Le cheval athlète est exposé à des lésions dégénératives ou traumatiques du cartilage prédisposant au développement de l’arthrose. Les arthropathies associées à une dégradation du cartilage se traduisent par une perte de fonctionnalité de l’articulation. Le cartilage ne possédant que de très faibles capacités de réparation intrinsèque, le développement de traitements de ces affections représente un enjeu thérapeutique. Le premier objectif de notre étude était la mise au point d’un substitut cartilagineux utilisant des cellules souches mésenchymateuses (CSMs) du sang de cordon ombilical (SCO). Après avoir été isolées et caractérisées, les CSMs de SCO ont été différenciées en chondrocytes au sein d’un biomatériau, en présence de facteurs de croissance (BMP-2 et TGF-β1 ou TGF-β3), en normoxie ou en hypoxie. L’utilisation de la BMP-2 et du TGF-β1 en normoxie permet une synthèse abondante des protéines caractéristiques de la matrice extracellulaire du cartilage avec néanmoins une expression persistante du collagène de type I, marqueur du fibrocartilage. La stratégie d’interférence par l’ARN ciblant Col1a2 permet de stabiliser le phénotype cellulaire obtenu au niveau transcriptionnel. Le deuxième objectif de notre étude était l’utilisation des CSMs de SCO indifférenciées lors d’injection intra-articulaire chez le cheval. La tolérance des CSMs allogéniques in vivo a été démontrée, ce qui n’a pu être le cas pour leur efficacité dans un modèle d’arthropathie induite expérimentalement. Ainsi, les CSMs de SCO constituent une source intéressante pour l’ingénierie tissulaire du cartilage mais leur utilisation thérapeutique sous forme indifférenciée doit être optimisée et validée. / The racehorse is exposed to degenerative or traumatic lesions of cartilage predisposing to the development of osteoarthritis. Arthropathies associated with cartilage degradation result in loss of joint functionality. The cartilage has only a very low intrinsic repair capacity ; that is why the development of new treatment represents a therapeutic challenge. The first objective of our study was to develop a cartilage substitute using mesenchymal stem cells (MSCs) from umbilical cord blood (UCB). After having been isolated and characterized, the MSCs of UCB were differentiated into chondrocytes in a biomaterial in the presence of growth factors such as BMP-2 and TGF-β1 or TGF-β3, in normoxia or in hypoxia. The use of BMP-2 and TGF-β1 in normoxia leads to an abundant synthesis of extracellular matrix proteins characteristic of hyaline cartilage but with persistent expression of type I collagen, a fibrocartilage marker. siRNAs targeting Col1a2 stabilize the cellular phenotype obtained at transcriptional level. The second aim of our study was the use of undifferentiated MSCs of UCB for intra-articular injection in horses. The tolerance of allogeneic MSCs was demonstrated in vivo, but not for now their efficacity in a model of joint arthropathy.Thus, the MSCs of UCB is a source of interest for tissue cartilage engineering but their therapeutic use in undifferentiated form must be optimized and validated.

Purpose: Despite its many health benefits, moderate exercise can induce joint discomfort when done infrequently or too intensely even in individuals with healthy joints. This study was designed to evaluate whether NEM (R) (natural eggshell membrane) would reduce exercise-induced cartilage turnover or alleviate joint pain or stiffness, either directly following exercise or 12 hours post exercise, versus placebo. Patients and methods: Sixty healthy, postmenopausal women were randomly assigned to receive either oral NEM 500 mg (n=30) or placebo (n=30) once daily for two consecutive weeks while performing an exercise regimen (50-100 steps per leg) on alternating days. The primary endpoint was any statistically significant reduction in exercise-induced cartilage turnover via the biomarker C-terminal cross-linked telopeptide of type-II collagen (CTX-II) versus placebo, evaluated at 1 and 2 weeks of treatment. Secondary endpoints were any reductions in either exercise-induced joint pain or stiffness versus placebo, evaluated daily via participant questionnaire. The clinical assessment was performed on the per protocol population. Results: NEM produced a significant absolute treatment effect (TEabs) versus placebo for CTX-II after both 1 week (TEabs - 17.2%, P=0.002) and 2 weeks of exercise (TEabs - 9.9%, P=0.042). Immediate pain was not significantly different; however, rapid treatment responses were observed for immediate stiffness (Day 7) and recovery pain (Day 8) and recovery stiffness (Day 4). No serious adverse events occurred and the treatment was reported to be well tolerated by study participants. Conclusion: NEM rapidly improved recovery from exercise-induced joint pain (Day 8) and stiffness (Day 4) and reduced discomfort immediately following exercise (stiffness, Day 7). Moreover, a substantial chondroprotective effect was demonstrated via a decrease in the cartilage degradation biomarker CTX-II.

L'arthrose est une maladie commune observée dans les populations vieillissantes caractérisée par une affection dégénérative du cartilage articulaire. Le diagnostic clinique de la maladie repose sur la radiographie conventionnelle. Cette technique permet de mettre en évidence les modifications de l'os liées à l'arthrose (géodes, condensation de l'os sous-chondral, et ostéophytes) mais n'offre pas une vision directe du cartilage. Grâce à sa résolution spatiale et son contraste tissulaire élevé, l'IRM individualise le cartilage et le différencie des structures adjacentes (os, tissu synovial, ménisques et liquide synovial). Nous avons mis à profit les potentialités de l'IRM à haut champ (7 Teslas) pour suivre, in vivo, les modifications du cartilage de l'articulation fémoro-tibiale chez le rat, au cours du processus de maturation/vieillissement ainsi que dans un modèle d'arthrose expérimentale (section du ligament croisé antérieur). Ces travaux ont montré une diminution du volume et des épaisseurs cartilagineuses liée à l'âge, tout comme des pertes chondrales fémorales et un œdème du cartilage tibial dans le genou arthrosique. Dans une seconde partie, nous avons appliqué les méthodes de cartographie T2 et de mesures volumiques (à 1,5 T) afin de déterminer les variations survenant au sein du cartilage rotulien humain vieillissant. Ces travaux attestent de la capacité de la cartographie T2 à détecter des modifications matricielles avant l'apparition de réelles pertes chondrales. Enfin, notre dernière étude, toujours à 1,5 T, concerne la quantification du volume et de l'activité de la membrane synoviale inflammatoire dans une cohorte de patients souffrant de gonarthrose. / Osteoarthritis (OA) is a common disease observed in elderly population and characterized by a progressive destruction of cartilaginous tissue. The clinical diagnosis of this disease is realized by conventional radiography. This method allows visualizing bone modifications related to OA disease (cysts, subchondral bone thickening, and osteophytes) but is unable to assess directly cartilage structure. Due to its high spatial resolution and high contrast between tissues, Magnetic Resonance Imaging (MRI) is able to visualize the cartilage structure and to differentiate it from adjacent structures (bone, synovial tissue, menisci, and synovial fluid). We have employed MRI potentialities at high magnetic field (7 Teslas) to follow, in vivo, cartilage modifications in the rat femoro-tibial articulation. This methodology was used to evaluate normal cartilage ageing-process and to assess an experimental OA model (anterior cruciate ligament transaction). These works showed an age-related cartilage volumetric and thickness decrease, as well as femoral cartilage damages and tibial cartilage oedema in OA knees. In a second part of our work, we applied T2 mapping and volumetric techniques (at 1.5 T) to determine variations which occur in the elderly human patellar cartilage. Results demonstrated the capacity of T2 mapping to early detect matricial modifications before any cartilage volumetric impact can be found. At least, our last study, always at 1.5 T, focused on the synovial membrane volume and inflammatory activity by taking into account a human population suffering from knee OA.

Abstract Collagen XI is a minor component of articular cartilage collagen fibrils together with collagen IX. They are in close functional relationship with the major cartilage collagen II. Collagen XI has been suggested to play a role in regulating the diameter of collagen II fibrils. Together these collagens form a supportive framework in the extracellular matrix. Besides articular cartilage, these three collagens can also be found in the vitreous body of the eye, the intervertebral disc, the inner ear and in various tissues during embryonic development. As the major cartilage collagen, collagen II has been studied quite extensively. Several syndromes ranging from lethal to milder ones have been shown to result from collagen II gene defects. Far less is known about defects in genes coding for the minor cartilage collagens, IX and XI. By identifying mutations in the coding genes and observing the resulting phenotypes, the function and importance of these genes start to unravel. The goal of this study was to provide more information about collagen XI. As a quantatively minor cartilage component, it is a good candidate for mild disease phenotypes. Collagen XI gene mutations have been shown to cause relatively mild phenotypes, such as Stickler and Marshall syndromes and non-syndromic hearing loss. Seven families with a recessive chondrodysplasia, otospondylomegaepiphyseal dysplasia (OSMED), were analysed for mutations in COL11A2. This study showed that OSMED is typically caused by the absence of the α2(XI) chains. Sixty-two patients with isolated Robin sequence, cleft palate or micrognathia were analysed for COL11A2 gene mutations. Six unique nucleotide changes were found that are likely to associate with the phenotype. The results showed that collagen XI gene defects can play a role in the etiology of oral clefting, but are not common causes of these phenotypes. Altogether 72 unrelated osteoarthrosis (OA) patients and one family with OA were analysed for mutations in genes coding for collagens II, IX and XI. Eighteen percent of them were found to have a unique sequence variation. An association analysis of OA patients failed to reveal any common predisposing alleles in these genes.

Objective: Physical exercise decreases disability and pain associated with chronic articular cartilage degradation. However, understanding of the pathology is lacking. In this study, the levels of 17 biomarkers of inflammation and cartilage degradation were measured in synovial fluid (SF) before and after a 30-minute run in able-bodied and previously-injured individuals. Materials & Methods: Four able-bodied recreational runners (3 men and 1 woman: 24 ± 2 years, 68 ± 7 kg, and 173 ± 9 cm) and 4 recreational runners who had undergone a unilateral anterior cruciate ligament reconstruction (ACLr) (2 men and 2 women: 23 ± 1 years, 71 ± 6 kg, and 175 ± 4 cm) were recruited to participate in this study. Using a saline-assisted method, SF was aspirated before and after both a 30-minute unloading and 30-minute exercise session. Samples were corrected for blood contamination and analyzed for 15 cytokines and 2 matrix metalloproteinases (MMPs). Mixed model analyses were used to determine the main effects of session, case/control status, pre/post aspirations, and the interactions between case/control status and pre/post aspirations. Results: Blood protein contamination was calculated and accounted for in 15 of 32 synovial fluid samples. Granulocyte colony stimulating factor (GCSF) was the only detectable cytokine of the 15 analyzed. No statistical differences were found in GCSF concentrations between pretreatment and posttreatment aspirations (p = 0.45), ACLr and able-bodied control groups (p = 0.60), or unloading and exercise sessions (p = 0.96). MMP-13 was undetectable. No statistical differences were found in MMP-3 between pretreatment and posttreatment aspirations (p = 0.15), ACLr and able-bodied control groups (p = 0.85), or unloading and exercise sessions (p = 0.14).Conclusions: Two (GCSF and MMP-3) of the 17 measured biomarkers were detectable. There were no significant differences in either GCSF or MMP-3 due to a 30-minute run or 30-minute unloading period in either the able-bodied or ACLr participants. Further, there were no significant differences between biomarker concentrations and case-control status. A novel method of controlling for blood contamination in synovial fluid samples was implemented.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18504号 / 医博第3924号 / 新制||医||1005(附属図書館) / 31390 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 中辻 憲夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Osteoarthritis is a progressive degenerative disease which affects the cartilage in articulating joints. The progression of osteoarthritis is known to be mechanically mediated, though specific mechanical factors have yet to be identified. In particular, the effects of frictional interactions and altered mechanical homeostasis remain unknown, and the inability to link specific mechanisms to disease advancement hinders the development of treatment strategies. The overarching objective of this dissertation is to study the mechanically-mediated fatigue failure process in articular cartilage through a validated computational model to ascertain the relative importance of mechanical factors, including surface friction and bulk cyclic stresses, on progression of osteoarthritis. Fatigue failure in cartilage progresses as a function of multiple mechanical and physicochemical interactions. Collagen fibrils are the primary constituents that fail under fatigue loading. As the collagen fails, homeostasis between osmotic pressure and collagen tension is disrupted and the cartilage imbibes water and swells, producing softening. This entire process occurs under frictional contact loading. From a modeling standpoint, several primary challenges arise: (1) Accounting for the osmotic swelling of cartilage, which has not been sufficiently characterized experimentally; (2) Developing finite element algorithms to handle frictional contact of charged multiphasic (solid-fluid-solute) materials such as cartilage; (3) Modeling fatigue mechanics with observable state variables representing measures of cartilage composition, such that imaging techniques may inform the theory; and (4) Formulating compatible plasticity theories to allow validation of the novel fatigue framework with the extensive literature on fatigue of metals. This dissertation addressed these challenges in pursuit of the overarching objective. Direct experimental measurements revealed the osmotic swelling pressure in cartilage does not obey ideal Donnan law, which significantly overestimates the measured pressure by approximately a factor of three. The aggregate modulus in triphasic theory was found to vary strongly with the external concentration, increasing three- to five-fold between hypertonic and hypotonic solutions. These results allow us to capture the interaction of swelling with damage. The fatigue process is coupled with swelling, and computer modeling must be performed in a multiphasic environment which accounts for flow of charged ions. To address this, a novel surface-to-surface finite element algorithm for frictional contact was developed, providing the capabilities of complex surface-smoothing algorithms while retaining the simplicity of node-to-segment methods. This powerful framework was adapted to model friction between porous-permeable tissues, resulting in the development, implementation, and validation of finite element algorithms for four different types (single-phase elastic, biphasic, biphasic-solute, and multiphasic) of frictional contact. For the latter three, our work represented the first algorithms of this type. These algorithms are applied to model cartilage friction. By using constrained reactive mixture theory, we developed a reactive plasticity framework that reduced to classical Prandtl-Reuss plasticity theory in the limit of infinitesimal deformation, using only scalar state variables representing composition measures. Applying this reaction kinetics-based approach to model fatigue mechanics provided a valid theoretical framework for treating evolving damage, where measures of the mass composition of cartilage served as observable state variables. By incorporating reactive plasticity, our reactive fatigue theory was thoroughly validated against experimental data from metals and biological tissues, including human tendon and human cartilage. These modeling efforts were then synthesized to develop a fully validated computational model of fatigue failure in articular cartilage. For the first time, the role of frictional interactions on the progression of fatigue in articular cartilage was quantified. Results demonstrate that friction has an effect, but it is relatively small compared to the magnitude of the damage which takes place due to contact loads raising the magnitude of stresses in the collagen matrix. The implication of this result is that fatigue accumulation in cartilage is more sensitive to contact loading rather than surface interactions such as friction. This key finding may have clinical implications regarding treatment strategies for early-stage osteoarthritis. This dissertation has generated a novel suite of theoretical and computational tools which have facilitated the development of a fully validated computational model of fatigue failure in articular cartilage. Replicating previous experimental fatigue studies with the model has confirmed that bulk matrix stresses are responsible for the majority of fatigue-induced damage, and that friction plays a relatively minor role. Future work will apply these computational models to further analyze fatigue failure in fibrous biological tissues and study experimentally-generated hypotheses.

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

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21657号 / 医博第4463号 / 新制||医||1035(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 松田 秀一, 教授 安達 泰治 / 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(医学) / 甲第21672号 / 医博第4478号 / 新制||医||1035(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 川上 浩司, 教授 古川 壽亮, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM