[Truncated abstract] An important field of research in orthopaedic biomechanics is the elucidation and mathematical modelling of the mechanical response of cartilaginous tissues. Such research has applications in the understanding of joint function and degenerative processes, as well as in surgical planning and simulation, and engineering of tissue replacements. In the case of surgical and tissue engineering applications especially, patient-specific mechanical properties are highly desirable. Unfortunately, obtaining such information would generally involve destructive mechanical testing of patient tissue, thus rendering the tissue functionally unusable. Development of a laser scanning confocal arthroscope (LSCA) within our School will soon allow non-invasive extraction of 3D microstructural images of cartilaginous tissues in vivo. It is also envisaged that, linked to a suitably formulated constitutive formulation, such information could allow estimation of tissue mechanical response without physical biopsy. This thesis describes the development of techniques to potentially allow non-invasive patient-specific estimation of tissue mechanical response based on confocal arthroscopy data. A microstructural constitutive model is developed which is capable of directly incorporating LSCA-derived patient-specific structural information. A fibre composite type homogenisation approach is used as the basis for the model. ... The result is a series of orientation tensors describing the 3D orientation of linear features in the image stack. The developed analysis techniques are used to estimate fibre volume fraction and orientation distribution for each of the meniscal specimens. The developed constitutive model and image-derived structural parameters are finally used to estimate the reaction force history of two meniscal cartilage specimens subjected to partially confined compression. The predictions are made on the basis of the specimens? individual structural condition as assessed by confocal microscopy and involve no tuning of material parameters. Although the model does not reproduce all features of the experimental curves, as an unfitted estimate of mechanical response the prediction is quite accurate. In light of the obtained results it is judged that more general non-invasive estimation of tissue mechanical properties is possible using the developed framework. The likely limitations and potential areas of improvement are discussed.

Abstract Osteoarthritis (OA) is a heterogenic joint disease significantly affecting the quality of life of a patient, causing pain and disability. OA causes degenerative changes to the structure and composition of articular cartilage and subchondral bone. Currently, effective treatments for OA are limited, partly due to limitations in defining the imaging biomarkers of early OA. Improvement of imaging modalities in OA research and clinical setup is a requirement for quantitating early OA-related tissue features. In the clinical and preclinical setup, computed tomography (CT) enables imaging of bone and, using specific contrast agents, articular cartilage. The aim of this study is to create and validate novel micro-computed tomography (μCT) methods to quantify OA-related features and modifications in articular cartilage and subchondral bone. Contrast-enhanced μCT methods for imaging the collagen (phosphotungstic acid (PTA) and phosphomolybdic acid (PMA)) and GAG (CA4+) content of the articular cartilage in vitro were validated against various reference methods measuring the biochemical composition of articular cartilage. To improve the μCT imaging of subchondral bone, grey-level co-occurrence matrix (GLCM) based analysis of sub-resolution features of subchondral bone was introduced. In addition, to test the translatability of the GLCM-based analysis to clinical resolution, sub-resolution features extracted from clinical cone-beam CT were validated against the subchondral bone morphometrics from the μCT. PTA showed stronger association with the collagen content of the articular cartilage compared to PMA. PTA was also associated with collagen content even in degraded articular cartilage. CA4+ distribution was found to accumulate in chondrons and surrounding areas, suggesting that it is a prominent contrast agent for high-resolution μCT studies of chondrocytes. The GLCM-based analysis of subchondral bone provided information on cellular structure from μCT images and trabecular bone micro-structures from clinical CT images. In conclusion, μCT imaging can provide quantitative information on the collagen content and chondrons of articular cartilage, as well as on osteocytes in subchondral bone. The methods presented here extend the tools for researchers to quantify osteochondral tissue modifications in OA. Furthermore, the developed image processing tools could be translatable to the clinical CT. / Tiivistelmä Nivelrikko on heterogeeninen niveltauti, joka huonontaa yksilön elämän laatua aiheuttaen kipua ja liikuntakyvyttömyyttä. Nivelrikko aiheuttaa nivelkudosten rappeumaa vaikuttaen mm. ruston ja rustonalaisen luun rakenteeseen ja koostumukseen. Nivelrikon kudosmuutosten kuvantamisen kehittäminen ja määrällinen tutkiminen taudin alkuvaiheissa tukisivat nykyisten nivelrikon hoitomenetelmien kehittämistä. Kliinisessä käytössä ja perustutkimuksessa, tietokonetomografia (TT) mahdollistaa luukuvantamisen ja varjoaineita käytettäessä myös rustokuvantamisen. Tämän väitöskirjan tavoitteena on esitellä ja validoida uusia mikrotietokonetomografia-menetelmiä (μTT) nivelrikon rusto- ja luumuutosten määrälliseen tutkimukseen. Varjoaineavusteisia μTT in vitro menetelmiä ruston kollageenin (fosfovolframihappoa (PTA) ja fosfomolybdeenihappoa (PMA)) ja GAG (CA4+) jakauman määrälliseen tutkimukseen validoitiin useilla eri ruston biokemiallista koostumusta mittaavilla vertailumenetelmillä. Rustonalaisen luun kuvantamista kehitettiin soveltamalla harmaasävyjen tekstuurianalyysiä, jolla pyrittiin tunnistamaan kuva-alkiota pienempiä luurakenteita. Rustonalaisen luun μTT-kuvien analyysien tulokset validoitiin synkrotronisäteilyyn perustuvan μTT:n avulla. Lisäksi tekstuurianalyysin soveltuvuutta testattiin kliinisen resoluution kartiokeilan TT-kuville. Kuvista analysoituja tekstuuriparametrejä verrattiin μTT:lla mitattuun todelliseen rustonalaisen luun rakenteeseen. Väitöskirjan tulokset osoittavat, että PTA on spesifimpi kollageenille testatuista varjoaineista ja sen jakauma on verrannollinen kollageenijakaumaan jopa rappeutuneessa nivelrustossa. GAG-spesifisen varjoaineen CA4+:n todettiin kerääntyvän myös kondroneihin, mikä viittaa siihen, että kyseinen varjoaine soveltuisi potentiaalisesti rustosolujen korkean resoluution μTT-tutkimuksiin. Rustonalaisen luun μTT-kuvista analysoitujen tekstuuriparametrien havaittiin olevan verrannollisia osteosyyttien tilavuusfraktion kanssa. Väitöskirjassa esitettyjen tulosten perusteella μTT-kuvantaminen tarjoaa kvantitatiivisen menetelmän nivelruston kollageenijakauman ja rustosolujen sekä rustonalaisen luun osteosyyttien tutkimuksiin. Väitöskirjassa esitetyt menetelmät laajentavat jo olemassa olevaa tutkimusmenetelmien kirjoa nivelrikon aiheuttamien nivelrusto- ja luumuutosten tutkimuksessa. Lisäksi kehitetyt kuva-analyysimenetelmät voivat tarjota tarkempaa tietoa kliinisestä TT:sta.

A aproximação cricotireóidea simula a contração do músculo cricotireóideo, aproximando as cartilagens cricóidea e tireóidea anteriormente. Distende e tensiona as pregas vocais, elevando a freqüência fundamental da voz. É indicada em pacientes que apresentam voz grave em conseqüência da paralisia do nervo laríngeo superior ou por doenças endócrinas e reumatológicas no sexo feminino. Com o objetivo de estudar os efeitos da aproximação cricotireóidea sobre a vibração das pregas vocais em ambos sexos, foram avaliadas 30 laringes excisadas de cadáveres humanos frescos pré e pós aproximação cricotireóidea. Esse trabalho experimental permitiu excluir as interferências dos mecanismos compensatórios da fonação presentes em um estudo 'in vivo'. As cartilagens aritenóideas foram aproximadas por sutura, fechando a glote. Um fluxo de ar comprimido umedecido foi injetado no interior da traquéia, e ao passar pela glote promoveu a vibração das pregas vocais. Foi então realizada a aproximação cricotireóidea através de suturas, aproximando as cartilagens tireóidea e cricóidea. O comprimento da porção membranosa das pregas vocais foi obtido através de um paquímetro. A vibração das pregas vocais foi gravada pré e pós a aproximação cricotireóidea utilizando-se uma câmera de videoquimografia. Após digitalização e análise quadro a quadro das imagens documentadas, foram realizadas medidas da freqüência fundamental, da amplitude de vibração das pregas vocais, do ciclo vibratório e suas fases (aberta, fechada, de abertura e fechamento) pré e pós aproximação cricotireóidea. Foram também calculados e analisados índices que correlacionam a duração das diferentes fases do ciclo vibratório, como o quociente de abertura, de fechamento e o quociente de velocidade. Estas variáveis foram analisadas estatisticamente pela técnica multivariada de análise de perfil e correlacionadas entre si. Observamos que houve alongamento significante das pregas vocais após a aproximação cricotireóidea, sendo mais evidente no sexo feminino. Ocorreu elevação significante da freqüência fundamental, mas esta não se correlacionou com o alongamento das pregas vocais. A amplitude de vibração diminuiu de forma significante, assim como a duração do ciclo vibratório e suas fases, correlacionando-se diretamente com o aumento da freqüência fundamental. A redução da fase de abertura foi significantemente maior no sexo masculino. Concluindo, a aproximação cricotireóidea provocou alongamento da prega vocal, redução da amplitude e duração do ciclo vibratório e suas fases, existindo correlação inversa com o aumento da freqüência de vibração. Embora as pregas vocais de laringes femininas tenham apresentado maior alongamento, e as masculinas maior redução da fase de abertura, não houve diferença significante do efeito da aproximação cricotireóidea sobre a freqüência vocal entre os sexos. / The cricothyroid approximation simulates the cricothyroid muscle contraction by approximating the cricoid and thyroid cartilage’s. By this surgery, the vocal folds are lengthened. Consequently, the fundamental frequency of the voice is increased. The main indications for this procedure are patients presenting voice hoarseness due to superior laryngeal nerve palsy or to rheumatoid and endocrinological diseases in women. Thirty fresh cadaver’s larynges were used for the purpose of studying the effects of the cricothyroid approximation on the vocal folds vibration in both genders. Ideally, this experiment would allow exclusion of the diverse phonation compensatory factors that can possible compromise the accuracy of an “in vivo” study. The surgical procedure was performed in the excised larynges. Initially, the arytenoids cartilages were approximated by suturing both together, closing the glottis. Humidified compressed air was injected inside of the tracheal tube, causing vocal folds vibration. The cricothyroid approximation was performed by sutures placed in the cricoid and thyroid cartilages. Calipers were used to measure the length of the membranous portion of the vocal folds. Pre and post cricothyroid approximation. Vocal folds vibration was recorded by a portable camera specific for videokymographic studies. After digitalizing the recorded images, each one of them was carefully evaluated. The fundamental voice frequency, the vocal folds vibratory amplitude, and the vibratory cycle with its phases (opened, closed, opening and closing phases) were measured pre and post operatively. Indexes related to the duration of the different phases of the vibratory cycle, such as open quotient, closed quotient and speed quotient were then calculated and analyzed. Multivariate statistical analysis and profile analysis were used for correlation and understanding of the results. There was a statistically significant lengthen of the vocal folds after cricothyroid approximation, more noticeable in female laryngeal specimens. The fundamental voice frequency was increased, but no relation with the increased length of the vocal cords could be observed. The vibratory amplitude was significantly reduced, as was the vibratory cycle duration. The latter was directly correlated with the increase of fundamental voice frequency. The male laryngeal specimens presented a significant higher reduction of the opening phase of the vibratory cycle. In this study, the cricothyroid approximation caused lengthening of the vocal folds. The vibratory cycle and its phases were also altered, presenting a reduced duration. An inverse correlation between these variables and the increase of the vibratory frequency was noted. Although the female laryngeal specimens had shown a longer length when compared with the male, and the male specimens had a higher decrease of the opening phase of the vibratory cycle, there was no statistically significant difference in the cricothyroid approximation effect on the vocal frequency according to gender.

Abstract Primary hallmark of osteoarthritis (OA) is the progressive degeneration of articular cartilage. An accurate estimation of cartilage mechanics is important when analyzing the subject-specific function of the knee joint and risks for the onset and development of OA due to cartilage damage. Finite element (FE) modeling can help to estimate peak joint stresses and strains and explain how they could lead to OA. FE models of the knee joint during simplified gait were developed to define the level of material complexity required for 3D FE modeling of the knee joint in estimation of reliable tissue stresses and strains within articular cartilage of the knee joint; and to investigate the predictive value of FE modeling of the knee joint on the development and progression of radiographic OA within obese and normal weight subjects. It was found that maximum principal stresses and strains within articular cartilage in the knee joint during walking are highly sensitive to the material parameters of cartilage. It was not possible to match simultaneously stresses, strains and contact pressures between simplified (non-fibrillar) and advanced (fibrillar) models. Though, it was possible to find parameters for transversely isotropic models that enable the estimation of stresses and strains throughout the depth of cartilage similarly to more advanced fibril reinforced models. Locations of peak cumulative stresses in obese subjects at the baseline without radiographic OA showed a good agreement with the locations of cartilage loss and magnetic resonance imaging (MRI) based scoring in four year follow-up when they had developed OA. Simulated weight loss in obese subjects significantly reduced the highest cumulative stresses in cartilage to the level of normal weight subjects. The cartilage degeneration algorithm was able to predict subject-specific progression of OA similarly with MRI follow-up data and separate subjects with radiographic OA from healthy subjects. The computational FE models developed in this thesis represent useful tools to identify possible risk locations within the knee joint and how they relate to OA onset and progression. The presented methods have clinical potential in the diagnostics of knee joint OA in a subject-specific manner and in simulating the effect of interventions on the progression of OA thus helping with an effective treatment planning. / Tiivistelmä Nivelrikon tunnusomaisin piirre on nivelrustokudoksen rappeutuminen ja kuluminen. Nivelruston tehtävänä on tasata niveliin kohdistuvaa kuormitusta. Rustokudoksen mekaanisten ominaisuuksien määrittäminen on tärkeässä roolissa, kun halutaan arvioida tarkemmin polvinivelen toimintakykyä sekä rustokudoksen rappeutumista. Magneettikuvantamisen pohjalta tehtävä polvinivelen biomekaaninen tietokonemallinnus mahdollistaa rustokudoksen jännitys- ja puristusjakauman arvioinnin simuloidun kuormituksen aikana, mikä edelleen voi antaa vastauksia siihen, kehittyykö niveleen tulevaisuudessa nivelrikko, tai miten tietyn nivelrikkopotilaan sairaus etenee. Tämän tutkimuksen päätavoitteena oli kehittää kolmiulotteisia polvinivelen biomekaanisia tietokonemalleja, joiden perusteella simuloitiin normaalia kävelyä. Polvinivelen kolmiulotteinen geometria luotiin terveiden koehenkilöiden sekä nivelrikkopotilaiden magneettikuvista. Malleilla selvitettiin aluksi, miten monimutkaisena materiaalina nivelrusto tulee mallintaa, jotta mallin ennustama jännitys- ja puristusjakauma on silti realistinen. Tämän jälkeen tutkittiin, miten hyvin tietokonemallinnus ennustaa polvinivelrikon kehittymistä ja etenemistä sekä nivelruston rappeutumista ylipainoisilla potilailla. Tutkimuksessa havaittiin, että tietokonemallin ennustamat jännitys- ja puristusjakaumat nivelrustossa kävelyn aikana riippuvat merkittävästi nivelrustolle valitusta materiaalimallista ja sen parametreista. Tietokonemallien ennustamat nivelruston jännityskeskittymien sekä ruston rappeutumisen sijainnit vastasivat erittäin hyvin nivelrustokudoksen todellisen kulumisen sijainteja magneettikuvasta arvioituna neljän vuoden seuranta-ajan jälkeen. Tietokonemalleilla oli myös mahdollista simuloida painon pudotuksen vaikutusta, jolloin nivelrustokudoksen jännitys- ja puristusjakaumat palautuivat normaalien koehenkilöiden tasolle. Tässä tutkimuksessa kehitetyt polvinivelen tietokonemallit tarjoavat tutkijoille uuden työkalun paikallistaa sellaiset kohdat nivelpinnalta, joissa kuormituksen aiheuttama mekaaninen jännitys on suurta; nämä kohdat ovat kaikista riskialtteimpia nivelrikon kehittymiselle. Kehitettyjä malleja voidaan perustutkimuksen lisäksi jatkokehittää edelleen kohti kliinistä sovellusta, jolloin niitä voitaisiin hyödyntää esimerkiksi simuloitaessa erilaisten hoitojen vaikutusta kuormitusjakaumiin ja rustokudoksen rappeutumiseen.

Les polluants environnementaux, en particulier les perturbateurs endocriniens (PE), agissent à très faibles doses sur des cibles multiples. Les effets rapportés portent en majorité sur les organes de reproduction. Très peu d’études ont porté sur le squelette alors que le cartilage et l’os sont sous un puissant contrôle hormonal, depuis le stade fœtal où le système hormonal se met en place jusqu’au vieillissement, en passant par la naissance (hormones thyroïdiennes, hormone de croissance), la puberté et la ménopause chez la femme (stéroïdes sexuels). L’objectif de ce travail est d’étudier les effets de polluants anti-androgènes (vinclozoline, V et métabolite actif M2) ou xenestrogènes (génistéine, G; bisphénol A, BPA), in vivo sur le développement du squelette du rat Wistar et in vitro sur les marqueurs de différenciation chondrogéniques. Les effets in vivo ont été étudiés à des doses inférieures aux “No Observed Adverse Effect Levels ” (NOAEL) fixés par les instances européennes (EFSA) et internationales (US EPA). Des rattes ont été exposées à V, G seuls, combinés (GV) et/ ou associés au BPA (BGV), et ce de la conception des petits jusqu’à leur sevrage (J30) ou leur sacrifice (J30, J110). Les effets ont été recherchés sur des petits de mères et portées différentes, quatre pour chaque traitement, âge et sexe. Les effets in vitro du métabolite M2 de la Vinclozoline, associé ou non avec G et BPA, ont été étudiés sur l’expression de marqueurs chondrogéniques en utilisant : 1) un modèle murin de cellules souches inductibles vers la voie chondrogénique (C1) pour les effets sur la différenciation chondrogénique précoce et 2) des chondrocytes de souriceaux nouveau-nés, différenciés en culture primaire ou dédifférenciés (passages répétés). Comparaison avec les effets du bFGF, facteur de dédifférenciation chondrogénique. Résultats : In vivo, l’exposition à V, seule ou associée à G ou au BPA induit chez les rattes F1 exposées, une cannelure de la queue, discrète mais perceptible à la palpation en regard de chaque articulation intervertébrale. Les xénestrogènes tendent à réduire cet effet de V. Les rats et les animaux F2 ne sont pas atteints. L’examen par micro CT-scan montre une augmentation significative de la largeur des apophyses transverses (ITA) des vertèbres, et une diminution de la hauteur des corps vertébraux chez les rattes F1 exposées en regard des contrôles. Ces modifications anatomiques rappellent certaines pathologies génétiques des collagènes (dysplasies épiphysaires) chez l’homme Elles sont absentes chez les rats F1 et les animaux F2. Elles sont en partie transitoires car présentes à J30 (effets sur ITA et longueur) quand seul l’effet sur l’ITA perdure à J110. L’examen histologique des cartilages de croissance des corps vertébraux montre un déséquilibre entre les zones de prolifération et d’hypertrophie qui évoque une modification de la maturation du cartilage de type estrogénique. Ces effets sont ici aussi transitoires et majoritairement observés chez les rattes. L’effet plus prononcé du BPA lisse toutes les autres activités. Cela suggère que les PE pourraient moduler la différenciation du cartilage de croissance. C’est ce qui a été étudié in vitro. In vitro. Le premier objectif était d’évaluer les effets des PE sur la dynamique d’induction chondrogénique (cellules C1). Nous montrons que l’addition de M2 seul ou avec G ou BPA modifie le processus de maturation du collagène2 sans effet sur les autres marqueurs (SOX9, Agrécane, Col10). M2 prolonge l’expression de COL2A immature et retarde son remplacement par l’isoforme COL2B. Le second objectif était d’étudier les effets des PE sur la régulation de l’expression de COL2A au cours du processus de dé-différenciation des chondrocytes in vitro. L’expression de COL2A augmente avec le degré de dédifférenciation cellulaire (passages successifs) et double en présence de M2, G et BPA. Cet effet dépend des récepteurs aux estrogènes (ER) et des voies p38-MAPK. (...) / Environmental pollutants, particularly Endocrine Disruptors (ED), show effects on multiple targets at very low doses. Mostly known effects target reproductive organs. Very few studies are conducted on skeleton, although cartilage and bone are under potent hormonal control, from fetal stage, where hormonal system takes place, until aging, through birth stage (thyroid hormones, growth hormones), puberty and menopause for women (steroid hormones). The aim of this work is to study effects of anti-androgenic pollutants (vinclozolin, V, and its active metabolite M2) or xenoestrogens (genistein, G; bisphenol A, BPA), in vivo on Wistar rat skeletal development and in vitro on chondrogenic differenciation markers.In vivo effects were studied at doses below the “No Observed Adverse Effect Levels” (NOAEL) established by European and American agencies (EFSA and US EPA respectively). Female Wistar rats were exposed to V and G alone, in combination (GV) and/or associated to BPA (BGV), from pups conception until weaning (d30) or sacrifice (d30, d110). Effects were investigated on offsprings from different mothers and litters, on four animals by treatment, age and gender. In vitro effects of M2 metabolite of Vinclozolin, combined or not to G and BPA, were studied on chondrogenic markers expression using : 1) inducible murine stem-cells model towards chondrogenesis (C1) to sudy effects on early chondrogenic differentiation and 2) post-natal mouse differentiated chondrocytes, in primary culture or dedifferenciated chondrocytes by successive passages. Comparison with bFGF, a dedifferentiation factor for chondrocytes.Results : In vivo, exposure to V, alone or combined to G or BPA, induce discrete but palpable annealing in F1 treated female rat tails, in front of each intervertebral articulation. Xenoestrogens tend to decrease V effect. Male rats and F2 offsprings were not affected. Micro-CT Scan analysis shows significative increase of vertebrae inter transverse apophyses (ITA) distance, and decrease of vertebral body height in F1 female rats comparing to control animals. Anatomical modifications recall human collagen genetic diseases (epiphyseal dysplasias). They are absent in F1 male rats and F2 offsprings. Furthemore they are partly transient, ITA and height effects being present at d30 whereas ITA effect alone remains until d110. Histological analysis of vertebral body growth plate shows unbalance between proliferative and hypertrophic zones, which evokes estrogenic acceleration of cartilage maturation. Those effects are still transient and mainly observed in female rats. BPA activity is dominant above G and V effects. This result suggests ED can modulate growth plate cartilage differentiation, which was studied in vitro. In vitro : First, we aimed to evaluate eventual role of ED on the dynamic of the chondrogenic differentiation process. We show that M2 addition, alone or in combination with G or BPA, modifies collagen 2 maturation process without any effect on other markers (SOX9, Agrecan, COL10). M2 addition extends immature isoform COL2A expression and delays its replacement by mature isoform COL2B. Second, we studied the effects of ED on the regulation of COLA expression through the dedifferentiating process of chondrocytes in vitro. COL2A expression increases with cell dedifferenciation degree (successive passages) and double with M2, G and BPA. No other chondrogenic marker was modified. This effect depends on estrogen receptors (ER) and p38-MAPK pathway. (...)

Le cartilage articulaire est un tissu avasculaire et non innervé, ce qui lui confère des capacités de réparation très limitées. Les traitements chirurgicaux actuels ne permettent pas d’obtenir un tissu de réparation similaire au cartilage natif. Les recherches s’orientent depuis de nombreuses années vers l’ingénierie cellulaire et tissulaire du cartilage selon le type de lésions à traiter, focale ou diffuse. Les cellules souches mésenchymateuses (CSMs) constituent une source cellulaire intéressante pour l’ingénierie du cartilage. Elles sont facilement accessibles et ont des potentialités de différenciation chondrogénique. Les CSMs issues de la moelle osseuse sont les plus étudiées et constituent la référence. D’autres sources de CSMs sont également très prometteuses. Notre choix s’est porté sur les CSMs issues de la membrane et du liquide synovial. Ces deux tissus articulaires présentent l’avantage de pouvoir être prélevés facilement lors d’un examen arthroscopique et leurs CSMs sont adaptées au microenvironnement (hypoxie, inflammation) de l’articulation. Ce travail a porté sur l’étude de deux sources cellulaires d’origine synoviale dans le traitement des lésions focales et diffuses du cartilage. Ces CSMs d’origine synoviale ont d’abord été caractérisées selon leurs phénotypes et leurs capacités de différenciation vers les voies ostéogénique, adipogénique et chondrogénique, par rapport aux CSMs issues de la moelle osseuse. Ensuite, les capacités chondrogéniques de ces CSMs synoviales destinées à produire un substitut cartilagineux consacré aux traitements des lésions focales du cartilage articulaire ont été étudiées. Les CSMs ont été ensemencées dans un biomatériau collagénique et différentes conditions environnementales (facteurs de croissance et oxymétrie) ont été évaluées afin de définir les conditions de culture les plus appropriées. La chondrogenèse a été induite avec succès par l’utilisation de facteurs de croissance tels que TGF-1 et TGF-3 seuls ou en association avec la BMP-2. L’hypoxie n’a pas montré d’effet bénéfique sur la synthèse matricielle au sein des substituts cartilagineux. Enfin, nous avons évalué les capacités des CSMs issues du liquide synovial à traiter les lésions diffuses du cartilage induites par un modèle de section du ligament croisé antérieur chez le rat athymique. Les deux injections intra-articulaires de CSMs issues du liquide synovial à 1 et 2 semaines après chirurgie n’ont pas permis de prévenir les lésions arthrosiques. / Joint cartilage is avascular and not innervated, which gives it very limited repair capabilities. Current surgical treatments do not provide repair tissue similar to native cartilage. For many years, research has been focused on cellular and tissue engineering of cartilage depending on the type of lesions to be treated, focal or diffuse. Mesenchymal stem cells (MSCs) are an interesting cellular source for cartilage engineering. They are easily accessible and have the potential for chondrogenic differentiation. MSCs from bone marrow are the most studied and are the gold-standard. Other MSCs sources of are also very promising. We chose MSCs from the synovial membrane and synovial fluid. These both joint tissues have the advantage of being easily retrievable during arthroscopic examination and their MSCs are adapted to the microenvironment (hypoxia, inflammation) of the joint. This thesis work focused on the study of two cellular sources of synovial origin in the treatment of focal and diffuse cartilage lesions. These synovial-derived MSCs were first characterized according to their phenotypes and their ability to differentiate to the osteogenic, adipogenic and chondrogenic pathways, compared to bone marrow derived MSCs. Then, the chondrogenic capacities of these synovial MSCs to produce a cartilage substitute for the treatment of focal lesions of joint cartilage were studied. The MSCs were seeded in a collagenic biomaterial and different environmental conditions (growth factors and oximetry) were evaluated to define the most appropriate culture conditions. Chondrogenesis has been induced with success by the use of growth factors such as TGF-β1 or TGF-β3 alone or in combination with BMP-2. Hypoxia has not exerted a beneficial effect on matrix synthesis in cartilage substitutes.Finally, we evaluated the ability of CSMs from human synovial fluid to treat diffuse cartilage lesions induced by an anterior cruciate ligament section model in athymic rats. The two intra-articular injections of synovial fluid MSCs, 1 and 2 weeks after surgery did not prevent osteoarthritic lesions.

La bioinspiration consiste à analyser les systèmes naturels qui se sont adaptés parfaitement à leurs environnements pour développer des solutions ingénieuses. Ce projet de thèse aborde la thématique de la lubrification articulaire dans le but de développer un traitement contre l'ostéoarthrite (OA). Nous nous sommes inspirés des articulations synoviales, systèmes tribologiques très performants grâce aux interactions synergiques entre la structure unique du cartilage et les molécules lubrifiantes (ML) du fluide synovial (SF). Cependant, lors de l'OA des mécanismes inflammatoires et d'érosion mécanique aboutissent à la dégénérescence progressive du cartilage et la dégradation spécifique des ML du SF (aggrécane et lubricine). Des mimes des ML du SF ont été synthétisés reprenant leur structure particulière dite en écouvillon moléculaire (BB), structure responsable de la lubrification. Des tests tribologiques (SFA, tribomètre) ont montré que les BB garantissent à la fois une faible friction et une résistance à l'usure sur des surfaces dures de mica. Ceci est dû à la présence, sur nos EM, de groupements d'ancrage spécifiques assurant l’adsorption sur la surface de mica et à la formation d'enchevêtrements et d’interactions intermoléculaires avec l'acide hyaluronique de haut poids moléculaire, composant essentiel du SF. Des mimes de cartilage à base d'hydrogels de chitosane multicouches ont été également réalisés reprenant les principales propriétés architecturales du cartilage. En combinaison avec nos EM, ces hydrogels, matériaux poroélastiques fragiles, sont capables d’être lubrifiés avec une friction dans la gamme physiologique et une nette amélioration de leur usure / Bioinspiration consists in the design of materials inspired by biological systems which have developed ingenious solutions to suit their environment. This project deals with bioinspiration for joint lubrication and in particular for the development of treatments for patients suffering from osteoarthritis (OA). To do so, we took our inspiration from joints which are amongst the most efficient aqueous tribological systems. Their unique properties arise from the complex synergistic interactions between cartilage structure and the lubricant macromolecules of the synovial fluid (SF). However, during OA, inflammatory mechanisms as long as mechanical erosion result in the degeneration of cartilage and lubricant macromolecules (aggrecan and lubricin). Polymeric mimes of the SF have been synthesized based on the bottle-brush (BB) architecture of LUB and AGG which is responsible for the joint lubrication. Tribological tests (SFA, tribometer) showed that BB polymers provided mica surfaces with a low friction and a wear protection up to several megapascals, typically in the range of natural joints. This wear protection was essentially due to the incorporation of anchoring groups specific to mica tribopairs on the BB polymers and the intermolecular bridging and entanglements emerging between BB polymers and high molecular weight HA, another main SF component. Cartilage mimes composed of multilayered chitosan hydrogels were designed to mimic the basic features of cartilage. Along with our BB polymers, the hydrogels, which are poroelastic and fragile materials, provided a low friction and a great decrease of wear

Tesis por compendio / [ES] El cartílago hialino es un tejido conectivo denso con poca capacidad de auto regeneración cuando es afectado por patologías degenerativas. Por lo tanto, la estimulación eléctrica se ha propuesto como una terapia alternativa no invasiva para mejorar la reparación del cartílago hialino. De acuerdo con esto, este trabajo presenta un enfoque computacional y experimental combinado para entender mejor la biología del cartílago hialino y su respuesta a la estimulación eléctrica usando diferentes modelos in vitro. En primer lugar, se ha desarrollado un modelo mecanobiológico para simular el proceso de osificación endocondral. Por otro lado, se ha evaluado el efecto de la estimulación eléctrica sobre el cartílago hialino en tres escenarios diferentes. Inicialmente se ha analizado la proliferación celular y la síntesis de glicosaminoglicanos de condrocitos cultivados en monocapa y estimulados con campos eléctricos. Luego, se ha realizado un análisis histomorfométrico a explantes de condroepífisis que fueron estimulados eléctricamente. Por último, se ha evaluado el efecto de los campos eléctricos sobre la diferenciación condrogénica de células madre mesenquimales cultivadas en hidrogeles. Los resultados indican que la estimulación eléctrica es un estímulo biofísico prometedor, ya que este tipo de estimulación mejora la viabilidad y la proliferación celular, induce cambios morfológicos en los condrocitos, y estimula la síntesis de las principales moléculas que componen el cartílago hialino, tales como SOX-9, glicosaminoglicanos y agrecan. Además, este proyecto es el primer paso hacia la implementación de un estímulo biofísico alternativo que modifica la dinámica celular de los condrocitos de la placa de crecimiento en condiciones ex vivo. Adicionalmente, este estudio resalta el efecto potencial de los campos eléctricos para inducir el proceso de condrogénesis de células madre mesenquimales cultivadas en condiciones basales. En general, la evaluación de la estimulación eléctrica sobre condrocitos, tejidos y andamios es una herramienta útil que puede contribuir al conocimiento actual de las terapias regenerativas enfocadas en la regeneración del cartílago hialino. / [CA] El cartílag hialí és un teixit connectiu dens amb poca capacitat d'auto regeneració quan es veu afectat per patologies degeneratives. Per tant, l'estimulació elèctrica s'ha proposat com una teràpia alternativa no invasiva per millorar la reparació del cartílag articular. D'acord amb això, aquest treball presenta un enfoc computacional i experimental combinat per entendre millor la biologia del cartílag hialí i la seva resposta a l'estimulació elèctrica usant diferents models in vitro. En primer lloc, s'ha desenvolupat un model mecanobiològic per simular el procés d'ossificació endocondral. D'altra banda, s'ha avaluat l'efecte de l'estimulació elèctrica sobre el cartílag hialí en tres escenaris diferents. Inicialment s'ha analitzat la proliferació cel·lular i la síntesi de glicosaminoglicans de condròcits cultivats en monocapa i estimulats amb camps elèctrics. Després, s'ha realitzat una anàlisi histomorfomètrica a explants de condroepífisis que van ser estimulats elèctricament. Finalment, s'ha avaluat l'efecte dels camps elèctrics sobre la diferenciació condrogénica de cèl·lules mare mesenquimals cultivades en hidrogels. Els resultats indiquen que l'estimulació elèctrica és un estímul biofîsic prometedor, ja que aquest tipus d'estimulació millora la viabilitat i la proliferació cel·lular, indueix canvis morfològics en els condròcits, i estimula la síntesi de les principals molècules que componen el cartílag hialí, com ara SOX-9, glicosaminoglicans i agrecan. A més, aquest projecte és el primer pas cap a la implementació d'un estímul biofísic alternatiu que modifica la dinàmica cel·lular dels condròcits de la placa de creixement en condicions ex vivo. Addicionalment, aquest estudi ressalta l'efecte potencial dels camps elèctrics per induir el procés de condrogènesi de cèl·lules mare mesenquimals cultivades en condicions basals. En general, l'avaluació de l'estimulació elèctrica sobre condròcits, teixits i scaffolds és una eina útil que pot contribuir al coneixement actual de les teràpies regeneratives enfocades a la regeneració del cartílag hialí. / [EN] Hyaline cartilage is a dense connective tissue with low self-healing capacity when is affected by degenerative pathologies. Therefore, electrical stimulation has been proposed as a possible non-invasive alternative therapy to enhance the restoration of the cartilaginous tissue. Accordingly, this work presents a combined computational and experimental approach to understand better the hyaline cartilage biology and its response to electrical stimulation using different in vitro models. On the one hand, a mechanobiological model was developed to simulate the endochondral ossification process. On the other hand, the electrical stimulation on hyaline cartilage was evaluated in three different scenarios. Initially, cell proliferation and glycosaminoglycans synthesis of chondrocytes, cultured in monolayer and stimulated with electric fields, was analyzed. Then, a histomorphometric analysis was performed to chondroepiphysis explants that were electrically stimulated. Finally, the effects of the electric fields on chondrogenic differentiation of mesenchymal stem cells cultured in hydrogels was assessed. The results indicated that electrical stimulation is a promising biophysical stimulus, due to the fact that this type of stimulation enhances the viability and the proliferation of cells, induces morphological changes in the chondrocytes, and stimulates the synthesis of the main molecules that compose the hyaline cartilage, such as SOX-9, glycosaminoglycans and aggrecan. Moreover, this project is the first step towards the implementation of an alternative biophysical stimulus that modifies the cellular dynamics of growth plate chondrocytes in ex vivo conditions. Additionally, this study highlights the potential effect of electric fields to induce the chondrogenesis process of mesenchymal stem cells cultured in basal conditions. Overall, the assessment of electrical stimulation on chondrocytes, tissues and scaffolds is a useful tool that may contribute to the current knowledge of regenerative therapies focused on hyaline cartilage healing. / To the financial support from COLCIENCIAS – COLFUTURO through the fellowship No. 647 for national doctorates. To the financial support from COLCIENCIAS through the research grant 712-2015 No. 50457. To the financial support from the Spanish Ministry of Economy and Competitiveness through the MAT2016-76039-C4-1-R project. / Vaca González, JJ. (2019). The effect of electric fields on hyaline cartilage: an in vitro and in silico study [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/120023 / Compendio

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Anabolic steroids are used extensively in beef cattle feeding management to take advantage of well-documented improvements in growth performance and efficiency of implanted cattle. In addition to muscle growth, steroids also impact changes in bone and cartilage formation. In general, these effects can be interpreted as hastening bone aging. The current study was designed to test the hypothesis that recently-identified peripheral indicators of bone and cartilage turnover could be detected in the peripheral circulation. Furthermore, it was hypothesized that these peripheral markers might reflect accelerated aging effects of the widely used steroidal implants trenbolone acetate (TBA) and estradiol-17β (E2). Circulating IGF-I was measured as a positive marker of steroid-induced enhancement of the somatotropic endocrine axis. Thirty-two crossbred yearling steers were blocked by BW and given one of four treatments: non-implanted controls; 25.7 mg estradiol-17beta (E2); 120 mg trenbolone acetate (TBA); or a combination of 120 mg TBA and 24 mg E2 (T+E). Blood samples were collected on d 0, d 7, d 14 and d 28 and serum was analyzed by ELISA for IGF-I concentrations, as well as osteocalcin, C-terminal telopeptides of Type I collagen (CTX-I) and C-terminal telopeptides of Type II collagen (CTX-II), which serve as markers of bone formation, bone resorption and cartilage resorption, respectively. Circulating IGF-I was similar among treatments on d 0 and 28. At d 7 and 14, steers receiving E2 or T+E had greater circulating IGF-I than non-implanted control steers (P < 0.05). In contrast, steers receiving only TBA tended to have elevated IGF-I compared to controls on d 7 and 14 (P = 0.10). Although treatment did not affect serum osteocalcin, concentrations were increased on d 7, 14, and 28 compared to d 0 (P < 0.005 for all). Implant treatment did not affect circulating CTXI, however CTX-II was affected by T+E treatment (P<0.05). The data suggest that, although selected markers of bone and cartilage turnover can be detected in circulation in cattle, implant-induced changes in the concentrations of these markers are not directly evident in the peripheral circulation at least through 28 d following treatment.

The effect of synovial membrane products and mechanical pressure upon the metabolism of articular cartilage proteoglycans has been studied in vitro. The degradation of cartilage proteoglycans was studied in an organ culture system and measured as the release of [35S ] sulphate from prelabelled cartilage. The effect of synovial membrane products upon the synthesis of proteoglycans was studied in a chondrocyte monolayer system and the effect of mechanical pressure upon the synthesis of proteoglycans in an organ culture system. In both types of experiments [35S] sulphate was used as precursor. The findings may be summarized as follows 1 Conditioned synovial medium (control-SM) enhanced the degradation and reduced the synthesis of cartilage proteoglycans. In addition the degradation was further enhanced when the synovial tissue had been cultured in the presence of dextran sulphate. 2 Conditioned medium from synovial tissue cultured in the presence of indo-methacin (indo-SM), significantly reduced the synthesis of cartilage proteoglycans in chondrocyte cultures and reduced, although non-significantly, the degradation of proteoglycans in whole cartilage cultures. 3 Addition o f the prostaglandins E1 or E2 (PGE1 or PGE2 ) together with indo-SM to the cartilage cultures greatly enhanced cartilage degradation whereas the addition of PGE1 or PGE2 together with control-SM had no effect compared with that of control-SM alone. 4 Conditioned medium from synovial tissue cultured in the presence of low doses of glucocorticoids reduced cartilage degradation compared with control-SM. However, addition of control-SM together w ith low concentrations of glucocorticoids to the cartilage cultures significantly enhanced cartilage degradation. 5 Conditioned medium from synovial tissue cultured with actinomycin D or cycloheximide did not enhance cartilage degradation compared with cartilage cultured alone. 6 A continuous pressure of approximately 30 kgfcm-2 on cultures of cartilage reduced both the synthesis and the degradation o f cartilage proteoglycans. Although it is difficult to extrapolate from the in vitro to the in vivo situation, it is proposed that some factor(s) from the synovial membrane have the capacity to enhance the degradation and reduce the synthesis o f articular cartilage proteoglycans. From these experiments it cannot be completely excluded that treatm ent of arthritic joints with non-steroidal or streroidal anti-inflammatory drugs may result under certain conditions in enhanced joint damage. It is also suggested that under certain conditions the metabolism o f cartilage proteoglycans could be directly affected by mechanical stress. / <p>Diss. Umeå, Umeå universitet, 1982, härtill 6 uppsatser</p> / digitalisering@umu