京都大学 / 新制・課程博士 / 博士(医学) / 甲第23604号 / 医博第4791号 / 新制||医||1055(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 戸口田 淳也, 教授 河本 宏, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18911号 / 人健博第25号 / 新制||人健||2(附属図書館) / 31862 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 坪山 直生, 教授 齋藤 邦明, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21012号 / 医博第4358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 戸口田 淳也, 教授 鈴木 茂彦, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

BACKGROUND/OBJECTIVE: Converging evidence in recent years suggests growth chondrocytes, involved in the integral process of endochondral bone formation and fracture healing, exhibit a dynamic bioenergetic profile despite residing in the nutrient poor cartilaginous environment. Specifically, chondrocytes show an increased dependence on mitochondrial derived oxidative phosphorylation during differentiating, collagen product, but to a differing extent when mineralizing. Therefore, quantitative analysis of mitochondrial dynamics during these varying processes serves to corroborate existing metabolic studies and further elucidate the role of oxidative metabolism during the endochondral process. METHODS: The murine chondroprogenitor cell line ATDC5 was used, and groups were cultured in differentiating, collagen promoted, and mineralizing conditions. Fluorescence confocal 3D image acquisition and bioimaging analysis was used to quantify changes in mitochondrial volume and branch length per mitochondria along with organization and colocalization changes of the actin cytoskeleton to mitochondria in the various conditions over 21 days. RESULTS: We showed that chondrocyte differentiation resulted in significantly increased mitochondrial volume and fusion when compared to non-differentiating groups, and in collagen promoted groups, mitochondrial volume was significantly higher. Additionally, we showed that the process of mineralization resulted in a significant decrease in mitochondrial volume and branch length per mitochondria by day 21 of the experiment. Finally, colocalization analyses of the actin cytoskeleton to mitochondria showed significantly increased overlap in non-differentiating cells when compared to differentiating conditions. CONCLUSIONS: These findings suggest that collagen production is likely an energetically taxing process and mineralization does not heavily rely on oxidative metabolism. Furthermore, the actin cytoskeleton likely plays a role in mitochondrial remodeling that coincides with mitochondrial fission and fusion; increased fission is associated with actin accumulation to mitochondria and fusion is associated with actin disassociation from the outer mitochondrial membrane.

Current articular cartilage tissue engineering endeavors, using synthetic polymers as scaffolds, have been somewhat successful. However, the use of these materials has not yielded a satisfactory, functional replacement for articular cartilage. Therefore, this project focuses on an alternative to these materials, chitosan, which is a naturally occurring biopolymer. The first project objective was to fabricate and analyze bulk, porous chitosan scaffolds, based on total porosity, average pore diameter, mechanical integrity, and degradation susceptibility. Secondly, scaffolds were evaluated in terms of their ability to support neochondrogenesis, including assessments of cell attachment and viability, cell morphology, and the biosynthesis of proteoglycan and type-II collagen-rich extracellular matrix. Results indicated that chitosan scaffolds possessing an interconnecting, porous structure could be easily created through a simple freezing and lyophilization process, and these scaffolds did support neochondrogenesis. Results suggest chitosan may be a useful alternative to synthetic polymers for use in cartilage tissue engineering applications.

Yes / Objective: Many patients with acromegaly, a hormonal disorder with excessive growth hormone (GH) production, report pain in joints. We undertook this study to characterize the joint pathology of mice with overexpression of bovine GH (bGH) or a GH receptor antagonist (GHa) and to investigate the effect of GH on regulation of chondrocyte cellular metabolism. Methods: Knee joints from mice overexpressing bGH or GHa and wild-type (WT) control mice were examined using histology and micro–computed tomography for osteoarthritic (OA) pathologies. Additionally, cartilage from bGH mice was used for metabolomics analysis. Mouse primary chondrocytes from bGH and WT mice, with or without pegvisomant treatment, were used for quantitative polymerase chain reaction and Seahorse respirometry analyses. Results: Both male and female bGH mice at ~13 months of age had increased knee joint degeneration, which was characterized by loss of cartilage structure, expansion of hypertrophic chondrocytes, synovitis, and subchondral plate thinning. The joint pathologies were also demonstrated by significantly higher Osteoarthritis Research Society International and Mankin scores in bGH mice compared to WT control mice. Metabolomics analysis revealed changes in a wide range of metabolic pathways in bGH mice, including beta-alanine metabolism, tryptophan metabolism, lysine degradation, and ascorbate and aldarate metabolism. Also, bGH chondrocytes up-regulated fatty acid oxidation and increased expression of Col10a. Joints of GHa mice were remarkably protected from developing age-associated joint degeneration, with smooth articular joint surface. Conclusion: This study showed that an excessive amount of GH promotes joint degeneration in mice, which was associated with chondrocyte metabolic dysfunction and hypertrophic changes, whereas antagonizing GH action through a GHa protects mice from OA development. / Dr. Zhu's work was supported by Ohio University, the Arthritis National Research Foundation (grant 833836), a FIRST award from the American Society for Bone and Mineral Research, the NIH (grant R15-AR-080813), and a Hevolution Foundation AGE grant (AGE-008). Dr. Davis’ work was supported by a medical student seed grant from Ohio University. Dr. Lotz's work was supported by the NIH (grant R37-AG-059418). Dr. Berryman was supported by the NIH (grant R01-AG-059779). Dr. Kopchick was supported by the State of Ohio's Eminent Scholar Program that includes a gift from Milton and Lawrence Goll and the AMVETS, and by the NIH (grant R01-AG-059779).

La régénération osseuse est un processus impliquant de nombreux types cellulaires comme les ostéoblastes, les chondrocytes ou les cellules stromales mésenchymateuses (CSM). Les CSM possèdent des capacités de différenciation suggérant leur implication dans ce processus de réparation. La régénération osseuse est le fruit de la coordination complexe de l'activité de nombreux facteurs de croissance. Parmi eux, l'« Heparin affin regulatory peptide » (HARP) est fortement exprimé dans le callus durant la régénération mais son rôle n'est pas clairement établi. Le but de ce travail de thèse a été (1) d'évaluer les effets de HARP sur les propriétés de migration, de prolifération et de différenciation des CSM in vitro ; (2) évaluer la capacité de HARP à induire une formation osseuse ou une régénération osseuse in vivo.Nos résultats démontrent que HARP est chémoattractant pour les CSM et potentialise leur prolifération. De plus, nous montrons pour la première fois que le traitement de CSM par HARP durant leur chondroinduction conduit à une différenciation chondrocytaire de type hypertrophique. Ce type cellulaire est primordial dans les derniers stades de la formation osseuse endochondrale qui se met en place durant la croissance osseuse, mais également durant la réparation. L'implantation de biomatériaux associés à HARP dans un défaut osseux de condyle fémoral a conduit à la formation de cartilage et d'os dans l'implant, reproduisant le mécanisme physiologique de formation osseuse endochondrale. Le biomatériau seul n'a été envahi que par du tissu fibreux.Durant les processus de réparation tissulaire, les glycosaminoglycannes (GAG), des chaînes polysaccharidiques sulfatées, composants majeurs de la matrice extracellulaire, participent à la modulation des effets des facteurs de croissance durant la réparation. Récemment, des mimétiques structuraux et fonctionnels des GAG ont été développés. Durant ma thèse, j'ai été associé au travail d'un doctorant de l'équipe de P.Albanese, qui a montré que des mimétiques de GAG induisent une différenciation ostéoblastique des CSM en l'absence de traitement ostéoinducteur. L'implantation sous-cutanée de biomatériaux covalemment associés aux mimétiques ont également été menées, et ont permis d'observer des potentialisations des processus de vascularisation de l'implant et de l'activité ostéoclastique. Ces resultats ont permis de valider l'interêt des GAG mimétiques dans le cadre des thérapies de régénération osseuse.Cette étude démontre pour la première fois les effets chondroinducteurs directs de HARP sur la production de molécules de la matrice cartilagineuse par les CSM in vitro, mais également sur la synthèse de tissu cartilagineux in vivo. Les effets de HARP observés sur la régénération osseuse confirment qu'il pourrait être un bon candidat en chirurgie orthopédique en permettant une régénération de type endochondrale typique de la réparation physiologique. De plus les nouvelles stratégies developpées dans le laboratoire sur la fonctionnalisation covalente de biomateriaux par des GAG mimétiques, meriteraient d'etre testées en association avec HARP, afin d'augmenter sa demi-vie et de controler son relarguage et ses activités biologiques in vivo. / Bone regeneration is a complicated process which involved many cellular types such as osteoblasts, chondrocytes and mesenchymal stromal cells (MSC). MSC can differentiate toward chondrocytes and osteoblasts, suggesting their implication in bone regeneration processes. Bone reparation involved a complex coordination of growth factors. Among them, heparin affin regulatory peptide (HARP) is found in callus during regeneration. However, its role is poorly understood. The aim of this thesis was (1) to evaluate HARP effects on proliferation, migration and differentiation of MSC in vitro, (2) to evaluate HARP ability to promote bone regeneration or bone formation.Our results demonstrate that HARP has chemoattractive and proliferative properties on human MSC. Moreover, we show for the first time that HARP commits human MSC toward hypertrophy during chondrogenesis. This is of great interest since hypertrophic chondrocytes are of primary importance in the late stage of endochondral bone formation. We further tested the association of HARP to scaffolds in a model of bone regeneration in femoral defect in rat. HARP associated scaffolds showed an invasion of cartilage and bony tissues, mimicking endochondral bone formation, whereas scaffold alone was just filled with fibrous tissue.During regenerative processes glycosaminoglycans, polysaccharides sulfated chains, are known as major components of the extracellular matrix and modulate the effects of growth factors during regenerative processes. Recently, structurally and functionally mimetics of GAG had been developed. During my PhD thesis, I was associated to the work of a doctoral student of P. Albanese who showed that GAG mimetics induce osteoblastic differentiation of MSC without any other osteoinductive treatment. The ectopic implantation of mimetic associated scaffolds didn't show effects on osteoformation but induced an enhancement of vascularization and of osteoclastic activity, both related to tissue remodeling. These results validate that GAG mimetics are of great interest in bone regenerative field.This study demonstrates for the first time the chondroinductive potential of HARP through its ability to induce cartilage specific matrix production by MSC in vitro but also by inducing cartilage tissue synthesis in vivo. The effects of HARP observed on bone regeneration, by inducing an endochondral bone formation similar to that observed in normal bone regeneration, confirm that HARP could be a good candidate in orthopedic surgery. Moreover, scaffold covalently linked with GAG mimetics should be tested in association with HARP. This strategy could increase the half life, control the release and potentiate HARP properties in vivo.

La dérégulation de la signalisation Wnt est impliquée dans les anomalies du développement et dans la pathogenèse de nombreuses maladies, y compris l'arthrose. Au cours de ce travail, nous avons étudié l'effet de Wnt-3a sur l’ADAMTS-4 dans les explants de cartilage et les chondrocytes primaires humains. Nous avons observé que Wnt-3a régule négativement l'expression de cette agrecanase et avons démontré que cette inhibition est médiée par Frizzled-8 via l’activation de la voie canonique et inhibition de l'activité de NFκB. En outre, nous avons montré que Wnt-3a est capable de s’opposer à l'induction de l’expression de l’ADAMTS-4 par l'IL-1ß, indiquant que la voie Wnt/ß-caténine peut jouer un rôle protecteur dans l'arthrose. D'autre part, la signalisation non canonique de Wnt induit une perte de la stabilité phénotypique des chondrocytes articulaires qui représente un événement précoce dans l'arthrose, cependant les mécanismes impliqués restent à élucider. Au cours de ce travail, nous avons identifié la cascade Wnt/CaMKII/B-raf/ERK1/2 comme voie de signalisation non-canonique modulant le phénotype chondrocytaire et avons montré que le syndécane4 est un composant essentiel. Nous avons démontré qu’en réponse à Wnt-3a, Frizzled-6 active la voie ERK1/2 en induisant la fixation de la kinase CaMKIIα au syndécane4 et celle de B-Raf à DVL-2 conduisant à l'activation de B-Raf. Dans une boucle de rétrocontrôle, Wnt-3a inhibe l’expression du syndécane4. Ce travail révèle le rôle du syndécane4 dans la régulation du phénotype chondrocytaire et met en évidence de nouvelles cibles qui peuvent avoir un potentiel thérapeutique contre l'arthrose / Dysregulation of Wnt signaling has been implicated in developmental defects and in the pathogenesis of many diseases, including osteoarthritis. Here, we studied the effect of Wnt-3a on ADAMTS-4 in human cartilage explants and primary chondrocytes and found that Wnt-3a negatively regulates the expression of this aggrecanase. We demonstrated that Wnt-3a inhibition of ADAMTS-4 expression is mediated by Frizzled-8 through activation of the canonical Wnt/ß-catenin pathway leading to inhibition of NFκB activity and down-regulation of ADAMTS-4. Furthermore, we showed that Wnt-3a is able to counteract the induction of ADAMTS-4 by IL-1ß, therefore indicating that Wnt/ß-catenin pathway may play a protective role in osteoarthritis. On the other hand, Non-canonical Wnt signaling induces loss of phenotypic stability of articular chondrocytes which represents an early event in osteoarthritis, but the underlying mechanisms are poorly understood. In this thesis, we identify Wnt/CaMKII/B-raf/ERK1/2 cascade as non-canonical signaling pathway that modulates chondrocyte phenotype and revealed that syndecan4 is an essential component of this pathway. We show that in response to Wnt-3a, Fz-6 activates non-canonical signaling by triggering the docking of CaMKIIα to syndecan4 and that of B-raf to DVL-2 leading to the activation of B-raf that transduces signals to ERK1/2 MAPK. In a feedback loop, non-canonical Wnt down-regulates the expression of syndecan4 to negatively regulate the signaling. Our finding uncovers a previously unanticipated role of syndecan4 as a regulator of chondrocyte differentiation. This study also provides new targets which may have therapeutic potential in osteoarthritis

Chez les patients atteints d'athérosclérose, les calcifications vasculaires sont une caracteristique des plaques d'athérome. Elles résultent de la trans-différenciation des cellules musculaires lisses (CMLs) en cellules de type ostéoblastique et/ou chondrocytaire, notamment en réponse à des cytokines inflammatoires. Les CMLs forment alors des cristaux par l'activité de la phosphatase alcaline non-spécifique du tissu (TNAP). A la lumière de résultats récents, nous avons émis l'hypothèse que la TNAP module la trans-différenciation des CMLs. Nos objectifs étaient donc de déterminer l'effet de la TNAP dans la trans-différenciation des CMLs, et d'étudier les mécanismes impliqués dans son induction. Nous avons observé que l'ajout de phosphatase alcaline purifiée ou la surexpression de TNAP stimule l'expression de marqueurs chondrocytaires en culture de CMLs et de cellules souches mésenchymateuses. De plus, l'inhibition de la TNAP bloque la maturation de chondrocytes primaires. Nous avons observé un rôle des cristaux formés par la TNAP, puisque l'ajout de cristaux seuls ou associés à une matrice collagénique a reproduit les effets de la TNAP. Nous suspectons que la TNAP agit en hydrolysant le PPi et en générant des cristaux. Ces cristaux ensuite induisent l'expression du facteur ostéogénique BMP-2 et l'inhibition des effets de la BMP-2 annule les effets de la TNAP. De plus, nous étions intéressés par les la localisation et la fonction de marqueurs de minéralisation comme les annexines en parallèle de la TNAP. Nous avons observé que l'activité TNAP des CMLs induit la minéralisation en grande partie quand la TNAP est associée aux vésicules matricielles et au fibres de collagène / Vascular calcification (VC) is a hallmark of atherosclerosis plaques. Calcification (formation of apatite) of advanced lesions share common features with endochondral ossification of long bones and appears to stabilize plaques. This process is associated with trans-differentiation of vascular smooth muscle cells (VSMCs) into chondrocyte-like cells. On the other hand, microcalcification of early plaques, which is poorly understood, is thought to be harmful. The two proteins necessary for physiological mineralization are tissue-nonspecific alkaline phosphatase (TNAP) and collagen. Under pathological conditions, TNAP is activated by inflammatory cytokines in VSMCs, whereas collagen is produced constantly. The activation of TNAP appears to induce calcification of these cells. Therefore, the objective of this PhD thesis was to study the role of TNAP and generated apatite crystals in the VSMC trans-differentiation and determine underlying molecular mechanisms. Based on the obtained results, we propose that activation of BMP-2, a strong inducer of ectopic calcification, and formation of apatite crystals generated by TNAP represents a likely mechanism responsible for stimulation of VSMC trans-differentiation. Moreover, we were interested in localization and function of mineralization markers such as TNAP and annexins in mineralization process mediated by trans-differentiated VSMCs and VSMC-derived matrix vesicles (MVs). We observed that, similarly as in the case of typical mineralizing cells, increased TNAP activity in VSMC-derived MVs and association with collagen were important for their ability to mineralize

L’ostéoarthrose (OA) est une maladie articulaire invalidante caractérisée par la perte de l’intégrité du cartilage articulaire. Les recherches tentent de comprendre les mécanismes moléculaires de la maladie afin de trouver des inhibiteurs efficaces pouvant prévenir la dégradation du cartilage articulaire. Les BMPs (bone morphogenic proteins) jouent un rôle dans le processus pathophysiologique de cette maladie. Cette étude cible le rôle d’un antagoniste des BMPs, le gremlin. Nous avons étudié la régulation de l’expression de gremlin par le clonage et la caractérisation de son promoteur et en déterminant si gremlin pouvait jouer un rôle autre qu’antagoniste des BMP, en affectant l’expression d’autres gènes par l’activation d’une cascade de signalisation dans la cellule. Les résultats ont identifié une région importante dans le promoteur de gremlin qui affecte son activité basale et induite, et ont montré que le gremlin ne pouvait pas affecter l’expression génique et l’activation de signalisation intracellulaire indépendamment des BMPs. Cette étude démontre que le rôle de gremlin dans l’OA en est un essentiellement d’antagoniste des BMPs. / Osteoarthritis (OA) is a disease that affects the integrity of the articular cartilage which leads to serious health issues for many individuals. Research is focused on understanding the molecular mechanisms which lead to this loss in integrity in the hopes of finding a way to turn the tide. The bone morphogenetic proteins (BMPs) have been shown to play a role in the progression of this disease and this study focuses on one of their antagonists, gremlin. We therefore decided to study what affects the expression of this protein through the cloning and characterization of its promoter region. We also studied the role of this protein in the disease, can it influence gene expression and can it initiate a signalling cascade within the cell on its own. The results identified a region important for basal and induced activity of its promoter .The results also demonstrated that the main role of this protein in the progression of OA is through BMP antagonism. Gremlin does not initiate a signalling cascade and affect gene expression on its own.