Thesis (Ph. D.)--Bioengineering, Georgia Institute of Technology, 2006. / Harish Radhakrishna, Committee Member ; Christopher Jacobs, Committee Member ; Andres Garcia, Committee Member ; Marc E. Levenston, Committee Chair ; Barbara Boyan, Committee Member.

En Chine, une grave maladie musculo-squelettique appelée la maladie de Kashin-Beck (KBD) se retrouve distribué sur une large zone géographique. Cette maladie touche plus de deux millions d'individus, notamment dans le centre de la Chine, et il est admis que plus de 30 millions d’individus seraient à risque. Des études géologiques et épidémiologiques ont montré une forte corrélation entre les zones de déficience en Se dans les sols et de KBD. KBD est une ostéoarthropathie, caractérisée par la destruction des chondrocytes du cartilage, très douloureuse et invalidantes, pouvant conduire à des déformations articulaires importantes. Le sélénium (Se) est présent partout dans l'environnement (eau, air, sols) et nos besoins physiologiques en Se sont couverts par notre alimentation quotidienne (eau, céréales). Bien que cet élément trace soit un nutriment essentiel pour la fonction cellulaire normale, ses mécanismes d’action ainsi que les transformations métaboliques de ses composés dans le corps humain ne sont toujours pas bien déterminés. Toutefois, à une dose un peu supérieure à la dose recommandée, il peut, selon la forme chimique ingéré, devenir toxique. Par conséquent, on retrouve le Se en très faible quantité (µg/L) dans l'organisme, ce qui rend difficile sa localisation et l’analyse de son rôle dans le métabolisme. Le Se fait partie de sites biologiquement actifs de protéines impliquées dans les mécanismes antioxydants de défense et le contrôle rédox des réactions intracellulaires. En outre, plusieurs études ont mis en évidence le rôle que joue de Se dans le développement des tissus tels que le cartilage articulaire. Cette action semble être médiée par l'intermédiaire de sélénoprotéines et seraient indirectement impliqués dans la croissance du cartilage normal et l'homéostasie. Aux Etats-Unis, une étude clinique a montré des preuves solides de l’influences d’un déficit en Se dans le métabolisme du cartilage conduisant un environnement favorable à l'apparition et la progression de l'arthrose. Même si le Se n’est pas le seul facteur dans le développement de maladies, il est fort probable que son absence impacte la croissance et le développement du cartilage articulaire. Un modèle in vitro de maturation accélérée du cartilage articulaire (explants) nous a permis d’analyser l'impact du sélénium dans la croissance et le développement de ce tissu. Des expériences biologiques, biophysiques et chimiques ont été réalisées pour comprendre comment la présence de Se affecte l'organisation des tissus. Un schéma récurrent de la distribution du Se dans le tissu a été découvert. Il semble être localisé au niveau des interfaces cellule-matrice, offrant des hypothèses intéressantes pour de futures études sur le rôle potentiel du Se dans la signalisation cellulaire ou transduction mécanique. Des analyses biomécaniques, structurelles et moléculaires ont été faites pour caractériser la matrice extracellulaire du cartilage articulaire traités avec différentes concentrations de Se. Il semble être localisé au niveau des interfaces cellule-matrice, ce qui suggère que le Se joue un rôle dans la signalisation cellulaire ou transduction mécanique. Des analyses biomécaniques, structurelles et moléculaires ont été faites pour caractériser la matrice du cartilage articulaire traités avec différentes concentrations de Se. Nous avons découvert qu’un déficit en Se peut induire à une morphologie proche de celle de l'arthrose lors de la maturation du cartilage immature. Cependant, le rôle exact de ce déficit en Se induisant ce type de phénotype reste inconnu. Ce projet contribue à une meilleure compréhension du Se dans le cartilage tout en montrant les difficultés d’étude du Se dans les milieux biologiques et les techniques permettant d’y répondre, mais aussi souligne l’importance de prendre en compte le Se comme élément important de traitements régénérateurs ou préventifs pour ce types de maladies. / In China, a severe musculoskeletal disease called Kashin-Beck disease (KBD) is largely endemic over a large geographical area. It has been reported that more than 2.5 million people in China suffer from KBD and about 30 million people are at risk. Geological and epidemiological investigations have shown that a strong correlation exists between the location of selenium (Se) deficient soils and the distribution of KBD in the population. The disease is manifested as degradation of the matrix, cell necrosis mainly in the articular and growth plate cartilage, which can result in growth retardation, secondary osteoarthrosis, and disability in daily life. The worst forms of this disease tend to start in childhood, which may lead to dwarfism. Selenium is present everywhere in the environment (water, air, soils) and it is mainly incorporated to the human organism through the daily diet (water, cereals). Although this trace nutriment element is essential for normal cellular function. Most of the selenium-related -functions and pathways remain incompletely understood. Whilst vital for normal function, it is toxic at concentration slightly higher than that required by the body. Consequently, it is present within the organism in parts per billion (microgram per liter) making it difficult to localize and analyze its role in metabolism. Despite being a trace element it is an essential component of antioxidant and anti-inflammatory-related proteins that protect cells against oxidative attack. Furthermore, several studies have exposed the role selenium plays in tissue development such as in articular cartilage. This action seems to be mediated via selenoproteins that are indirectly involved in normal cartilage growth and homeostasis. In the USA, a clinical study has shown strong evidence that Se-deficiency influences cartilage metabolism inducing a favorable environment for the onset and the progression of osteoarthritis. Even if the selenium is not the only factor in the development of degenerative joint disease, it is highly likely that its absence impacts its growth and development of articular cartilage. The main focus of this study was then to understand better the role of Se in the normal metabolic processes of articular cartilage. Cultures of articular cartilage explants were used on a previously validated in vitro model of tissue maturation to analyze the role of selenium in growth and development. Physical and chemical experiments were preformed to understand how the presence of selenium affects tissue organization. It has been possible to determine a fundamental recurrent pattern of Se-distribution in the tissue. It appears to be localized at cell-matrix interfaces and it can be hypothesized that Se plays role in cell signaling or mechanotransduction. Biomechanical, structural and molecular analyses have been made to characterize the extracellular matrix of articular cartilage treated with different concentrations of Se-level. We discovered that Se-deficiency induces morphological changes in the cartilage matrix during the fast maturation-like process, which could be related to degenerative-like morphology of the cartilage. This could potentially be associated with degenerative changes that occur in KBD patient during childhood. This project is a prospective work for a potential future enhancement of the regenerative or preventive treatments for specific musculoskeletal diseases with a metabolic component.

Joint disease is detrimental to basic quality of life. Articular cartilage is responsible for reducing friction and distributing loads in joints as they undergo large, repetitive load cycles each day, but damaged tissue has very limited intrinsic regenerative ability. Osteoarthritis (OA), the most common joint disease, affects over 500 million people worldwide, contributes more than $27 billion dollars in annual healthcare expenditures, and has increased in prevalence by nearly 50% since 1990 with our aging population. In spite of all this, OA remains a chronic degenerative condition lacking in effective treatment strategies. For cartilage repair in late-stage disease, synthetic joint replacements carry risk of altered loading and metal hypersensitivity, while clinically approved autografts or autologous chondrocyte implantation procedures suffer from lack of donor tissue and donor site morbidities. Prior to surgical intervention, OA management is focused on analgesia rather than preventing or slowing early-stage disease. Disease-modifying OA drugs are yet to successfully complete clinical trials, in part due to the widespread use of animal models for therapeutic discovery rather than high-fidelity human models. Alleviating the burden of cartilage damage will require improvements in both early-stage therapeutic interventions and late-stage repair. Tissue engineering has the potential to offer more biologically faithful cartilage derived with minimal invasiveness, but the resulting cartilage currently lacks the organization or maturity of native tissue. Thus, the central concept of my thesis work was to introduce biologically inspired spatiotemporal cues to guide engineered cartilage formation, establishing novel methods for cartilage tissue engineering that would provide (i) cartilage-bone grafts for regenerative implantation and (ii) advanced in vitro models for studying osteochondral disease. United by the central theme of cartilage, this dissertation spanned three complementary and interacting areas of tissue engineering: regenerative medicine in Aim 1, tools and technological development in Aim 2, and organs on a chip in Aim 3. In Aim 1, we created patient-specific cartilage-bone constructs with native-like features at a clinical scale, using decellularized bone matrix, autologous adipose-derived stem/stromal cells, and dual-chamber perfusion bioreactors to recapitulate the anatomy and zonal organization of the temporomandibular ramus-condyle unit with its fibrocartilage. We validated key tissue engineering strategies for achieving in vivo cartilage regeneration, with the cartilage-bone grafts serving as templates for remodeling and regeneration, rather than providing direct replacements for the native tissue. To enable precise in vitro manipulation of TGF-β signaling, a key pathway in cartilage development, in Aim 2 we developed an optogenetic system in human induced pluripotent stem cells and used light-activated TGF-β signaling to direct differentiation into smooth muscle, tenogenic, and chondrogenic lineages. This optogenetic platform served as a versatile tool for selectively activating TGF-β signaling with precise spatiotemporal control. Using optogenetic recapitulation of physiological spatiotemporal gradients of TGF-β signaling in Aim 3, we formed stratified human cartilage integrated with subchondral bone substrate, towards in vitro engineering of native-like, zonally organized articular cartilage. Collectively, these studies established novel cartilage tissue engineering approaches which can be leveraged to alleviate the burden of joint disease.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21019号 / 医博第4365号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 安達 泰治, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

published_or_final_version / Biochemistry / Master / Master of Philosophy

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

During open orthopaedic surgical procedures, the articular cartilage covering the exposed joint surfaces can be exposed to air for prolonged periods. This exposure facilitates cartilage drying, characterised by changes to the extracellular matrix and chondrocyte death. Due to cartilage’s limited capacity for regeneration, it has been proposed that this may lead to post-operative joint degeneration. It has been proposed that chondrocyte death occurs as a result of both drying and nutritional deficiency. It is known that death during drying correlates with the drying interval and is initiated in the superficial chondrocytes, progressing to deeper layers at higher intervals. Additionally, it is well established that periodic rewetting (e.g. using 0.9% saline solution) can reduce chondrocyte death. The work presented in this thesis aimed to characterise chondrocyte death during drying, with particular reference to the chondrotoxic/protective effect of environmental variables (airflow) and surgical interventions (irrigation solutions), mechanism of injury and cell death, and the effect of in vivo drying on joint health. An ex vivo model of cartilage drying was developed and carried out on bovine and human intact cartilage and osteochondral explants, while varying environmental factors (drying interval, airflow velocity, oxygen concentration) and interventions (irrigation solutions and protective coverings. Throughout the study, cartilage drying was assessed in terms of 1) cartilage macroscopic appearance, 2) percent chondrocyte death (PCD), 3) cartilage water content, and 4) chondrocyte morphology. Histologically and fluorescently labelled samples were imaged using light and confocal laser scanning microscopy respectively, which formed the basis of the qualitative and quantitative assessments. Experimental drying at high airflow velocities had a more severe effect on cartilage appearance, PCD, and water content than in static air. This relationship was apparent in dried intact joints and osteochondral explants and in bovine and human samples. This suggests that the effects of surgical drying (where ventilation systems and airflow are routine) may be more pronounced than previously suggested and demonstrates a correlation between PCD and water-loss. Irrigation solutions supplemented with glucose (25-100 mM) had no significant effect on the PCD or water content in dried samples. Additionally, PCD was minimal in osteochondral explants cultured in the absence of glucose, even after 24 hr. This suggests that nutritional deficiency is unlikely to contribute to PCD during drying. However, chondrocyte death (in intact bovine cartilage) was reduced when drying was carried out at an oxygen concentration more reflective of the in vivo environment (5 %), which suggests that cell death during drying may be facilitated by a hyperoxic shock. Finally, in vivo cartilage drying was carried out on murine cartilage. Compared to sham operated controls, dried cartilage demonstrated a loss of surface integrity (4 weeks post-surgery) and fibrillations (8 weeks) and an increased modified Mankin score (at 4 and 8 weeks). Microscopically, an altered cartilage thickness, and chondrocyte density and arrangement were visible. These changes are comparable with changes in osteoarthritis. The results of this study demonstrate the importance of maintained cartilage hydration in order to avoid unnecessary chondrocyte death articular cartilage degeneration.