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.

Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

ERP systems implementation and organizational culture : the case of Jordan

Jordan is adopting Enterprise Resource Planning (ERP) systems in both its public and private sectors. Jordan's emerging private sector has historically close ties to the public sector; though a global market orientation requires a shift in its organizational culture. ERPs however embed business processes which do not necessarily fit with traditional cultural practices, and implementation success is not assured. This study looks at the perceptions of both public and private sector ERP implementations in Jordan and assesses these on various measures of success. There were few differences between public and private sectors, but the benefits actually realized in Jordanian ERPs fell short of claims made for the technology in other cultures.

The relationship between culture, attitude, social networks and quality of life in midlife Australian and Taiwanese citizens

Objectives: The objectives of this study were to specifically investigate the differences in culture, attitudes and social networks between Australian and Taiwanese men and women and identify the factors that predict midlife men and women’s quality of life in both countries. Methods: A stratified random sample strategy based on probability proportional sampling (PPS) was conducted to investigate 278 Australian and 398 Taiwanese midlife men and women’s quality of life. Multiple regression modelling and classification and regression trees (CARTs) were performed to examine the potential differences on culture, attitude, social networks, social demographic factors and religion/spirituality in midlife men and women’s quality of life in both Australia and Taiwan. Results: The results of this study suggest that culture involves multiple functions and interacts with attitudes, social networks and individual factors to influence a person’s quality of life. Significant relationships were found between the interaction between cultural circumstances and a person’s internal and external factors. The research found that good social support networks and a healthy optimistic disposition may significantly enhance midlife men and women’s quality of life. Conclusion: The study indicated that there is a significant relationship between culture, attitude, social networks and quality of life in midlife Australian and Taiwanese men and women. People who had higher levels of horizontal individualism and collectivism, positive attitudes and better social support had better psychological, social, physical and environmental health, while it emerged that vertical individualists with competitive characteristics would experience a lower quality of life. This study has highlighted areas where opportunities exist to further reflect upon contemporary social health policies for Australian and Taiwanese societies and also within the global perspective, in order to provide enhanced quality care for growing midlife populations.

The inherent equitable jurisdiction and the plenary power of the Supreme Court of New South Wales to order the winding up of companies

Not all companies in Australia are amenable to a winding up order pursuant to the Corporations Act 2001 (Cth). The Supreme Court of New South Wales has previously dealt with such winding up applications by apparently focusing on the inherent jurisdiction of the court to consider whether the court has jurisdiction to firstly consider the winding up application. This article proposes an original alternative paradigm: the plenary power provided to the court by s 23 of the Supreme Court Act 1970 (NSW) can be utilised to initially attract the jurisdiction of the court and subsequently the inherent jurisdiction specifically utilising the equitable “just and equitable” ground is available to the court to consider and make such a winding up order if appropriate. Variation of such a paradigm may also be available to the court when considering the inherent jurisdiction in relation to corporation matters more generally.