Articular cartilage provides a near frictionless surface for the articulating ends of bones. Cartilage functions to lubricate and transmit compressive forces resulting from joint loading and impact. If damaged, whether by traumatic injury or disease, cartilage lacks the ability for self-repair. This study explores the production of scaffoldree cartilage and investigates the effect of Tissue Growth Technologies’ CartiGen Bioreactor on the cartilage. Chondrocyte and bone marrow-derived stem cell (BMSC) attachment to chitosan is also investigated in hopes of producing a bilayered construct for osteochondral repair. Results demonstrate that culturing of scaffoldree cartilage in the CartiGen bioreactor resulted in an enhancement of the scaffoldree cartilage’s biomechanical and biochemical properties and that the chitosan microspheres were able to successfully support porcine chondrocyte and BMSC attachment. Results from both studies are encouraging for future work involving tissue engineered cartilage.

Conventional landfills are built to dispose of the increasing amount of municipal solid waste (MSW) generated each year. A relatively new type of landfill, called a bioreactor landfill, is designed to optimize the biodegradation of the contained waste to stabilized products. Landfills with stabilized waste pose little threat to the environment from ozone depleting gases and groundwater contamination. Limited research has been done to determine the importance of biodegradation enhancement techniques and the analytical methods that are used to characterize waste stability. The purpose of this research was to determine the effectiveness of several biodegradation enhancements and to evaluate the analytical methods which predict landfill stability. In the first part of this study leachate recirculation, and moisture and temperature management were found to significantly affect the biodegradation of MSW. Leachate recirculation, increased moisture, and higher temperatures increased the first order degradation rates of cellulose and volatile solids. Of the three enhancements, temperature was shown to have the biggest impact on the biodegradation of waste, but sufficient moisture is critical for degradation. Plastic material was also shown to significantly impact the measurements for volatile solids and lignin, which is important if these measurements are used to establish waste stability. In the second part of the study the analytical methods used to characterize waste were evaluated to determine if relationships existed between the methods and which methods were the best predictors of waste stability. Volatile solids and cellulose were found to be the best parameters to monitor waste in landfills. These parameters correlate well with each other, age of the waste, and other parameters. Volatile solids and cellulose are also relatively easy to determine, quick, and show little variation. / Master of Science

This was a study undertaken to investigate municipal solid waste (MSW) landfill stability parameters and landfill leachate properties to determine how solid waste and leachate characteristics can be used to describe stability. The primary objective was to determine if leachate properties could be used to determine stability of the overlying refuse. All landfills studied were engineered landfill bioreactors giving insight to how leachate recirculation affects stability. This study investigated the correlation between cellulose, lignin, volatile solids, and biochemical methane production (BMP). These parameters can been used to characterize landfill stability. The BMP tests indicate that a saturated waste can produce methane. Cellulose is an indicator of landfill stability. Wastes high in cellulose content were found to have high BMP. Paper samples studied indicated gas production from high-cellulose paper was higher compared to low-cellulose samples. Lignin has been found to correlate fairly well with BMP. Increasing cellulose to lignin ratios correlate well with increasing BMP levels, further supporting the use of the BMP test to indicate solid waste stability. In the BMP test for leachate, a mixture of the standard growth medium (less 80% distilled water) and 80% v/v leachate incubated for 15 days produced the most consistent BMP results. Leachate cellulose and BMP correlated well. The chemical oxygen demand (COD) and biochemical oxygen demand (BOD) also had some correlation to BMP tests. Leachate COD was found to decrease over time in landfill bioreactors. The use of leachate rather than MSW to determine stability would be more efficient. / Master of Science

No description available.

While tissue-engineering approaches of heart valves have made great strides towards creating functional tissues in vitro, the instruments used, named bioreactors, cannot efficiently integrate multiple stimuli to accurately emulate the physiological microenvironment. To address this, we conceptually designed and built a bioreactor system that applied a range of mechanical tension conditions, modulated matrix stiffness, and introduced biochemical signals in a combinatorial and high-throughput manner. Proof-of-concept experiments on PAVIC-seeded hydrogels were performed to assess the independent and combined effects of tensile strain, matrix stiffness and TGF-β1 on myofibroblast differentiation by measuring α-SMA expression, a marker that indicates a disease-associated phenotype. We found that matrix stiffness and TGF-β1 significantly increased α-SMA levels (p < 0.001), while the effect of mechanical strain was only significant on soft gels (~12 kPa) without TGF-β1. This study therefore demonstrated independent and integrated effects of multiple stimuli in regulating key cellular events in the aortic valve.

Nanoporous alumina is a rather newly characterized material that so far has found limited use in the construction of bioreactors. The material has many advantages compared to conventional immobilization matrices. I have investigated its use in flow-through bioreactors. The rigidity and porous structure of the material makes it an excellent choice for multienzyme reactor construction. The total activity in a reactor is easily controlled by the number of membranes since the porosity makes the material less prone to increase flow system pressure. This bioreactor is suitable for characterization of new enzymes since the amount of immobilized enzyme is standardized and the enzyme may be reused many times. We designed a simple stepwise technique for covalent immobilization on this matrix in a monolayer to minimize mass transfer effects in the reactor function. The kinetic parameters for ten different substrates were investigated for immobilized alcohol oxidase and, as a second step, a two-step reactor was also designed by addition of horseradish peroxidase. This bienzymatic reactor was, in turn, employed for measuring injected alcohol concentrations. The use of the matrix for substrate specificity screening was proven for two new epsilon-class glutathione transferases from Drosophila melanogaster. Immobilized trypsin showed a substantially prolonged lifetime and its potential use as an on-line digestion unit for peptide mass fingerprinting was also demonstrated. Finally, I investigated the immobilization of the model enzyme lactate dehydrogenase by adsorption mediated by metal ion chelation similar to IMAC. Regeneration was here possible multiple times without loss of capacity. In conclusion, immobilization of enzymes on nanoporous alumina is a convenient way to characterize, stabilize and reuse enzymes.

Human embryonic stem cells (hESCs) can provide a unique approach for novel tissue engineering applications. Previous groups have shown that hESCs can differentiate into specialized cell types through the generation of human embryoid bodies (hEBs). These multi-cellular constructs are then subjected to suspension culture for several weeks. Traditional hESC differentiation techniques have yielded non-homogeneous EBs derived in standard static cultures providing an inefficient platform for cellular viability and embryonic modeling. Here, our study aimed at systematically comparing the formation, growth, and differentiation capabilities of hESC-derived hEBs in dynamic and static suspension cultures. We used a continuous flow perfusion slow turning lateral vessel, STLV, system (Synthecon) to model after an in vivo environment. This study is in part of a larger study investigating the role of HOXB5 in the human endothelial differentiation pathway. Embryoid bodies were created by hanging drops and then subjected to static or dynamic culture for 10 days. Cells were harvested and a simple Alkaline Phosphatase assay was used to determine if the system was viable for propagating hEB. We show that the STLV system is viable for our future studies and this system more efficient at maintaining hEBs.

Student Number : 9902262G - MSc Dissertation - School of Molecular and Cell Biology - Faculty of Science / Anaerobic fermentative bacteria degrade waste components in landfills where methane (CH4) and carbon dioxide (CO2) are the primary biogases emitted and methanotrophic bacteria in the cover soil oxidise the emitted CH4. Three bi-phasic bench-scale landfill bioreactors were commissioned to evaluate soil nutrient addition effects on CH4 formation and oxidation and to isolate inherent soil methanotrophs using Nitrate Mineral Salts (NMS) medium. Set A soil contained no nutrient additions, Set B soil contained 50 μM nitrate and 150 μM phosphate and Set C soil contained dried sewage cake. Bioreactors were run for a 4 week period and pH, anaerobic gas emissions, volatile fatty acids (VFA), bacterial counts and scanning electron microscopy (SEM) analyses were performed. A pilot study revealed that pH dictated the stability of methanogenesis, where increased VFA levels inhibited methanogenesis. Furthermore, it was revealed that modifications of the NMS medium were needed to enrich for methanotrophs. An in depth study showed that the Set C anaerobic reactor produced the most methane with Set B the least. The hypothesis that methane oxidation in the soil could regulate methane formation in the waste could not be conclusively observed, as a lack of aeration in the soil reactors is believed to have prevented the proliferation of methanotrophs here. No methanotrophs were successfully isolated from soil, but rather major heterotrophic bacterial interference was observed. SEM revealed the presence of rod and cocci forms of bacteria in both leachate and soil, consistent with literature reports, which indicated that the bench-scale landfill bioreactors were capable of promoting bacterial growth.

The anterior cruciate ligament (ACL) is a major ligament within the knee joint. Its role is to provide stability and maintain the physiological kinetics and kinematics of the joint. ACL injuries are common as a result of sporting and traffic accidents and current therapeutic options do not fully restore the joint kinetics and kinematics. As such, patients often suffer from increased joint laxity and joint pain following an ACL reconstruction and this can lead to secondary problems such as osteoarthritis. It is believed that improving the ACL graft could help restore the normal kinetics and kinematics of the knee joint and hence postpone or prevent the onset of primary and secondary problems. Tissue engineering has the potential to provide functional tissue to repair or replace injured or diseased tissues in the patient. The ACL is a tissue which could benefit from such developments and thus improve the success of the reconstruction. However, the ACl is a complex structure made up of a highly orientated collagen hierarchy which experiences three dimensional loading in vivo. For an engineered tissue to be functional it is necessary for this orientated structure to be replicated. The appropriate structure is achieved by replication of the in vivo ACL strain pattern which requires combined tensile and torsional loading. Current custommade and commercially available bioreactors have not been able to fully replicate this motion with the necessary feedback and monitoring of mechanical parameters. The aim of this project was to develop a novel bioreactor with physiological mechanical conditioning for the tissue engineering of an anterior cruciate ligament. A bioreactor capable of applying complex tensile and torsional loading to a developing ACL was designed, manufactured and validated. The bioreactor which has been developed is a novel research tool which allows the effect of a number of parameters to be investigated in a 3D loading environment. It can be used for the engineering of connective tissues such as ligaments and tendons and has the potential to be adapted for use with other musculoskeletal tissues such as bone. It could also be used for research to understand the processes involved in the growth and development of tissues.

The membrane bioreactor (MBR) is becoming increasingly popular for wastewater treatment, mainly due to its capability of producing high quality effluent with a relatively small footprint. However, high plant maintenance and operating costs due to membrane fouling limit the wide spread application of MBRs. Membrane fouling generally depends on the interactions between the membrane and, the activated sludge mixed liquor, which in turn, are affected by the chosen operating conditions. The present research study aimed to explore the process performance and membrane fouling in the membrane enhanced biological phosphorus removal (MEBPR) process under different operating conditions by, (1) comparing two MEBPRs operated in parallel, one with constant inflow and another with a variable inflow, and by, (2) operating the MEBPRs with different solids retention times (SRT). On-line filtration experiments were conducted simultaneously in both MEBPR systems by using test membrane modules. From the transmembrane pressure (TMP) data of the test membrane modules, it was revealed that fouling propensities of the MEBPR mixed liquors were similar in both parallel reactors under the operating conditions applied, although the fouling propensity of the aerobic mixed liquors of both reactors increased when the SRT of the reactors was reduced. Routinely monitored reactor performance data suggest that an MEBPR process with a varying inflow (dynamic operating condition) performs similarly to an MEBPR process with steady operating conditions at SRTs of 10 days and 20 days. Mixed liquor characterization tests were conducted, including critical flux, capillary suction time (CST), time to filter (TTF) and, bound and soluble extracellular polymeric substances (EPS) were quantified, to evaluate their role on membrane fouling. The tests results suggest that the inflow variation in an MEBPR process did not make a significant difference in any of the measured parameters. With decreased SRT, an increase in the concentrations of EPS was observed, especially the bound protein, and the bound and soluble humic-like substances. This suggests that these components of activated sludge mixed liquors may be related to membrane fouling. No clear relationship was observed between membrane fouling and other measured parameters, including critical flux, normalized CST and normalized TTF.