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.

published_or_final_version / Dentistry / Master / Master of Orthodontics