Thesis (M.S.)--University of Michigan, 1986.

Thesis (M.Phil.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 125-141) Also available in print.

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2007. / Radhakrishna, Harish, Committee Member ; LaPlaca, Michelle, Committee Member ; Nerem, Robert, Committee Member ; Garcia, Andres, Committee Member ; Levenston, Marc, Committee Chair.

Thesis (M.S.)--Brigham Young University. Dept. of Microbiology and Molecular Biology, 2006. / Includes bibliographical references (p. 46-47).

Thesis (M. Orth.)--University of Hong Kong, 2005.

Thesis (Ph. D.)--Bioengineering, Georgia Institute of Technology, 2008. / Guldberg, Robert, Committee Chair ; Boyan, Barbara, Committee Member ; O'Keefe, Regis, Committee Member ; Vito, Ray, Committee Member ; Bellankonda, Ravi, Committee Member.

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A hallmark of the joint disease osteoarthritis (OA) is the degradation of the articular cartilage in the affected joint, debilitating pain and decreased mobility. At present there are no disease modifying drugs for treatment of osteoarthritis. This represents a significant, unmet medical need as there is a large and increasing prevalence of OA. Using a systems biology approach, we aimed to better understand the pathogenic mechanisms of OA and ultimately aid development of therapeutics. This thesis focuses on the analysis of gene expression data from human OA cartilage obtained at total knee replacement (TKR). This transcriptomics approach gives a genome-wide overview of changes, but can be challenging to interpret. Network-based algorithms provide a framework for the fusion of knowledge so allowing effective interpretation. The PhenomeExpress algorithm was developed as part of this thesis to aid the interpretation of gene expression data. PhenomeExpress uses known disease gene associations to identify relevant dysregulated pathways in the data. PhenomeExpress was further developed into an 'app' for Cytoscape, the widely used network analysis and visualisation platform. To investigate the processes that occur during the degradation of cartilage we examined the gene expression of damaged and intact OA cartilage using RNA-Seq and identified key altered pathways with PhenomeExpress. A regulatory network driven by four transcription factors accounts for a significant proportion of the observed differential expression of damage-associated genes in the PhenomeExpress identified pathways. We further explored the role of the cytokines IL-1 and TNF that have been reported to β drive the progression of OA. Comparison of the expression response of in vitro cytokine-treated explants with the in vivo damage response revealed major differences, providing little evidence for any significant role of IL-1 and TNF as drivers of OA β damage in vivo. Finally, we examined the heterogeneity of OA through analysis of cartilage expression profiles at TKR. Through a network-based clustering method, we found two subgroups of patients on the basis of their gene expression profiles. These subgroups were found to have distinct OA expression perturbations and we identified TGF and S100A8/9 β signalling as potentially explaining the observed differential expression. We developeda RT-qPCR based classifier that allowed classification of new samples into these subgroups so allowing future assessment of the clinical significance of these subgroups. The work presented in this thesis includes a novel, widely-accessible tool for the analysis of disease gene expression data, which we used to give new insights into the pathogenesis of osteoarthritis. We have produced a rich dataset for future research and our analysis of this data has increased our understanding of cartilage damage processes and the heterogeneity of OA.

Septic arthritis has the potential to be a highly destructive joint disease. Although numerous bacterial species are capable of inducing septic arthritis, Staphylococcus aureus is most commonly implicated, accounting for up to 65% of cases. Whilst this organism is known to produce a diverse array of potential virulence factors, studies investigating a variety of S. aureus-related infections have implicated alpha(Hla)-, beta(Hlb)- and gamma(Hlg)-haemolysins as key damaging toxins, with the ‘pore-forming’ Hla considered to be the most potent. The work presented in this study focused on gaining further insight into the interaction between S. aureus toxins and in situ chondrocytes during an episode of septic arthritis. An in vitro bovine osteochondral explant model of S. aureus-induced septic arthritis was developed in this study. Utilising fluorescence-mode confocal laser scanning microscopy (CLSM), the model, which avoided the complexities of a host immune response, permitted an assessment of the following: (1) the spatial and temporal quantification of in situ chondrocyte viability following exposure to both a laboratory ‘wild-type’ (S. aureus 8325-4) and clinical strains of S. aureus; (2) the influence of Hla, Hlb and Hlg on in situ chondrocyte viability through the use of specific ‘haemolysin-knockout’ mutant strains; (3) the influence of altered culture medium osmolarity and extracellular Ca2+ on Hla-induced in situ chondrocyte death; and (4) dynamic changes in intracellular Ca2+ within in situ chondrocytes following Hla exposure. S. aureus 8325-4 and S. aureus clinical strains rapidly reduced in situ chondrocyte viability ( > 45% chondrocyte death at 40hrs). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) of cartilage and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins (achieved with the use of subchondral bone-free explants) demonstrated that SZ chondrocytes were more susceptible to the toxins than DZ chondrocytes. When explants were cultured in the presence of a selection of isogenic S. aureus mutants, with varying Hla, Hlb and Hlg production capabilities (all originating from S. aureus 8325-4), Hla-producing mutants induced significant in situ chondrocyte death compared to toxin deficient controls (Hla-Hlb-Hlg-). In contrast, mutants producing Hlb and Hlg in the absence of Hla were unable to induce significant chondrocyte death. Hla alone was therefore identified as the key damaging toxin to in situ chondrocyte viability. Raised culture medium osmolarity had no influence on Hla-induced in situ chondrocyte death. In the absence of Hla, a high extracellular Ca2+ concentration (20mM) had no influence on chondrocyte viability during the experimental period. Hla-induced chondrocyte death increased in the presence of raised extracellular Ca2+ concentrations thereby confirming a role of Ca2+ in the chondrocyte death pathway. There was no significant difference between S. aureus growth in high and low Ca2+ culture media. Finally, when live osteochondral explants stained with the Ca2+-sensitive fluorophore Fluo-4 were cultured with an Hla-containing S. aureus supernatant (S. aureus 8325-4 (Hla+Hlb+Hlg+)) there was a significant rise in intracellular Ca2+ in comparison to those explants exposed to a non-Hla-containing supernatant (S. aureus DU5938 (Hla- Hlb-Hlg-)). The Hla-induced Ca2+ transients were always followed by chondrocyte death. Thus, it is likely that Hla-induced chondrocyte death was associated with a rise in intracellular Ca2+. These findings are of translational relevance. Firstly, toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes, thereby advocating the prompt and thorough removal of bacteria and their toxins during the treatment of septic arthritis. Secondly, the identification of Hla alone as the key damaging toxin to in situ chondrocyte viability, with its destructive action being associated with a rise in intracellular Ca2+, may enable the development of future targeted therapeutic strategies in order to reduce the extent of cartilage destruction during and after an episode of septic arthritis.

Le cheval athlète est exposé à des lésions dégénératives ou traumatiques du cartilage prédisposant au développement de l’arthrose. Les arthropathies associées à une dégradation du cartilage se traduisent par une perte de fonctionnalité de l’articulation. Le cartilage ne possédant que de très faibles capacités de réparation intrinsèque, le développement de traitements de ces affections représente un enjeu thérapeutique. Le premier objectif de notre étude était la mise au point d’un substitut cartilagineux utilisant des cellules souches mésenchymateuses (CSMs) du sang de cordon ombilical (SCO). Après avoir été isolées et caractérisées, les CSMs de SCO ont été différenciées en chondrocytes au sein d’un biomatériau, en présence de facteurs de croissance (BMP-2 et TGF-β1 ou TGF-β3), en normoxie ou en hypoxie. L’utilisation de la BMP-2 et du TGF-β1 en normoxie permet une synthèse abondante des protéines caractéristiques de la matrice extracellulaire du cartilage avec néanmoins une expression persistante du collagène de type I, marqueur du fibrocartilage. La stratégie d’interférence par l’ARN ciblant Col1a2 permet de stabiliser le phénotype cellulaire obtenu au niveau transcriptionnel. Le deuxième objectif de notre étude était l’utilisation des CSMs de SCO indifférenciées lors d’injection intra-articulaire chez le cheval. La tolérance des CSMs allogéniques in vivo a été démontrée, ce qui n’a pu être le cas pour leur efficacité dans un modèle d’arthropathie induite expérimentalement. Ainsi, les CSMs de SCO constituent une source intéressante pour l’ingénierie tissulaire du cartilage mais leur utilisation thérapeutique sous forme indifférenciée doit être optimisée et validée. / The racehorse is exposed to degenerative or traumatic lesions of cartilage predisposing to the development of osteoarthritis. Arthropathies associated with cartilage degradation result in loss of joint functionality. The cartilage has only a very low intrinsic repair capacity ; that is why the development of new treatment represents a therapeutic challenge. The first objective of our study was to develop a cartilage substitute using mesenchymal stem cells (MSCs) from umbilical cord blood (UCB). After having been isolated and characterized, the MSCs of UCB were differentiated into chondrocytes in a biomaterial in the presence of growth factors such as BMP-2 and TGF-β1 or TGF-β3, in normoxia or in hypoxia. The use of BMP-2 and TGF-β1 in normoxia leads to an abundant synthesis of extracellular matrix proteins characteristic of hyaline cartilage but with persistent expression of type I collagen, a fibrocartilage marker. siRNAs targeting Col1a2 stabilize the cellular phenotype obtained at transcriptional level. The second aim of our study was the use of undifferentiated MSCs of UCB for intra-articular injection in horses. The tolerance of allogeneic MSCs was demonstrated in vivo, but not for now their efficacity in a model of joint arthropathy.Thus, the MSCs of UCB is a source of interest for tissue cartilage engineering but their therapeutic use in undifferentiated form must be optimized and validated.