Osteoarthritis (OA) is a debilitating disease that affects millions of people. It is characterized by the degeneration of articular cartilage, narrowing of the joint cavity, damage to the subchondral bone, loss of synovial fluid, and osteophyte formation. These symptoms can cause muscle weakness, decrease in range of motion at the joint, and pain. Pain is the symptom that is most frequently treated. However, pinpointing the exact origin and location that is causative to the pain can be difficult. Patients of OA commonly have areas of subchondral edema identified on an MRI as marrow bone lesions (BML). On a CT image, these BML are found to be areas of bone resorption within the subchondral bone of the affected joint called subchondral bone cysts (SBC). These cysts are hypothesized to be a source of pain in OA as well as progression of the disease. The synovial intrusion theory for cyst formation states that there is a physical connection made between the joint cavity and the SBC through which synovial fluid travels. The bony contusion theory describes micro cracks developing below the articular cartilage within the subchondral bone causing bone necrosis and SBC formation. This study was undertaken to investigate the histological presentation of the contents of the SBC and the surrounding area.Seven human femoral head samples, ranging from age 49-72 years old, from patients who received total hip arthroplasty due to OA were examined. Three areas were sectioned from each femoral head including an area containing a cyst, an area of primary compressive bone, and an area at the medial side of the femoral head. These sections were then stained for bone, cartilage, and nerves and examined histologically. Sclerotic bone was shown to surround each cyst cavity, while cysts were composed of a mixed connective tissue infiltrated with multiple blood vessels and potential nerve fibers. With further investigation of these structures, the location of the nerve fibers within the SBC could be a possible source of pain in OA and a target for future treatments and therapies. Within the femoral head, cysts were found to be both shallow and deep to the articular cartilage. Shallow cysts may support the synovial intrusion theory for cyst formation while deep cysts could support the bony contusion theory. In relation to articular cartilage, cysts were not only found below a degraded articular cartilage surface as expected but also below intact, less degraded cartilage. This in depth look at the cells and tissues present within and surrounding the cyst provides information to better understand the pathology of OA and possibly an alternative method for treating the disease.

Osteoarthritis is a chronic degenerative joint disease that affects the whole joint, including the articulating surfaces and capsular tissues in and around the joint (Hügle and Geurts 2016). Osteoarthritis currently impacts 27 million Americans (Racine 2015). Two prominent features in osteoarthritis that are correlated with greater pain and reduced function are MRI-identified bone marrow lesions and CT-identified subchondral bone cysts (Kumar et al. 2013). At this time, the relationship between bone marrow lesions and subchondral bone cysts has not been confirmed. Nor has the nature of the tissues associated with these clinical signs been fully characterized, pointing to the need for further research to identify the composition of subchondral bone cysts as these cysts could be a potential target for therapeutic intervention. This study focused on characterizing the tissue content of subchondral bone cysts in osteoarthritic femoral heads. Osteoarthritic femoral heads were collected from male and female patients, ranging in age from 43-72 years old, who underwent total hip replacement. After surgery, the femoral heads were fixed in 4% paraformaldehyde and scanned via micro-computed tomography (MicroCT). The MicroCT images were used to identify regions in each head containing large subchondral bone cysts, the primary compressive bone, and anterior cartilage. These regions were mapped to the actual femoral head and guided the dissection of the femoral head. Samples from each of the regions were decalcified, embedded in paraffin, and cut into 5-micron-thick histological sections which were then mounted onto slides. Each section was stained in Safranin-O/Fast green and hematoxylin and eosin to visualize the tissues present. Immunohistochemistry with anti-CD31 was carried out on selected slides to identify blood vessels. The samples within all three regions of the femoral head were graded histologically for the presence of subchondral bone cysts, whether cartilage was within the cyst region, if the histological section matched its corresponding MicroCT image, if the articular cartilage was fibrillated, the presence of sclerotic bone, the presence of osteophytes, and the presence of blood vessels. It was clear that many of these samples were in later stages of osteoarthritis considering most samples exhibited all of the above characteristics, contained fibrous tissue, and had little normal fatty marrow. Typically, subchondral bone cysts presented beneath fibrillated and degenerated articular cartilage, contained fibrous tissue that was much more intensely vascularized and innervated as compared to normal fatty marrow, and was surrounded by sclerotic trabecular bone. In some cases, osteophytes also formed at the articular surface beneath areas of degenerated cartilage. Some subchondral bone cysts contained cartilage and even smooth muscle cells in addition to fibrous connective tissue. The varied location of the subchondral bone cysts shows the need for further research as to how their etiologies develop. The content of the subchondral bone cysts suggests that areas with disrupted trabeculae become intensely vascularized to allow the influx of inflammatory materials and mesenchymal stem cells to lay down fibrous tissue and thicken the surrounding trabeculae to stabilize the weakened microstructure of the femoral head. By gaining a comprehensive understanding of the pathology of osteoarthritis and of subchondral bone cysts in particular, the progression of the disease can be more firmly established and potential new treatments can be developed.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第21460号 / 人健博第67号 / 新制||人健||5(附属図書館) / 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 杉本 直三, 教授 藤井 康友, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Background: Osteoarthritis (OA) is a debilitating joint disease marked by cartilage and bone changes. Morphological and mechanical changes to bone, which are thought to increase overall bone stiffness, result in distorted joint mechanics and accelerated cartilage degeneration. Using a parametric finite element (FE) model of the proximal tibia, the primary objective of this study was to determine the relative and combined effects of OA-related osteophyte formation, and morphological and mechanical alterations to subchondral and epiphyseal bone on overall bone stiffness. The secondary objective was to assess how simulated bone changes affect load transmission in the OA joint. Methods: The overall geometry of the model was based on a segmented CT image of a cadaveric proximal tibia used to develop a 2D, symmetric, plane-strain, FE model. Simulated bone changes included osteophyte formation and varied thickness and stiffness (elastic modulus) in subchondral and epiphyseal bone layers. Normal and OA related values for these bone properties were based on the literature. “Effective Stiffness (K)” was defined as the overall stiffness of the proximal tibia, calculated using nodal displacement of the loaded area on the subchondral cortical bone surface and the load magnitude. Findings: Osteophyte formation and thickness or stiffness of the subchondral bone had little effect on overall bone stiffness. Epiphyseal bone stiffness had the most marked effect on overall bone stiffness. Load transmission did not differ between OA and normal bone. Interpretation: Results suggest that epiphyseal (trabecular) bone is a key site of interest in future analyses of OA and normal bone. Results also suggest that observed OA-related alterations in epiphyseal bone may result in OA bone being more flexible than normal bone.

The mandibular condyles represent important growth sites within the facial skeleton. Condylar growth is not a pacemaker of mandibular development, but it provides regional adaptive growth that is of considerable clinical significance, as the condyle’s upward and backward growth movement regulates the anteriorly and inferiorly directed displacement of the mandible as a whole. Orthopedic problems of the temporomandibular joint (TMJ), such as displacement of the TMJ disk, are common in the adolescent population. Clinical studies of mandibular asymmetry and mandibular retrognathia in adults as well as in children and adolescents, have reported an association with coexisting non-reducing displacement of the TMJ disk without identifying the cause and effect. Through experimental studies causality has been established, and unilateral affliction during growth has been shown to retard ipsilateral mandibular development with facial asymmetry as the sequel. It was hypothesized that bilateral non-reducing TMJ disk displacement during growth would impair mandibular development bilaterally, resulting in mandibular retrognathia. TMJ disk displacement has repeatedly been demonstrated to induce histological reactions of the condylar cartilage. An additional assumption was therefore that a non-deranged TMJ disk function is crucial for the maintenance of the growing condyle’s biophysical environment, and that a connection ought to exist between the amount of condylar cartilage changes caused by TMJ disk displacement and the amount of subsequent adverse mandibular growth. It was also hypothesized that non-reducing displacement of the TMJ disk in growing individuals would result in qualitative and quantitative changes of the condylar subchondral bone. An improved experimental cephalometric method was developed in order to optimize the reliability of longitudinal radiographic evaluation of fast growing small animals. Bilateral non-reducing TMJ disk displacement was surgically created in ten growing New Zealand White rabbits, with ten additional rabbits serving as a sham operated control group. The amount and direction of craniofacial growth was followed over time in serial cephalograms, aided by tantalum implants in the jaws. The study period was chosen to correspond to childhood and adolescence in man. The assessed growth of each side of the mandible was correlated to the histological feature of ipsilateral condylar cartilage at the end of the growth period. The amount and composition of subchondral bone from three regions of interest in the condyle, and the expression of local growth factors in the adjacent condylar cartilage was evaluated. The results verified that bilateral non-reducing TMJ disk displacement retarded mandibular growth bilaterally; the extent corresponding to mandibular retrognathia in man. Displacement of the TMJ disk during the growth period induced condylar cartilage adaptive reactions that were associated with both an adverse amount and direction of mandibular growth, manifesting in a retrognathic mandibular growth pattern. Growth impairment fluctuated over time, with the most striking retardation occurring during periods of increased general growth, implying a local growth reduction explicitly counteracting general hormonal growth acceleration. A significant decrease of the total amount of subchondral bone, in spite of a general increase of new bone formation in the experimental condyles, pointed to a reparative compensation for an extensive resorption of subchondral bone due to displacement of the TMJ disk, but not to the extent that normal growth would be maintained. These results constitute an explanation for the adverse mandibular development following non-reducing TMJ disk displacement in growing individuals. This project has shown that non-reducing displacement of the TMJ disk during growth has significant consequences on facial development. The findings strongly advocate early and accurate diagnosis and treatment of TMJ disk displacement in the adolescent population, thereby presumably reducing the need for future orthodontic and surgical craniofacial corrective therapy. The results furthermore enhance the need for full appraisal of TMJ disk function in the adolescent population during orthodontic functional therapy, as the condylar cartilage and subchondral bone reactions to a concomitantly displaced non-reducing TMJ disk must be expected to interfere with the intended growth stimulating treatment. The findings of intact articular layers in spite of gross histological and morphological soft and hard tissue changes as a sequel to TMJ disk displacement in growing individuals, implicate a clinical risk of false positive radiographic diagnosis of degenerative changes of the TMJ in children and adolescents.

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

L'importance de l'os sous-chondral dans la pathogenèse et la prise en charge de l'arthrose intéresse les cliniciens et la communauté scientifique. En effet, il existe des liens forts entre l'os sous-chondral et le cartilage, maintenant l'intégrité de ce dernier reposant sur l'os sous-chondral pour fournir un support mécanique et un soutien nutritionnel. Ici, nous avons étudié la relation entre les structures osseuses et cartilagineuses et l'approvisionnement vasculaire dans l'arthrose de la humaine.Nous avons recueilli 37 plateaux tibiaux arthrosiques prélevés après arthroplastie totale du genou. Dans ces mêmes plateaux, plusieurs carottes ont été prélevées et scannés par microtomographie. Les projections résultantes ont été reconstruites, puis segmentées manuellement pour séparer l'os sous-chondral de l'os trabéculaire et une analyse microarchitecturale a été développée sous les deux structures osseuses. Les échantillons ont été décalcifiés, coupés en sections de 16 heures, colorés dans de l'HES et classés en 6 groupes selon l'échelle OARSI. La surface de l'os sous-chondral et l'épaisseur et la surface du cartilage articulaire ont été cultivées. Le nombre de vaisseaux dans le sous-chondral a été compté par deux opérateurs différents et une coloration immunofluorescente avec du VEGF a été effectuée. Enfin, le cartilage, l'os sous-chondral et trabéculaire ont été utilisés pour mesurer les marqueurs ribonucléiques et protéiques liés à la vascularisation, l'innervation et l'inflammation.La microstructure de l'os a évolué au fur et à mesure que l'arthrose s'aggrave. L'os sous-chondral s'est épaissi et est devenu plus poreux. La fraction volumique osseuse, l'épaisseur trabéculaire, l'espacement et le nombre de trabécules ont été corrélés positivement avec le score OARSI. Une diminution significative du nombre de vaisseaux sanguins a été observée au dernier stade de l'arthrose. Enfin, les marqueurs ribonucléiques et protéiques liés à la vascularisation, à l'innervation et à l'inflammation ont été modulés au cours du développement de la pathologie. Pris ensemble, nos données montrent une interaction et des structures de soutien dynamiques entre l'os sous-chondral et le cartilage. La compréhension des voies de signalisation, l'unité biochimique du cartilage dans les articulations et la communication intercellulaire entre le cartilage et l'os sous-chondral peuvent mener à l'élaboration de stratégies plus efficaces pour traiter les patients souffrant d'arthrose. / The importance of subchondral bone in the pathogenesis and management of osteoarthritis retain the interest of clinicians and the scientific community. Indeed, there are strong links between the subchondral bone and the cartilage, maintaining the integrity of the latter resting on the subchondral bone to provide mechanical and nutritional support. Here, we investigated the relationship between bone and cartilage structures and vascular supply in human osteoarthritis.We collected 37 osteoarthritic tibial plates taken after total knee arthroplasty. In these same plates, several carrots were removed and scanned by microtomography. The resulting projections were reconstructed, then manually segmented to separate the subchondral bone from the trabecular bone and a microarchitectural analysis was done on both bone structures. The samples were decalcified, cut into 4 μm sections, stained in HES and classified into 6 groups according to the OARSI scale. The surface of the subchondral bone and the thickness and surface of the articular cartilage were measured. The number of vessels in the subchondral region was counted by two different operators and a VEGF immunofluorescent staining was performed. Finally, cartilage, subchondral and trabecular bone were used to measure ribonucleic and protein markers related to vascularization, innervation and inflammation.The microconstructure of the bone has evolved as osteoarthritis worsens. The subchondral bone has thickened and become more porous. Bone volume fraction, trabecular thickness, spacing and number of trabeculae were positively correlated with the OARSI score. A significant decrease in the number of blood vessels was observed in the last stage of osteoarthritis. Finally, ribonucleic and protein markers related to vascularization, innervation and inflammation were modulated during the development of the pathology. Taken together, our data show dynamic interaction and support structures between subchondral bone and cartilage. Understanding of signaling pathways, the biochemical unity of cartilage in the joints and intercellular communication between cartilage and subchondral bone can lead to the development of more effective strategies for treating patients with osteoarthritis.

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L’arthrose (OA) est une maladie articulaire dégénérative à l’étiologie complexe et diverse. Les travaux de ces dernières années ont démontré que l’OA est une pathologie affectant tous les tissus de l’articulation incluant le cartilage, la membrane synoviale et l’os sous-chondral. L’OA se traduit par une déstructuration et une perte de fonctionnalité de l’articulation, et est principalement caractérisée par une perte de cartilage articulaire. L’inflammation de la membrane synoviale joue un rôle déterminant dans la progression de l’OA, toutefois elle serait secondaire à la dégradation du cartilage. De plus, l’os sous-chondral est également le siège de nombreuses transformations lors de l’OA. Il est fortement suggéré que ces changements ne correspondent pas seulement à une conséquence, mais pourraient être une cause du développement de l’OA impliquant une communication entre ce tissu et le cartilage. Il est maintenant bien établi que les voies inflammatoires et cataboliques jouent un rôle crucial dans l’OA. C’est pourquoi, nous avons étudié l’implication d’une nouvelle famille de récepteurs membranaires, les PARs, et plus particulièrement le PAR-2 dans les voies physiopathologiques de l’OA. Notre hypothèse est que l’activation de PAR-2 au cours de l’OA est un phénomène majeur du développement/progression de la maladie faisant du récepteur PAR-2 un candidat privilégié pour le développement de nouvelles approches thérapeutiques ciblant non seulement le cartilage mais aussi l’os sous-chondral. Pour cette étude, nous avons travaillé in vitro avec des chondrocytes (Cr) et des ostéoblastes (Ob) OA respectivement du cartilage et de l’os sous-chondral du condyle fémoral humain. Nos résultats ont démontré que PAR-2 était plus exprimé dans les Cr et les Ob OA que dans les cellules normales. Par ailleurs, PAR-2 est régulé positivement par certains facteurs retrouvés au cours de l’OA comme l’IL-1β, le TNF-α et le TGF-β dans les Cr OA, et par l’IL-1β, le TNF-α et la PGE2 dans les Ob OA. De plus, les principaux facteurs cataboliques et inflammatoires, soit la MMP-1, la MMP-13 et la COX-2 sont produits en quantité plus élevée suite à l’activation du récepteur dans le cartilage OA. De même, l’activation de PAR-2 dans les Ob OA conduit à une production accrue de facteurs pro-résorptifs tels que RANKL, l’IL-6, la MMP-1 et la MMP-9, et à l’augmentation de l’activité pro-résorptive de ces cellules. En outre, dans les deux types tissulaires étudiés, l’activation de PAR-2 augmente l’activité de certaines protéines de la famille des MAPKinases comme Erk1/2, p38 et JNK. Finalement, nous avons conclu notre étude en employant un modèle in vivo d’OA induite chez la souris sauvage et déficiente pour le gène PAR-2. Nos résultats ont démontré que l’absence d’expression et de production de PAR-2 influençait le processus inflammatoire et les changements structuraux affectant à la fois le cartilage et l’os sous-chondral, conduisant à un ralentissement du développement de l’OA. Nos travaux de recherche ont donc permis de montrer que le récepteur PAR-2 est un élément majeur du processus OA en agissant sur les voies cataboliques et inflammatoires du cartilage, et sur le remodelage tissulaire de l’os sous-chondral. Mots-clés : Arthrose, chondrocyte, cartilage, ostéoblaste, os sous-chondral, PAR-2, MMPs, COX, ILs, RANKL, résorption osseuse, MAPKinase, catabolisme, inflammation / Osteoarthritis (OA) is a complex degenerative articular disease. Recent studies have shown that OA is a pathology affecting all the tissues of the joint including the cartilage, the synovial membrane and the subchondral bone. OA is regarded as destruction and a loss of functionality of joint, and is mainly characterized by a loss of articular cartilage. The synovial inflammation also plays a key role in the progression of OA; however, it is believed to be secondary to cartilage degradation. In addition, there are also in the subchondral bone numerous changes which occur during the course of the disease. It is strongly suggested that these changes in subchondral bone are not just a consequence but could be a cause of the development of OA, thus involving a cross-talk between this tissue and the cartilage. It is now well established that inflammatory and catabolic pathways play a crucial role during OA. This is why we have engaged the study of the involvement of a new family of membranous receptors, the PARs, and more particularly PAR-2 during the pathophysiological process of OA. Our hypothesis is that PAR-2 activation during OA course is a major phenomenon for the development/progression of the disease, and that PAR-2 seems a suitable candidate for the development of new therapeutic approaches targeting not only the cartilage but also the subchondral bone. Thus, we have performed in vitro studies on OA chondrocyte (Cr) and osteoblasts (Ob) cells issued respectively from human femoral condyle cartilage and subchondral bone. Our results showed that PAR-2 was expressed at a higher level in the OA Cr and Ob compared to normal cells. Moreover, PAR-2 is found to be positively regulated by some factors present during the course of OA, such as IL-1β, TNF-α and TGF-β in the OA Cr, and IL-1β, TNF-α and PGE2 in the OA Ob. In addition, the main catabolic and inflammatory factors including MMP-1, MMP-13 and COX-2 are enhanced following PAR-2 receptor activation in the OA cartilage. Similarly, the activation of PAR-2 in OA Ob lead to an increased production of pro-resorptive factors such as RANKL, IL-6, MMP-1 and MMP-9, and an increased pro-resorptive activity of these cells. In addition, in both tissue types, PAR-2 activation increases the activity of certain protein MAPKinases family such as Erk1/2, p38 and JNK. Finally, we have performed an in vivo study using an OA induced model in wild-type and PAR-2 gene knock-out mice. Our results demonstrated that the absence of PAR-2 expression and production influenced the inflammatory process and structural changes affecting the cartilage and the subchondral bone, leading to a slowing down of OA development. Our research investigation have bring to light that PAR-2 receptor is a key element during OA process by acting on the cartilage catabolic and inflammatory pathways as well as the tissue remodelling of the subchondral bone. Keywords : Osteoarthritis, chondrocyte, cartilage, osteoblast, subchondral bone, PAR-2, MMPs, COX, ILs, RANKL, bone resorption, MAPKinase , catabolism, inflammation