A Tissue Engineering (TE) approach to heart valve replacement has the aim of producing an implant that is identical to healthy tissue in morphology, function and immune recognition. The aim is to harvest tissue from a patient, establish cells in culture from this tissue and then use these cells to grow a new tissue in a desired shape for the implant. The scaffold material that supports the growth of cells into a desired shape may be composed of a biodegradable polymer that degrades over time, so that the final engineered implant is composed entirely of living tissue. The approach used at Swinburne University was to induce the desired mechanical and functional properties of tissue and is to be developed in an environment subjected to flow stresses that mimicked the haemodynamic forces that natural tissue experiences. The full attainment of natural biomechanical and morphological properties of a TE structure has not as yet been demonstrated. In this thesis a review of Tissue Engineering of Heart Valves (TEHVs) is presented followed by an assessment of biocompatible materials currently used for TEHVs. The thrust of the work was the design and development of a Bioreactor (BR) system, capable of simulating the corresponding haemodynamic forces in vitro so that long-term cultivation of TEHVs and/or other structures can be mimicked. A full description of the developed BR and the verification of its functionality under various physiological conditions using Laser Doppler Anemometry (LDA) are given. An analysis of the fluid flow and shear stress forces in and around a heart valve scaffold is also provided. Finally, preliminary results related to a fabricated aortic TEHV-scaffold and the developed cell culture systems are presented and discussed. Attempts to establish viable cell lines from ovine cardiac tissue are also reported.

Acrylonitrile (AN) is a major volatile waste generated in the production of acrylamide and often associated with aromatic contaminants (toluene and styrene) in plant effluents. We examined Rhodococcus rhodochrous DAP 96622 to determine if it could be adapted to efficient biodegradation of acrylonitrile (AN) in a bioreactor. A model bioreactor with granular activated carbon (GAC) as a substratum for Rhodococcus with AN as sole carbon or in combination with toluene was established. The kinetics of AN biodegradation by immobilized and planktonic cells were evaluated and compared. Inlet load and empty retention time were varied to test the removal efficiency in fed-batch and single-pass mode reactor. In addition, the three dimensional structure and characteristics of the biofilm were followed using confocal scanning laser microscopy (CSLM) and relative software. Immobilized cells in the bioreactor, at starting concentrations of AN up to 1150 mg l-1 in the presence of Tol, had at least 13 fold higher AN degradation rates than that seen of planktonic cells. A near steady state of AN degradation was maintained at 75-85% for AN and 80%-90% for Tol within the parameter of EBRT=8 min and AN and Tol inlet loads between 50-200 mg l-1 h-1 and 200-500 mg l-1h-1, respectively. However, when the inlet load of AN was increased to more than 200mg l-1 h-1 and 500 mg l-1 h-1 for Tol, a reduction in efficiency of AN degradation was observed. Biofilms with discrete microcolonies interspersed with voids and channels were observed. Precise measurement of biofilm characteristics agreed with the assumption that the biomass and thickness of the biofilm increased along the carbon column depth. With a porous attachment material like GAC, substrate diffusion is most likely not a limiting factor for AN degradation. Rhodococcus rhodochrous DAP 96622 in a non-sterile activated charcoal column showed efficient degradation of AN in the presence of Tol. The Rhodococcus bioreactor may provide a potential practical waste gas and water treatment system.

En aquest memòria es recullen els resultats més rellevants del treball experimental realitzat amb l'objectiu d'avançar en el coneixement per a desenvolupar un procés de tractament en continu de decoloració d'aigües residuals tèxtils utilitzant fongs ligninolítics. S'inicia el treball realitzant un estudi sobre les condicions d'operació per al tractament en continu d'una solució del colorant tèxtil Gris Lanaset G (complex organometal·lic) en un bioreactor fluiditzat per polsos d'aire amb el fong Trametes versicolor en forma de pèl·lets, establint la concentració de nutrients i el temps de residència hidràulic al qual s'ha d'operar per assolir percentatges de decoloració que compleixen la normativa d'abocament.Es proposa el mecanisme de decoloració, que té lloc en varies etapes en sèrie, primer es produeix l'adsorció inicial del colorant sobre la biomassa, posteriorment aquest es transfereix cap a l'interior de les cèl·lules, on es produeix el trencament del complex metàl·lic i finalment s'excreten al medi dels productes de degradació.Es determina el temps de residència cel·lular màxim en 40 dies i s'estableix la metodologia de renovació de biomassa en forma de pèl·lets per al tractament en continu. La renovació total de la biomassa es realitza en tres etapes, renovant un terç de la biomassa total del bioreactor en cada etapa, de manera que transcorregut un temps de residència cel·lular s'hagi renovat tota la biomassa del sistema. Es dissenya un reactor fluïditzat per polsos d'aire a escala pilot de 10 litres de volum útil i es realitza un estudi del comportament fluidodinàmic determinant el temps de mescla del sistema, i s'opera en discontinu i continu sense problemes operacionals. Un cop comprovada la bondat de l'escalat del bioreactor, s'introdueixen canvis paulatinament per adaptar el procés a condicions d'operació industrials, con són la no esterilitat de l'efluent a tractar, la supressió de macronutrients i micronutrients, la substitució de glucosa de qualitat anàlisi per glucosa de qualitat industrial, i així acabar tractant aigües residuals reals d'una indústria tèxtil sense esterilitzar on es posen de manifest alguns problemes que requeriran estudis posteriors.Donat que el procés d'eliminació de color més utilitzat a escala industrial és l'adsorció, s'estudia a escala laboratori l'adsorció del colorant Gris Lanaset G amb carbó actiu granular per tal de realitzar posteriorment un anàlisi comparatiu tant econòmic com ambiental dels dos processos, el d'adsorció i el biotecnològic. Aquests anàlisis posen de manifest que els costos d'operació del tractament biològic són únicament un 20% superiors que els costos d'operació del procés d'adsorció, i que aquest percentatge es podria reduir encara més trobant substrats alternatius a l'extracte de malta per la formació dels pèl·lets. També es posa de manifest que el procés biotecnològic és més sostenible que el procés d'adsorció, i per tant el procés biotecnològic és una alternativa interessant des de el punt de vista ambiental al tractament d'adsorció. / In this work are shown the most important results obtained in the experimental studies done with the goal of improving in the knowledge to develop a continuous process treatment for the decolourisation of textile wastewaters by ligninolytic fungi. The work starts with an operation conditions study for the continuous treatment of the dye Gris Lanaset G, which is a textile metal-complex dye, in an air pulses fluidised bioreactor by the white root fungi Trametes versicolor in pellet morphology. The nutrients concentration is established, and the effect of the hydraulic residence time in the decolourisation percentages is evaluated, taking into account the accomplishment of the standards established.It is proposed the decolourisation mechanism, which presents tree steeps: first of all, the dye is adsorbed in the biomass, after the dye is transferred into the cells, where takes place the break down of the metal complex, and finally the degradation products are released to the liquid medium.The maximum cellular residence time is determined in 40 days, and the biomass renovation methodology is established for the continuous treatment. The total biomass renovation takes place in three steeps, renovating one third of the total biomass in the bioreactor in every steep, in such a way that in the course of one cellular residence time, all the biomass in the bioreactor has been renovated. A pilot scale air pulses fluidised bioreactor is designed with a 10 L working volume, and the fluidodimanic behaviour is studied, determining the mixing time of the system. Without having any operational problem, the batch and continuous operation is carried out successfully. Once it is checked the goodness of the system, some changes are introduced gradually to adapt the system to industrial conditions. The changes are: the non-sterility of the effluent, the suppression of macronutrients and micronutrients, and the substitution of analysis quality glucose by industrial quality glucose. Finally is carried out the treatment of non-sterile real textile wastewaters.The most used removal colour process at industrial scale is the adsorption, so the adsorption of the dye Gris Lanaset G in granular activated carbon (GAC) is studied. The goal of this study is to make a comparative economical and environmental analysis of the two processes, adsorption and biodegradation. These analysis show that the operational cost of the biologic treatment is only 20% more than the operational cost for the adsorption process. This percentage could be lower if an alternative substrate is found for the pellets formation. Also is showed that the biotechnological rocess is more sustainable than the adsorption one, so the biotechnological process is an interesting choice from the environmental point of view to the adsorption treatment.

Grandes defectos óseos constituyen un reto para el campo clínico, ya que no puede ser reparado por el propio organismo, sino que requieren la implantación de injertos de hueso adecuado. Para superar los inconvenientes de los injertos procedentes de fuentes autólogas o allogeneicas, la ingeniería de tejidos óseos pretende sustituir el tejido perdido utilizando el cultivo de células in vitro sobre biomateriales porosos. El cultivo de células en grandes andamios porosos ha demostrado ser difícil, que requiere bioreactores, que se utilizan para el cultivo de tejidos y el estudio del comportamiento de células en 3D de los andamios. De interés especial es el condicionamiento mecánico de los tejidos cultivados por bioreactor de la ingeniería del tejido óseo, que es capaz de aumentar el potencial osteogénico de los injertos sintéticos.En este trabajo, dos sistemas de bioreactores fueron desarrollados para permitir comprender las propiedades bioactivas de andamios de diferentes materiales y la mecanoregulación del comportamiento de células o tejidos. Un sistema de bioreactor de perfusión in vitro fue desarrollado para el sembrado y cultivo de células incorporadas en cilindros de un biomaterial poroso. Varios estudios para la determinación de los parámetros del sembrado de células aplicable se llevaron a cabo, así como experimentos de cultivo de células bajo flujo de fluido constante con una estimulación mecánica adicional por alternancia del flujo.Un sistema de cámara ósea fue desarrollado como un bioreactor in vivo. El sistema produjo un defecto óseo grande en tibias de perros y permitió la implantación repetida de grandes andamios porosos de materiales diferentes. El tejido creciendo en los andamios permite extraer conclusiones sobre las propiedades de osteoconductividad u osteinductividad de los andamios. Además, un dispositivo de compresión se ha desarrollado para aplicar cargas cíclicas en los andamios en vivo para estudiar el efecto de la estimulación mecánica en el desarrollo de los tejidos.Los estudios con el sistema de perfusión desarrollado han demostrado que el sembrado de células en grandes andamios porosos es posible, lo que se considera crucial para el cultivo celular. El largo tiempo de cultivo de células mostró la proliferación de las células madre mesenquimales hasta dos semanas. El patrón de estimulación utilizado en el estudio aumentó la expresión de la osteocalcina, lo que indica una mayor actividad de las células, pero la ausencia de expresión de RunX2 y colágeno I impidió la determinación concluyente de la diferenciación.El sistema desarrollado de la cámara ósea demostró su funcionalidad en el entorno quirúrgico durante los experimentos in vivo. Complicaciones durante los experimentos no permitieron la aplicación de las cargas cíclicas de los andamios implantados. La formación de hueso retrasada debido al defecto óseo creado y material de andamios restantes no permitieron conclusiones definitivas acerca de las propiedades del material del andamio. Sin embargo, el estudio proporciona datos para el desarrollo futuro del dispositivo y protocolo clínico.Los estudios realizados constituyen una novedad en respecto a la creación de bioreactores para el estudio de la andamios porosos sintéticos de grandes dimensiones in vitro e in vivo. Los sistemas desarrollados constituyen la base para otros estudios en mecanobiología de las células óseas y los tejidos. / Large bone defects constitute a challenge for the clinical field, because they cannot be repaired by the body itself, but require the implantation of suitable bone grafts. To overcome the drawbacks of grafts from autologous or allogous sources, modern bone tissue engineering aims to replace lost tissue by cultivating cells in vitro on porous biomaterials. The cell culture on large porous scaffolds has shown to be difficult, requiring bioreactors, which are used for tissue culture and the study of cell behaviour in 3D scaffolds. Of special interest is the mechanical conditioning of the cultured tissue for bioreactor-based bone tissue engineering, which is able to enhance the osteogenic potential of the synthetic grafts.In this work two bioreactor systems were developed to allow insight into bioactive properties of different scaffold materials and the mechanoregulation of cell or tissue behaviour. An in vitro perfusion bioreactor system was developed for the cell seeding and culture on porous biomaterial cylinders. Several studies for the determination of applicable cell seeding parameters were conducted, as well as experiments of cell culture under steady fluid flow with additional mechanical stimulation by alternating fluid flow. A bone chamber system was developed as an in vivo bioreactor. The system produced a large bone defect in dog tibia and allowed the repeated implantation of large porous scaffolds of different material compositions.The ingrowing tissue was observed to allow conclusions about osteoconductive or osteinductive properties of the scaffolds. Additionally a compression device was developed to apply cyclic loading on the scaffolds in vivo to study the effect of mechanical stimulation on tissue development.The studies with the developed in vitro perfusion bioreactor system have shown that it is possible to seed cells throughout large porous scaffolds, which is deemed crucial for the further cell culture. The long time cell culture showed the proliferation of mesenchymal stem cells up to two weeks. The stimulation pattern used in the study enhanced the expression of osteocalcin, indicating an enhanced cell activity, but the absence of RunX2 and collagen I expression rendered the determination of differentiation inconclusive.The developed bone chamber system proved to be functional in the surgical environment during the in vivo experiments. Occurring complications during the experiments did not allow the application of the cyclic loading of implanted scaffolds. Delayed bone formation due to created bone defect and remaining scaffold material did not allow final conclusions about the scaffold material properties. Nevertheless the study provides input for further development of the device and clinical protocol.The conducted studies constitute a novelty regarding the creation of bioreactors for the study of synthetic porous scaffolds of large dimensions in vitro and in vivo. The developed systems form the basis for further studies in mechanobiology of bone cells and tissue.

Biological materials are complex hierarchical systems subjected to external stimuli like mechanical forces, chemical potentials and electrical signals. A deeper understanding of the behavior of these materials is required for the response characterization of healthy and diseased conditions. The primary aim of this dissertation is to study the mechanics of biological materials like cells and tissues from a computational perspective and relate its behavior with experimental works, so as to provide a framework for the identification and treatment of pathological conditions like cancer and vascular diseases. The first step towards understanding the behavior of a biological cell is to comprehend its response to external mechanical stimuli. Experimentally derived material properties of cells have found to vary by orders of magnitude even for the same cell type. The primary cause of such disparity is attributed to the stimulation process, and the theoretical models used to interpret the experimental data. The variations in mechanical properties obtained from the experimental and theoretical studies can be overcome only through the development of a sound mathematical framework correlating the derived mechanical property with the cellular structure. Such a formulation accounting for the inhomogeneity of the cytoplasm due to stress fibers and actin cortex is developed in this work using Mori-Tanaka method of homogenization. Mechanical modeling of single cells would be extremely useful in understanding its behavior in an experimental setup. Characterization of in-vivo response of cells requires mathematical modeling of the embedding environment like fibers and fluids, which forms the extra cellular matrix. Studies on fluid-tissue interactions in biomechanics have primarily relied on either an iterative solution of the individual solid or tissue phases or a sequential solution of the entire domain using a coupled algorithm. In this dissertation, a new computational methodology for the analysis of fluid-tissue interaction problem is presented. The modeling procedure is based on a biphasic representation of fluid and tissue domain, consisting of fluid and solid phases. The biphasic-fluid interaction model is also implemented to study the transfer of low-density lipoprotein from the blood to the arterial wall, and also the nutrient transfer in the tissue scaffolds of a bioreactor.

The drinking water treated by water treatment plant (WTP) usually has an excess of assimilable organic carbon (AOC) in distribution systems in south Taiwan. They will cause the growth of heterotrophic plate count (HPC) and deterioration of water quality in pipeline of distribution systems. Recently, part of traditional purification processes were changed into advanced processes in WTP. The past researches showed the combined advanced processes ultrafitration (UF) / reverse osmosis (RO) in south WTP in Taiwan has the removal problems of AOC in above UF / RO processes because the organic compounds in raw water caused a fouling layer which was formed on the membranes surface. These problems made the back-wash frequency increasing, short membrane life and raising cost. The study combines activated carbon and membrane bioreactor (MBR) to explore the removal efficiency of drinking water in laboratory. The system showed the removal efficiencies of dissolved organic carbon (DOC) and AOC were 57% and 36%, respectively in average. More, the system showed the removal efficiencies of DOC and AOC were 81% and 66%. The results of this research showed good removal efficiency was found in AOC and DOC. Good quality of biological stability, removal of organic compounds, low cost in building and maintaining were reached.

Embryonic stem (ES) cells provide a potentially unlimited cell source for cellular therapies; however, reliable methods must be developed to provide clinically-relevant numbers of homogeneous therapeutic cell populations. Dynamic cultures may encourage ES cell differentiation and amenable to large-scale cell production. Our goal was to optimize dynamic culture parameters (bioreactor type, speed, cell seeding density, conditioned medium, and hypoxia) to maximize the generation of hematopoietic stem and progenitor cells (HSPCs) from ES cells and also to investigate the ability of dynamic culture-derived HSPCs to generate terminally differentiated hematopoietic cells. Our results indicate that varying cell seeding density and speed in two different bioreactors significantly affects embryoid body formation and ES cell differentiation efficiency into progenitor cells. In general, increased cell seeding density generated higher percentages of HSPCs in both bioreactors. In addition, rotary (Synthecon) bioreactors produced more sca-1⁺ progenitors, and spinner flasks generated more c-kit⁺ progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that unique gene expression profiles were observed in the two bioreactors with the expression of specific hematopoietic genes more up regulated in the Synthecon cultures compared to spinner flasks. Combining bioreactor cultures with directed differentiation strategies via conditioned medium and hypoxic culture may further encourage hematopoietic differentiation. Dynamically cultured ES cell-derived hematopoietic stem and progenitor cells were further differentiated into a phenotype typical of dendritic cells which had the ability to process antigen. Additionally, microarray analysis of isolated ES cell-derived HSPCs demonstrated differences in the gene expression from native HSCs isolated from the fetal liver or bone marrow of mice. Insight gained from this work should be continued by comparing the differentiation efficiency of HSPCs derived in dynamic and traditional static culture methods into functional, terminally differentiated hematopoietic cells to generate clinically-relevant numbers of transplantable, therapeutic cells. / text

Mining-Influenced Water (MIW) poses major environmental issues in New Zealand and worldwide due to a legacy of unmitigated mining activities. As conventional MIW treatment technologies can be very costly in terms of chemical and energy inputs, cheaper and environmentally-friendly alternative remediation strategies have been developed. These so-called passive treatment technologies include a range of engineered systems relying on biogeochemical processes able to mitigate the acidity and to immobilize the metals in MIW. The present research, built on previous work conducted at the University of Canterbury, investigated the use of waste materials in mesocosm lab-scale sulfate-reducing bioreactors (SRBR) to treat actual mining-influenced water (MIW) sourced at an active coal mine in New Zealand. Specifically, this study investigated using waste mussel shells as an alkaline amendment (instead of the more conventional material limestone), with organic waste materials such as wood byproducts and compost in complex substrate mixtures in upward-flow SRBR. The influence of hydraulic retention times of approximately 3 and 10 days (HRT; i.e. the contact time between the MIW and the substrate mixtures in the SRBR) on the treatment performances was also evaluated. Overall, each system successfully treated the MIW (e.g. increased the pH > 6 and removed >78 % of the metals, except Mn) during the first 5-month treatment period, while during the second 5-month period, the treatment systems containing limestone and/or operating at a short HRT started to show signs of decreased efficiency. Generally, the system containing mussel shell and operating at a long HRT was constantly the most efficient system. Over the whole 41-week period of treatment, key metal removal efficiencies ranged between 97.6 and 99.7 % (Al), 83.9 and 95.2 % (Fe), and 9.2 and 38.8 % (Mn). Sulfate removal, in terms of moles of sulfate removed per cubic meter of substrate per day, was on average below the design values of 0.3 mol/m3/d, and ranged between 0.03 and 0.55 mol/m3/d (median values were 0.26 to 0.3 mol/m3/d during the first 5-month period but dropped to 0.094 to 0.1 mol/m3/d during the second 5-month treatment period). The SRBR containing mussel shell instead of limestone resulted in significantly higher alkalinity generation (between 32 to 85 % higher) and higher metal removals (between 0.6 % higher for Al and 14 % higher for Ni). These results were mainly attributed to the unique mineralogy of the mussel shell which comprises of aragonite with traces of calcite, while limestone comprises of pure calcite with traces of quartz. The statistical analyses showed that the sulfate reduction was not significantly affected by the alkalinity source. Similarly, systems operating at a longer HRT (10 days instead of 3 days) showed better treatment performances than systems operating at a short HRT in terms of alkalinity generation (44 to 62% higher), metal removal (between 0.5 % higher for Al to 15 % higher for Ni, and between 17 to 23 % higher for Mn), and sulfate reduction (50 to 77 % higher). Overall, the systems operation on a longer HRT were dominated by a more reduced environment facilitating the precipitation of metal sulfides, while the reactors running on a shorter HRT were constantly maintained out of equilibrium by the continuous addition of fresh MIW. Chemical and mineralogical analyses performed on the spent substrates suggested that the metals were removed through precipitation as, and adsorption onto, metal sulfides (Fe, Zn, Ni, Cu), (oxy)hydroxides (Al, Fe, Zn), and carbonates (Mn, Zn). Mn, a metal known to be harder to remove from solution was likely removed through the precipitation of rhodochrosite (MnCO3) and via adsorption onto the organic matter. These results generally corroborated the results obtained using the geochemical modeling PHREEQC. Overall, this study showed that mussel shells are not only a sustainable and effective alternative to mined limestone, but their use in SRBR would also result in a better treatment of MIW. Additionally, even though an increase in HRT resulted in a better contaminant removal, a HRT of approximately 3 days was sufficient to remove about 80% of all metals (except Mn). Therefore, the difficult choice of an optimal HRT must balance the need to meet a specific effluent quality while keeping the treatment time reasonably short, and an intermediate retention time of approximately 6 days could be optimal.

A conventional membrane bioreactor (MBR) and two moving bed bioreactors coupled with ultrafiltration membrane filtration were operated for close to six months to investigate biological nutrient removal and potential fouling inducing parameter mitigation. Unique to one of the moving bed membrane bioreactors (MBMBR) was a newly designed media that incorporated a hydrodynamic exterior carrier with a highly porous interior packing. Preliminary investigation indicates that nitrogen compounds were superiorly removed in the two MBMBRs when compared with the MBR. This is a result of denitrification processes occurring in anoxic micro-zones found within the depths of the biofilm affixed to media. Fouling propensity was found to be increased by over four times in the MBMBR systems as compared to the MBR. Mixed liquor, permeate and filtrate analysis, membrane fibre examination and permeability tests indicated that colloidal organic carbon, as well as soluble microbial products were the dominant fouling inducing compounds. / Manuscript format / The Natural Sciences and Engineering Research Council of Canada

This research was completed to assess passive treatment methods for mitigation of acid mine drainage (AMD) at a former mine site in British Columbia. The objectives were to determine if suitable passive treatment methods were available, and if concentrations of Cd, Zn, and other key contaminants in groundwater could be reduced to below regulatory standards during bench-scale testing. Biological treatment with sulphate reducing bacteria (SRB) was selected, and bench-scale treatment testing was conducted using columns amended with low cost organic sources. Removal of more than 99% Cd, 93% Co, 96 % Cu, 86% Ni and 98% Zn was observed, resulting in metals concentrations in treated effluent consistently lower than applicable groundwater standards. Sustainability attributes of treatment with SRB and the potential to recover valuable metals are discussed, and recommendations for further testing and implementation are provided.