Experimental studies indicate that culturing chondrocytes on biodegradable polymeric scaffolds may yield“engineered" cartilage for the replacement of tissue lost to injury or diseases such as osteoarthritis. A method of estimating the outcome of cell-polymer cultures would aid in the design and evaluation of engineered tissue for therapeutic use. The goals of this project were to develop, validate, and apply first-generation mathematical models that describe the kinetics of extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs cultured in vitro. The ECM deposition model is based on a product-inhibition mechanism and predicts an asymptotic, exponential increase in the concentration of ECM molecules found in cartilage, including collagen and glycosaminoglycans (GAG). The scaffold degradation model uses first-order kinetics to describe the hydrolysis of biodegradable polyesters in systems not limited by diffusion. Each model was fit to published data describing the accumulation of GAG and collagen, as well as the degradation of poly glycolic acid (PGA) and poly lactic acid (PLA), respectively. As experimental validation, cell-polymer constructs (n = 24) and unseeded scaffolds (n = 24) were cultured in vitro, and biochemical assays for GAG and collagen content, as well as scaffold mass measurements, were performed at 1, 2, 4, 6, 8, or 10 weeks of culture (n = 8 per time point). The mathematical models demonstrate a moderate to strong goodness of fit with the previously published data and our experimental results (R2=0.75-0.99). These models were also combined to predict the temporal evolution of total construct mass with reasonable accuracy (30% RMS deviation). In ongoing work, estimates of biochemical composition derived from these models are being proposed to predict the mechanical properties and functionality of the constructs. This modeling scheme may be useful in elucidating more specific mechanisms governing ECM accumulation. Given their potential predictive power, these models may also reduce the cost of performing long-term culture experiments.

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.