression motions seen during daily activities. Results indicate that this complex dynamic bioreactor may be appropriate for extended pre-clinical testing of vertebral mounted spinal devices and therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22503号 / 農博第2407号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5283(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

A new and promising trend in solid waste management is to operate the landfill as a bioreactor. Bioreactor landfills are controlled systems in which moisture addition and/or air injection are used as enhancements to create a solid waste environment capable of actively degrading the biodegradable organic fraction of the waste. Although there are many advantages associated with bioreactor landfills, some challenges remain. One such challenge is the ammonia-nitrogen concentration found in the leachate. The concentrations of ammonia-nitrogen tend to increase beyond concentrations found in leachate from conventional landfills because recirculating leachate increases the rate of ammonification and results in accumulation of higher levels of ammonia-nitrogen concentrations, even after the organic fraction of the waste is stabilized. Because ammonia-nitrogen persists even after the organic fraction of the waste is stabilized, and because of its toxic nature, it is likely that ammonia-nitrogen will determine when the landfill is biologically stable and when post-closure monitoring may end. Thus an understanding of the fate of nitrogen in bioreactor landfills is critical to a successful and economic operation. Ammonia-nitrogen is typically removed from leachate outside of the landfill. However, additional costs are associated with ex-situ treatment of ammonia, as separate treatment units on site must be maintained or the leachate must be pumped to a publicly owned wastewater treatment facility. Therefore, the development of an in-situ nitrogen removal technique would be an attractive alternative. Several recent in-situ treatment approaches have been explored, but lacked the information necessary for field-scale implementation. The objectives of this study were to develop information necessary to implement in-situ ammonia removal at the field-scale. Research was conducted to evaluate the kinetics of in-situ ammonia removal and to subsequently develop guidance for field-scale implementation. An aerobic reactor and microcosms containing digested municipal solid waste were operated and parameters were measured to determine nitrification kinetics under conditions likely found in bioreactor landfills. The environmental conditions evaluated include: ammonia concentration (500 and 1000mg N/L), temperature (25o, 35o and 45oC), and oxygen concentration in the gas-phase (5, 17 and 100%). Results suggest that in-situ nitrification is feasible and that the potential for simultaneous nitrification and denitrification in field-scale bioreactor landfills is significant due to the presence of both aerobic and anoxic areas. All rate data were fitted to the Monod equation, resulting in an equation that describes the impact of pH, oxygen concentration, ammonia concentration, and temperature on ammonia removal. In order to provide design information for a field-scale study, a simple mass balance model was constructed in FORTRAN to forecast the fate of ammonia injected into a nitrifying portion of a landfill. Based on model results, an economic analysis of the in-situ treatment method was conducted and compared to current ex-situ leachate treatment costs. In-situ nitrification is a cost effective method for removing ammonia-nitrogen when employed in older waste environments. Compared to reported on-site treatment costs, the costs associated with the in-situ ammonia removal process fall within and are on the lower end of the range found in the literature. When compared to treating the leachate off-site, the costs of the in-situ ammonia removal process are always significantly lower. Validation of the laboratory results with a field-scale study is needed.

ABSTRACT Implementation of Physiologic Pressure Conditions in a Blood Vessel Mimic Bioreactor System Kevin Mark Okarski Tissue engineering has traditionally been pursued as a therapeutic science intended for restoring or replacing diseased or damaged biologic tissues or organs. Cal Poly’s Blood Vessel Mimic Laboratory is developing a novel application of tissue engineering as a tool for the preclinical evaluation of intravascular devices. The blood vessel mimic (BVM) system has been previously used to assess the tissue response to deployed stents, but under non-physiologic conditions. Since then, efforts have been made to improve the vessel and bioreactor’s ability to emulate in vivo conditions. The ability to tissue engineer constructs similar to their native tissue counterparts is heavily reliant upon controlling the environment and mechanical stimuli the construct is exposed to. Mimicking physiologic conditions influences cellular growth, proliferation, and differentiation. Two important mechanical stimuli are cyclic strain and wall shear stress. Previous work sought to improve these factors within the BVM bioreactor and resulted in the implementation of pulsatile perfusion and increased fluid viscosity. These previous bioreactor design modifications generated pulsatile pressures of approximately 80 mmHg and a wall shear stress of 6.4 dynes/cm2. However, physiologic pressure waveforms were not achieved. Studies in this thesis were carried out to implement an effective means of establishing a more physiologic pressure wave within the bioreactor that is accurate, consistent, and easily adjustable. As a result of conducting the present studies, modifications to the bioreactor system were made that uphold the overall goals of efficacy and efficiency. The desired pressure wave was created by setting the degree of pump tubing occlusion on the 3-roller peristaltic pump head and using a water column to backpressure the bioreactor chamber. Maintaining a desired backpressure within the system necessitated the development of a new bioreactor chamber with increased extraluminal leak pressure resistance. The opportunity was also used to further improve upon the bioreactor chamber design to allow for 360° rotation to reduce cell sedimentation. Modifications to the bioreactor system required quantitative evaluation to assess their impact upon local flow dynamics to the tissue construct. A system model was created and evaluated using computational modeling. Through the work performed in this thesis, pulsatile pressure waves of approximately 120/80 mmHg were successfully established within the bioreactor. The ability to accurately model physiologic pressures will ultimately help yield tissue constructs more similar to native tissues – both healthy and pathological. The newly designed bioreactor chamber and computational model for the system will be helpful tools for implementing or evaluating future bioreactor developments or improvements. While the main objective of the thesis has been completed by creating a system capable of emulating physiologic pressure fluctuations, there still remains room for further improvements in back-pressuring and scaling the system, refining the rotational bioreactor chamber design, and building upon the complexity and accuracy of the computational model.

Mammary epithelial cells are highly efficient secreting cells. With genetic engineering, the uses of these cells could be endless. Research is being conducted on these cells to determine their full potential to the biotech industry. This paper investigates whether bovine epithelial mammary cells can survive in glutaraldehyde-treated gelatin tubes in an operating bioreactor. Many bioreactors were developed and tested to suit the needs of the cells. Procedures were created and carried out to ensure sterility of the bioreactors. Bovine mammary epithelial cells were implanted in the bioreactors and samples of their growth were taken over time.

We present research investigating how, with in depth knowledge of the community, microbial communities may be harnessed for bioremediation of hazardous water contaminants. We focused on the bacterial reduction of perchlorate, a common water contaminant. For this we studied the structure and capabilities of a novel sulfur-utilizing, perchlorate-reducing bacterial (SUPeRB) consortium. Initially, we characterized the minimal consortium that retained functional capabilities, using 16S rRNA and functional gene analysis. A diverse functional consortium dominated by Beta-Proteobacteria of the family Rhodocyclaceae and sulfur-oxidizing Epsilon-Proteobacteria was found. We also examined the optimal growth conditions under which perchlorate degradation occurred and uncovered the upper limits of this function. Bacterial isolates were screened for function and the presence of functional genes. We expanded to bioreactor studies at bench- and pilot-scale, and first used a perchlorate-reducing, bench-scale bioreactor to probe the stability of the microbial ecosystem. During stable reactor function, a core consortium of Beta- and Epsilon-Proteobacteria reduced perchlorate and the co-contaminant nitrate. A disturbance of the vi consortium led to a failure in function and to higher system diversity. This suggests that the SUPeRB consortium was not metabolically flexible and high population diversity was necessary for a return to stable function. In a pilot-scale bioreactor we determined that the SUPeRB consortium could stably degrade low levels of perchlorate to below the EPA maximum recommended limit. Field conditions, such as temperature extremes and intermittent perchlorate feed, did not negatively impact overall function. When all reactor consortia were compared we observed that the volume of the reactor and the initial inoculum were not as important to stable reactor function as the acclimatization of the consortium to the system and maintenance of favorable conditions within the reactor. In summary we found that the SUPeRB consortium successfully degraded perchlorate in multiple systems. The study of this novel consortium expands our knowledge of the metabolic capabilities of perchlorate-reducing bacteria and suggests potential evolutionary pathways for perchlorate-reduction by microorganisms. The SUPeRB consortium may be used to establish bioremediation systems for perchlorate and other environmental contaminants.

Conventional nonpoint source pollution policies encourage the adoption of conservation practices to reduce nonpoint source pollutants by paying a portion of the cost to install best management practices. Alternative financial incentive programs, such as payment for environmental services (PES) programs, aim to improve program effectiveness by paying directly for the quantity of environment services provided, but implementing PES programs to reduce nonpoint source pollution has been challenging given the costs and technical feasibility of measuring pollutant outcomes. Bioreactors, engineered sinks that convert biologically available forms of nitrogen into an inert form (N_2), have recently been proposed to treat and remove legacy nitrogen from springs (Easton et al., 2019). Since nitrogen removal can be directly measured, there is potential to implement an outcome-based PES program. Little information exists on the costs and risks sellers face under such a program or the impact of contractual conditions. This research applies Monte Carlo simulation to a case study bioreactor in the Chesapeake Bay Watershed to estimate the financial risks and rewards to N removal service providers under different outcome-based PES contractual conditions. Results indicate that under a fifteen-year contract term and price of $25/lb/yr of nitrogen removal, outcome-based PES for denitrifying bioreactors has a high chance of generating positive financial outcomes for a commercial size case study bioreactor that removes an average of 1,279 lbs of N annually. / Master of Science / Conventional policies to reduce diffuse water pollutants encourage the adoption of conservation practices to reduce diffuse water pollutants by paying a portion of the cost to install remedial practices or technologies. Payment for environmental services (PES) programs, an alternative to conventional policies, aims to improve program effectiveness by paying directly for the quantity of environment services provided. However, implementing PES programs to reduce diffuse water pollution has been challenging given the costs and technical feasibility of measuring pollutants and outcomes of remedial efforts. Bioreactors, engineered sinks that convert the diffuse water pollutant nitrogen into a non-pollutive form, have recently been proposed to remove legacy nitrogen from springs (Easton et al., 2019). Using bioreactors, nitrogen removal can be directly measured, so there is potential for an outcome-based PES program. Little information exists on the costs and risks sellers face under such a program or the impact of contractual conditions. This research applies financial simulation to a case study bioreactor in the Chesapeake Bay Watershed to estimate the financial risks and rewards to N removal service providers under different outcome-based PES contractual conditions. Results indicate that under a fifteen-year contract term and price of $25/lb/yr of nitrogen removal, outcome-based PES for denitrifying bioreactors has a high chance of generating positive financial outcomes for a commercial size case study bioreactor that removes an average of 1,279 lbs of N annually.

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