OBJECTIVE: Osteoarthritis (OA) is a leading cause of disability worldwide. This disease is characterized by the inflammation and degradation of the cartilage and surrounding tissue in a joint. The disease manifests as either a result of years of wear and tear or after a joint injury. Post-traumatic osteoarthritis, as this latter case is named, is frequently studied since the exact trigger of the disease is known. In addition to several changes within the joint space, a significant alteration is the degradation of cartilage caused primarily by the release of inflammatory cytokines including interleukin-1 and 6 and tumor necrosis factor α. One current pharmacological treatment for the pain caused by OA is an intra-articular injection of glucocorticoids such as dexamethasone. As this is a common treatment, the goal of this research was to determine if, at the cellular level, this treatment impacts cell viability in the presence of pro-inflammatory cytokines. Another goal was to investigate how such treatment affects the progression of cartilage degradation caused by cytokines. OA results in the loss of the key extracellular matrix molecule, aggrecan, which contains negatively charged glycosaminoglycan (GAG) chains. Measurement of the amount of GAGs lost is an early indicator of cartilage degradation. In addition, biosynthesis of GAG chains can be measured to estimate the overall metabolic health of the cells. We hypothesized that dexamethasone blunts the harmful effects of proinflammatory cytokines and improves GAG biosynthesis and chondrocyte viability. METHODS: Cylindrical cartilage explants were collected from bovine knee joints and trimmed to a uniform 3 millimeters in diameter and 1 millimeter thick. Each treatment group consisted of n=6 explants from the same knee joint. In one set of experiments, these explants were subjected to two different doses of interleukin-1α (1 ng/mL and 10 ng/mL) with and without dexamethasone at 100 nM. In another set of experiments, explants were subjected to both interleukin-1α and tumor necrosis factor-α (1 ng/mL and 25 ng/mL respectively). The explants were cultured in medium for 6 days and were digested for outcome measurements on the final day. On day 4, 35S-sulfate was added to the explant medium for later measurement of radiolabel incorporation as a measure of GAG biosynthesis. Cell viability was measured on day 5 using red/green fluorescent viability dyes fluorescein diacetate (FDA) which stains live cells green and propidium iodide (PI) which stains dead cells red. RESULTS: Compared with untreated controls, explants subjected to the pro-inflammatory cytokines interleukin-1α and tumor necrosis factor-α exhibited greater glycosaminoglycan loss and a decrease in GAG biosynthesis. These treatments also decreased cell viability. Addition of dexamethasone improved cell viability compared to treatment with the cytokines. In addition, dexamethasone prevented glycosaminoglycan loss and increased GAG biosynthesis in the presence of interleukin-1α. However, dexamethasone did not prevent tumor necrosis factor-α mediated loss of GAGs. CONCLUSION: These studies demonstrated that dexamethasone inhibited specific aspects of cartilage degradation associated with inflammation in early OA. This therapeutic counteracts the degradative changes initiated by inflammatory cytokines such as interleukin-1α without compromising cell viability. Future studies are needed to identify the mechanisms of dexamethasone action and the ideal concentration to use if it is to be used as a treatment for OA following acute joint injury.

Cartilage has limited intrinsic healing potential upon injury, due to the low cell density and the lack of blood supply. Degenerative disease of the cartilage, such as osteoarthritis (OA), is challenging to treat without clear mechanistic understandings of cartilage development. With over 90% of the cartilage tissue occupied by extracellular matrix (ECM), understanding the cellular and molecular effects of cartilage ECM on chondrogenesis and chondrocyte behavior is crucial for therapeutic development. The focus of this work is to study the regulation of chondrogenesis and hypertrophic maturation of human mesenchymal stem cells (MSCs) by cartilage ECM in the context of potential therapeutic applications. To study the cartilage ECM, we created a decellularized ECM digest from native porcine cartilage and examined its effects on MSCs. Since native cartilage ECM maintains chondrocyte homeostasis without progressing to hypertrophic degeneration, we hypothesized that the decellularized ECM would promote MSC chondrogenesis and inhibit hypertrophy. Indeed, we showed that ECM promoted MSC chondrogenesis and matrix production, and inhibited hypertrophy and endochondral ossification. The chondrogenic effect was shown to potentially involve the PI3K-Akt-Foxo1 and Hif1 pathways. By recapitulating the activated Hif1 pathway, roxadustat, a small molecule stabilizer of Hif, was able to reproduce the chondrogenic and anti-hypertrophic effects of the cartilage ECM. It also reduced the expression of matrix metalloproteases (MMPs) in MSCs, healthy chondrocytes, and OA chondrocytes, and alleviated matrix degradation in bovine cartilage explants. We also attempted to identify ECM components that display chondrogenic properties. Collagen XI, a minor component of articular cartilage, was shown to promote cartilage matrix formation in MSCs and healthy chondrocytes, and to reduce matrix degradation in bovine cartilage explants. Taken together, this study reveals the dual roles of cartilage ECM in promoting chondrogenesis and matrix production and inhibiting cartilage hypertrophy. Importantly, small molecule drugs that recapitulate the signaling pathways of ECM regulation, and collagen XI, a component of the ECM, may serve as leads for further therapeutic development for cartilage injury and degenerative disease.

Arthritis is a pathological condition whereby a persistent inflammatory response leads to breakdown of articular cartilage in synovial joints. Cartilage is a specialised avascular tissue containing chondrocytes embedded in an extracellular matrix. The cartilage matrix is composed of collagen to provide strength with aggregated proteoglycan to facilitate hydration. Cartilage has been reported to loose proteoglycans with concordant loss of integrity observed in arthritic disease pathology. Proteoglycans loss from cartilage has also been reported in in vitro models. Application of interleukin-1 (lL-1β) to cartilage in vitro has been demonstrated to increase loss of proteoglycans and modulate production of inflammatory mediators such as Nitric Oxide (NO) and Prostaglandin-E2 (PGE2). NO and PGE2 have also been associated with cartilage breakdown. Other cytokines such as colony stimulating factors (CSFs) may regulate cartilage function. The aim of this study was to select a cartilage explant system and compare the effects of interleukin-1 (lL-1) with those of colony stimulating factors (CSFs) by measuring the production of NO and PGE2 and release of proteoglycans. It was found that IL-1β increased PGE2 and NO production, but not loss of proteoglycans from rat cartilage explants. Granulocyte-CSF (G-CSF) and lL-3 increased production of NO and PGE2, respectively. When combined, IL-1β / Granulocyte-Macrophage (GM-CSF) increased production of PGE2 and G-CSF / IL-1β produced increased proteoglycan loss from explants. The model was then modified by integrating Swiss 3T3 Fibroblasts monolayers with explants. Fibroblasts were initially screened to determine their separate response to these cytokines. Fibroblasts did not release proteoglycans into the culture media, but produced elevated concentrations of NO and PGE2 in response to IL-lβ.·Fibroblast-cartilage co-cultures treated with IL-lβ produced increased NO, PGE2 and proteoglycan release. G-CSF, GM-CSF and IL-3 caused increased levels of PGE2 in co-cultures, however, IL-1β was required to generate significant proteoglycan loss from cartilage explants. Finally, extra-cellular signal related protein kinases I and 2 (ERK 1&2) and p38 intracellular signalling pathways were shown to be involved in IL-1β mediated production of NO fibroblasts and explants. These studies show that IL-1β has increased potential to mediate cartilage breakdown when interacting with other cytokines, such as G-CSF, and other cell types, such as Swiss 3T3 fibroblasts. IL-1β has defined intracellular signalling pathways that may produce a range of responses in cartilage explants and fibroblasts. These studies may relate to production of inflammatory processes and loss of cartilage integrity and function in pathological conditions.

It has been shown that reactive oxygen species (ROS) are released in response to articular cartilage injury. The excessive release of ROS has been shown to be mitochondrial in nature and leads to chondrocyte death which in turn can lead to post-traumatic osteoarthritis (PTOA). Evidence suggests that mitochondria are attached to chondrocytes' cytoskeleton. Upon tissue level deformation, it is believed that mitochondria also experience deformation in response to cytoskeletal strain, releasing ROS. Therefore, it was hypothesized that inhibition of chondrocytes' cytoskeleton would prevent mitochondrial distortion rendering them unable to release ROS in response to the applied strain, saving chondrocytes. Osteochondral explants treated with cytoskeletal inhibitors were found to reduce mitochondrial ROS production directly after impact and increase chondrocyte viability 24 hours after impact. The release of mitochondrial ROS is an important mechanotranduction pathway in the initiation of PTOA.

Reproduction de : Thèse d'exercice : Pharmacie : Nantes : 2008. / Bibliogr.

Thèse doctorat : Sciences : Paris 12 : 2003. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 82 réf.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2009. / Title from title screen (site viewed January 5, 2010). PDF text: xiv, 328 p. : ill. (some col.) ; 7.48 Mb. UMI publication number: AAT 3373081. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Drs. Sheila Grant and James Cook. Includes bibliographical references.