In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

The development of an on-line module for students on “Working in Inter-cultural Teams”

Since 2005 QUT through a number of large Teaching and Learning Grants has sponsored a range of teamwork learning initiatives to assist students to develop the teamwork skills demanded by industry. After a suite of six online team learning modules was developed, first year unit coordinators requested an additional module to address the challenges of working with the diverse range of social, cultural and personal values that students from different backgrounds bring to student teams. The Intercultural Teams module asks students to map themselves against a Cultural Orientations Framework so they can understand their own cultural beliefs. By learning about other cultural orientations and comparing and analysing their effects, team members can develop communication and team process management strategies to leverage their differences to realise effective and creative outcomes. The interactive session will demonstrate the elements of the Intercultural Teams module and ask participants to consider ways the module can be integrated into classroom learning to support the development of students’ intercultural competencies.

The importance of a triple bottom line approach for safeguarding urban water quality

Water environments are greatly valued in urban areas as ecological and aesthetic assets. However, it is the water environment that is most adversely affected by urbanisation. Urban land use coupled with anthropogenic activities alters the stream flow regime and degrade water quality with urban stormwater being a significant source of pollutants. Unfortunately, urban water pollution is difficult to evaluate in terms of conventional monetary measures. True costs extend beyond immediate human or the physical boundaries of the urban area and affect the function of surrounding ecosystems. Current approaches for handling stormwater pollution and water quality issues in urban landscapes are limited as these are primarily focused on ‘end-of-pipe’ solutions. The approaches are commonly based either on, insufficient design knowledge, faulty value judgements or inadequate consideration of full life cycle costs. It is in this context that the adoption of a triple bottom line approach is advocated to safeguard urban water quality. The problem of degradation of urban water environments can only be remedied through innovative planning, water sensitive engineering design and the foresight to implement sustainable practices. Sustainable urban landscapes must be designed to match the triple bottom line needs of the community, starting with ecosystem services first such as the water cycle, then addressing the social and immediate ecosystem health needs, and finally the economic performance of the catchment. This calls for a cultural change towards urban water resources rather than the current piecemeal and single issue focus approach. This paper discusses the challenges in safeguarding urban water environments and the limitations of current approaches. It then explores the opportunities offered by integrating innovative planning practices with water engineering concepts into a single cohesive framework to protect valuable urban ecosystem assets. Finally, a series of recommendations are proposed for protecting urban water resources within the context of a triple bottom line approach.

Investigation of 'off-line‘ relaxation phenomena in oil filled transformers

Power transformers are one of the most important and costly equipment in power generation, transmission and distribution systems. Current average age of transformers in Australia is around 25 years and there is a strong economical tendency to use them up to 50 years or more. As the transformers operate, they get degraded due to different loading and environmental operating stressed conditions. In today‘s competitive energy market with the penetration of distributed energy sources, the transformers are stressed more with minimum required maintenance. The modern asset management program tries to increase the usage life time of power transformers with prognostic techniques using condition indicators. In the case of oil filled transformers, condition monitoring methods based on dissolved gas analysis, polarization studies, partial discharge studies, frequency response analysis studies to check the mechanical integrity, IR heat monitoring and other vibration monitoring techniques are in use. In the current research program, studies have been initiated to identify the degradation of insulating materials by the electrical relaxation technique known as dielectrometry. Aging leads to main degradation products like moisture and other oxidized products due to fluctuating thermal and electrical loading. By applying repetitive low frequency high voltage sine wave perturbations in the range of 100 to 200 V peak across available terminals of power transformer, the conductive and polarization parameters of insulation aging are identified. An in-house novel digital instrument is developed to record the low leakage response of repetitive polarization currents in three terminals configuration. The technique is tested with known three transformers of rating 5 kVA or more. The effects of stressing polarization voltage level, polarizing wave shapes and various terminal configurations provide characteristic aging relaxation information. By using different analyses, sensitive parameters of aging are identified and it is presented in this thesis.

A new method for improving reliability and line loss in distribution networks

In this paper, both Distributed Generators (DG) and capacitors are allocated and sized optimally for improving line loss and reliability. The objective function is composed of the investment cost of DGs and capacitors along with loss and reliability which are converted to the genuine dollar. The bus voltage and line current are considered as constraints which should be satisfied during the optimization procedure. Hybrid Particle Swarm Optimization as a heuristic based technique is used as the optimization method. The IEEE 69-bus test system is modified and employed to evaluate the proposed algorithm. The results illustrate that the lowest cost planning is found by optimizing both DGs and capacitors in distribution networks.

QoS-oriented sesource allocation and scheduling of multiple composite web services in a hybrid cloud using a random-key genetic algorithm

In cloud computing resource allocation and scheduling of multiple composite web services is an important challenge. This is especially so in a hybrid cloud where there may be some free resources available from private clouds but some fee-paying resources from public clouds. Meeting this challenge involves two classical computational problems. One is assigning resources to each of the tasks in the composite web service. The other is scheduling the allocated resources when each resource may be used by more than one task and may be needed at different points of time. In addition, we must consider Quality-of-Service issues, such as execution time and running costs. Existing approaches to resource allocation and scheduling in public clouds and grid computing are not applicable to this new problem. This paper presents a random-key genetic algorithm that solves new resource allocation and scheduling problem. Experimental results demonstrate the effectiveness and scalability of the algorithm.

Virtual diasporas and the dilemma of multiple belongings in cyberspace

One of the most celebrated qualities of the Internet is its enabling of simultaneity and multiplicity. By allowing users to open as many windows into the world as they (and their computers) can withstand, the Internet is understood to have brought places and cultures together on a scale and in a manner unprecedented. Yet, while the Internet has enabled many to reconnect with cultures and places long distanced and/or lost, it has also led to the belief that these reconnections are established with little correspondent cost to existent ties of belonging. In this paper, I focus on the dilemma multiple belongings engender for the ties of national belonging and question the sanguinity of multiple belongings as practised online. In particular, I use Lefebvre's notion of lived space to unpack the problems and contradictions of what has been called 'Greater China' for the ethnic Chinese minority in nations like Malaysia, Singapore and Australia.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.