No description available.

No description available.

Articular cartilage (AC) morbidity represents a substantial burden to global and public health, and continues to afflict millions of people in the UK. This costs the economy more than £3 billion annually and requires approximately 80,000 knee replacements every year. To avoid these radical surgery procedures, various strategies have been recently developed to repair or restore AC through implantation of osteochondral grafts. However, their success remains limited, mainly with respect to the quality of the newly formed cartilage. To contribute to the development of improved treatment options, this thesis explored a tissue engineering approach towards the potential use of the electrospinning technique to build osteochondral grafts. The main objectives of the research were to produce and characterise a range of electrospun membranes with various compositions, to construct three-dimensional (3D) scaffolds seeded with cells, and to study the release of therapeutic agents from electrospun materials. Poly(lactic-co-glycolic acid) (PLGA) and Poly-e-caprolactone (PCL) were the two FDA-approved polymers used to create the electrospun membranes. Collagen and hy- droxyapatite were also added to increase the biocompatibility of the scaffolds. Tissue constructs were created by layering electrospun membranes with seeded sheets of human mesenchymal stem cells. Bovine Serum Albumin (BSA) and recombinant Transforming Growth Factor (33 (TGF-(33) were also incorporated into the fibres and used as model proteins to study the release of therapeutic agents from the core of co-axial electrospun fibres. Results suggest that minor alterations in composition cause gradual but significant changes in morphology, surface properties, mechanical characteristics and biocompatibil- ity of the scaffold. The combination of electrospinning and cell sheet engineering presents a unique and effective strategy that can be used to create 3D tissue constructs with high cell density and viability. Differentiation results also indicate that intrinsic properties of PLGA and PCL affect the chondrocyte phenotype. With regard to drug delivery, BSA and TGF-(33 were successfully incorporated into electrospun materials and subsequently released into an aqueous environment. The release profiles recorded exhibited a pro- nounced initial burst release that was significantly reduced by mineral deposition onto the membranes. To conclude, this thesis presents several contributions that demonstrate the use of mul- tilayered electrospun scaffolds as a successful approach for the generation of tissue engi- neered osteochondral grafts. Furthermore, this work supports the use of electrospinning as a key technology for future AC repair.

The unique structure and biochemical composition of articular cartilage determine its biomechanical properties and in turn, its functionality. Current clinical techniques available to repair damaged cartilage are unable to reliably regenerate mechanically stable, functional tissue. Hence there is a need to identify new and effective means to repair damaged cartilage. The underpinning concept and long term aim of this research is that of "acellular repair", to produce a direct replacement for damaged cartilage using acellular cartilage-like constructs developed via tissue engineering principals: I) seeding of suitable cells onto scaffolds and culture in chondrogenic medium; 2) application of cyclic compressive loading to promote cell differentiation and deposition of cartilage-like matrix; 3) achievement of construct mechanical properties comparable to native cartilage and 4) construct decellularisation to avoid any immune response following implantation in to the patient. The aim of this thesis is to begin optimisation of the parameters required to deliver the acellular repair. Human foetal osteoblastic cells (1.19 cell line; hFOB), bovine synoviocytes and human bone marrow mesenchymal stem cells were investigated by seeding onto polyethylene terephthalate non-woven fibre scaffolds of differing porosities and cultured for 4 weeks in chondrogenic medium. Protein and DNA assays, plus scanning electron microscopy revealed that synoviocytes were the most effective in producing filled scaffolds with 90.2 % being the optimum scaffold porosity. Constructs were then subjected to 13 to 23 % cyclic compressive strain at 1 Hz for 1 hr per day, using an in-house bioreactor. After 56 days (sustained at Day 84) loaded constructs had compressive moduli comparable to the higher ranges of native cartilage and histological properties similar to cartilage itself. It is concluded that optimisation of the parameters to deliver the acellular repair concept has been achieved. Reliable production of mechanically functional constructs containing cartilage-like matrix will allow for the following stage, decellularisation, to be undertaken. The development of the acellular cartilage-like constructs, thus far, provides promise for potential future clinical application.

No description available.

One of the author's previously published articles is inserted. / Bibliography: leaves 174-191. / xi, 213 leaves, [15] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A clinical and molecular study of patients with unequivocal Marfan sydnrome, or with an undiagnosed connective tissue disorder with some features in common with Marfan syndrome. Presents the phenotype of six Marfan patients with an FBN1 mutation, patients with Shprintzen-Goldberg syndrome or furlong syndrome, and two children with congenital aneurysms. Details the molecular screening of 44% of the FBN1 gene coding sequence for putative mutations. / Thesis (M.D.)--University of Adelaide, Dept. of Paediatrics, 1995

Les protéoglycanes (PGs) jouent un rôle important dans de multiples processus cellulaires tels que la prolifération, la différenciation et la migration cellulaires. Les PGs sont constitués d’une protéine porteuse sur laquelle sont fixées de façon covalente des chaînes hétéropolyssacharidiques de glycosaminoglycanes (GAGs). L’initiation de la biosynthèse des GAGs sur les PGs implique une glycosyltransférase, la ß1,3-galactosyltransférase 6 (ß3GalT6) qui catalyse l’addition d’un résidu galactose sur un disaccharide accepteur (Gal-Xyl) fixé au niveau de motifs d’ancrage des GAGs sur la protéine porteuse du PG. Des mutations de la ß3GalT6 ont été récemment associées à une forme pléiotropique du syndrome d’Ehlers-Danlos (SED), un groupe hétérogène de maladies génétiques rares touchant les constituants matriciels des tissus conjonctifs. L’implication de la ß3GalT6 dans la pathogénie du SED n’est cependant pas encore connue à ce jour, point qui sera exploré au cours de ce travail de thèse. Nous avons montré que la mutation du gène B3GALT6 conduit à une diminution de la biosynthèse des GAGs matriciels et membranaires, associée à une réduction de la capacité migratoire des fibroblastes de derme humain issus de patients atteints de SED par rapport aux fibroblastes contrôle, non porteurs de l’altération génétique. Une étude “gain et perte de fonction” a montré que l’extinction du gène B3GALT6 dans des fibroblastes contrôle impacte la biosynthèse des GAGs. De façon complémentaire, la restauration de l’expression de la ß3GalT6 dans les fibroblastes des patients a eu pour conséquences une augmentation du taux de synthèse des GAGs matriciels et membranaires, associée à une augmentation significative de la capacité de migration des cellules équivalente à celle des cellules non déficientes. Les résultats obtenus nous permettent de mieux comprendre le rôle de la ß3GalT6 dans la pathogénie du SED. Ces travaux ciblant la ß3GalT6 peuvent ouvrir la perspective de proposer des stratégies thérapeutiques visant à s’opposer à la perte d’anabolisme des GAGs et au défaut de migration observés dans le SED. / Proteoglycans (PGs) play important roles in many physiological processes, including cell proliferation, differentiation and migration. PGs are composed of linear heteropolysaccharide chains, called glycosaminoglycans (GAGs), which are covalently attached to a core protein through a tetrasaccharide linkage. The addition of the third residue (galactose) of the linkage is catalyzed by ß1,3-galactosyltransferase 6 (ß3GalT6), a key glycosyltransferase in GAG initiation. Recently, mutations of ß3GalT6 have been associated to Ehlers-Danlos Syndrome (EDS), a group of rare and severe genetic connective tissue disorders. However, the role of ß3GalT6 defects in EDS pathogeny remains unknown. In my thesis, we showed that ß3GalT6 defective dermal fibroblasts of affected patients exhibited a marked reduction in GAG anabolism associated to a significant delay in wound closure compared to control cells. The ß3GalT6 gain- and loss-of-function studies demonstrated that B3GALT6 gene deletion in control fibroblasts affects the synthesis of GAGs chains. Interestingly, GAG anabolism and cell migration were restored when ß3GalT6 is overexpressed in patient fibroblasts, which could be the starting point to the development of therapeutic strategies against the loss of GAG synthesis and defect of cell migration observed in EDS. This work provides a better understanding of the crucial role of ß3GalT6 in EDS pathogeny

Το αντικείμενο της παρούσας μεταπτυχιακής διατριβής ειδίκευσης ήταν η μελέτη των φυσικοχημικών και μηχανικών ιδιοτήτων παθολογικά αλλοιωμένων ανθρώπινων μηνίσκων. Οι μηνίσκοι είναι ινοχόνδρινοι μηνοειδείς δίσκοι που παρεμβάλλονται μεταξύ των αρθρικών επιφανειών του μηριαίου κονδύλου και της κνημιαίας γλύνης. Αποτελούνται ως επί το πλείστον από ίνες κολλαγόνου και χονδροκύταρα. Περιέχουν ακόμα γλυκοσαμινογλυκάνες, ελλαστίνη και DNA. Συμβάλλουν καθοριστικά στη σωστή λειτουργία της άρθρωσης καθώς είναι επιφορτισμένοι με λειτουργίες, όπως η προστασία των αρθρικών επιφανειών από τη φθορά, η λίπανση και η σταθεροποίηση της άρθρωσης. Η σημαντικότερη ίσως μεταβολική νόσος των μηνίσκων είναι η χονδρασβεστίωση (chondrocalcinosis) κατά την οποία παρατηρείται ανάπτυξη ανόργανων αλάτων στην επιφάνεια ή/και στο σώμα του ιστού. Για τη μελέτη των ιδιοτήτων των παθολογικά αλλοιωμένων ανθρώπινων μηνίσκων χρησιμοποιήθηκαν τεχνικές μηχανικής και φυσικοχημικής ανάλυσης. Με Δυναμική Μηχανική Ανάλυση επιχειρήθηκε να αξιολογηθεί η επίδραση των διαφόρων τύπων παθολογικών αλλοιώσεων στις δυναμικές μηχανικές ιδιότητες των ιστών. Οι ασβεστοποιημένοι ιστοί και οι εναποθέσεις τους μελετήθηκαν με φασματοσκοπικές τεχνικές RAMAN, FT-RAMAN, FT-IR καθώς επίσης και με απεικονιστικές τεχνικές οπτικής μικροσκοπίας, σαρωτικής μικροσκοπίας ηλεκτρονίων (SEM) και μικροσκοπίας ατομικής δύναμης (AFM). Επιπλέον, για πρώτη φορά έγινε διασύγκριση της ικανότητας τριών διαφορετικών διατάξεων RAMAN (FT-RAMAN: 1064 nm, Dispersion RAMAN: 633 nm και Dispersion RAMAN: 325 nm) να εντοπίσουν την παρουσία εναποθέσεων στους ιστούς. Στην παρούσα μελέτη εξετάστηκαν 63 μηνισκικά μοσχεύματα από οστεοαρθριτικούς ασθενείς οι οποίοι υποβλήθηκαν σε ολική αρθροπλαστική γόνατος στο Περιφερειακό Πανεπιστημιακό Νοσοκομείο Πατρών. Τα μοσχεύματα αυτά εξετάστηκαν από ιστοπαθολόγο και διαγνώστηκαν οι παθολογικές αλλοιώσεις που έφερα καθένας από αυτούς. Με βάση τα ιστοπαθολογικά αποτελέσματα έγινε κατηγοριοποίηση των δοκιμίων και συσχέτιση των αποτελεσμάτων αυτών με τα αποτελέσματα των προαναφερθέντων τεχνικών. Από τα αποτελέσματα της δυναμικής μηχανικής ανάλυσης φάνηκαν μικρές αλλά όχι στατιστικά σημαντικές διαφοροποιήσεις μεταξύ ασβεστοποιημένων μηνίσκων και μηνίσκων που παρουσίαζαν μυξωματοειδή εκφυλισμό και κύστες (σύμφωνα με τα αποτελέσματα της ιστοπαθολογικής εξέτασης). Τα αποτελέσματα της περιθλασιμετρίας ακτίνων Χ των ασβεστοποιημένων ιστών έδειξαν παρουσία μείγματος τρικλινούς και μονοκλινούς διένυδρου πυροφωσφορικού ασβεστίου (CPPD) υψηλής κρυσταλλικότητας καθώς και μικροκρυσταλλικές εναποθέσεις απατιτικής σύστασης (HAP). Οι τεχνικές μικροσκοποίας (οπτική, SEM και AFM) επιβεβαίωσαν τον παραπάνω ισχυρισμό αποκαλύπτοντας το χαρακτηριστικό τρόπο ανάπτυξης κάθε τύπου εναπόθεσης. Τα αποτελέσματα της φασματοσκοπικής ανάλυσης με χρήση των τεχνικών RAMAN – FT-RAMAN – και FT-IR έδειξαν την παρουσία επίσης χαρακτηριστικών κορυφών του CPPD και του HAP. Οι εναποθέσεις από CPPD ανιχνεύθηκαν στο 82.6 % των ασβεστοποιημένων δειγμάτων (19 στα 23) ενώ από απατίτη στο 17.4% (4 στα 23) Aναφορικά με τα RAMAN, επισημαίνεται ότι ανεξάρτητα από το μήκος κύματος που χρησιμοποιήθηκε και παρά την ισχυρή μπάντα φθορισμού που επεδείκνυαν τα φάσματα, οι χαρακτηριστικές κορυφές των εναποθέσεων ήταν εμφανείς. / The aim of the present master thesis study was the physicochemical and biomechanical characterization of pathological human menisci. Menisci are semilunar shaped fibrocartilaginous tissues that lie between the femoral condyle and tibia plateau. They mainly consist of collagen fibers and chondrocytes. Additionally glycosaminoglycans, elastin and DNA are present in the tissue. Menisci play an important role in the good function of human knee joint by distributing the applied loads, contributing to the joint’s stability and lubricating the joint’s area. The most common metabolic meniscal lesion is the chondrocalcinosis where inorganic phases of calcium or sodium salts are deposited in/on the tissue. Pathologically altered human menisci were studied by mechanical and physicochemical analysis techniques. Dynamic Mechanical Analysis was used to investigate the effects of degeneration lesions on the dynamic mechanical properties of the tissue. Calcified tissues and the corresponding encrustations were studied by spectroscopic techniques such as RAMAN, FT-RAMAN, FT-IR as well as imaging techniques such as optical microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Moreover, for the first time a comparison of the ability of three different RAMAN setups (FT-RAMAN: 1064 nm, Dispersion RAMAN: 633 nm and Dispersion RAMAN:325 nm) to detect calcification on the tissues was performed. The clinical sample comprised of 63 menisci with histopathologicaly confirmed alterations that were surgically extracted from osteoarthritic patients due to total knee arthroplasty in the University Hospital of Patras. Samples were classified according to the histopathological evaluation and a correlation of histopathological with mechanical or spectroscopic findings was attempted. Dynamic mechanical analysis results showed small but no statistically significant difference between the group of calcified and the group of mucoid and cystic degenerated tissues (as determined after histopathological examination) X-ray diffraction analysis of the menisci encrustations revealed highly crystallized depositions of triclinic and monoclinic Calcium Pyrophosphate Dihydrate (CPPD) and some samples with poorly crystallized apatitic depositions (HAP). Microscopic techniques (optical, SEM and AFM) confirmed the presence of CPPD and HAP and in addition revealed information regarding their growth mechanism.The results of spectroscopical analysis using RAMAN – FT-RAMAN and FT-IR showed also the characteristic peaks of the inorganic phase (CPPD or HAP) presented in/on the tissue. CPPD encrustations were detected in 82.6 % (19 of 23) of the calcified samples while apatite in 17.4 % (4 of 23). Moreover, it is notable that all RAMAN setups showed the characteristic peaks of CPPD and HAP regardless of the excitation source and the strong fluorescence band.

Le Syndrome d’Ehlers-Danlos vasculaire (SEDv) est une pathologie génétique rare à transmission autosomique dominante. Les patients sont prédisposés à la survenue de ruptures vasculaires, digestives et utérines induisant une létalité précoce. Cette pathologie est due à des mutations du gène COL3A1 (codant le collagène de type III), majoritairement de type faux-sens, agissant via un mécanisme dominant négatif. Plus rarement, de larges délétions ou des mutations non-sens induisent une haplo-insuffisance. In fine, les conséquences de ces mutations portent essentiellement sur la synthèse du collagène de type III, aboutissant à une fragilité artérielle importante. A ce jour, aucun traitement curatif n’est disponible. L’inactivation du gène Col3a1 chez la souris entraine une mortalité in utero et néonatale quasi-systématique en cas d’homozygotie, mais les souris Col3a1+/- sont exemptes de phénotype vasculaire évident. Afin d’obtenir un modèle de dissection artérielle liée à un défaut de collagène de type III, une perfusion chronique d’Angiotensine II (Ang II) à dose pressive a été utilisée. Nos résultats montrent que l’haplo-insuffisance en collagène de type III confère une sensibilité artérielle majeure à l’Ang II. Cette fragilité est caractérisée par le développement de dissections/ruptures de l’aorte ascendante et induit des décès prématurés de ces animaux. Ce phénotype est lié non seulement à l’élévation de pression artérielle mais aussi à l’activation des voies de signalisation de l’Ang II. Enfin, nous montrons qu’un traitement associant un bêtabloquant (propranolol) et un vasodilatateur artériel (hydralazine) permet de réduire la mortalité induite par l’Ang II. Ces résultats suggèrent l’intérêt de l’ajout d’un traitement préventif par inhibiteur de l’Ang II au traitement bêtabloquant (Celiprolol) recommandé dans la pathologie humaine.Parallèlement, nous avons généré un modèle murin knock-in génétiquement plus proche des patients SEDv, par l’introduction d’une mutation ponctuelle faux-sens (Gly183Ser) observée chez un patient. L’analyse préliminaire de ce modèle montre que les souris Col3a1+/G183S décèdent spontanément, dès 4 semaines, de ruptures/dissections de l’aorte ascendante. Cependant, ces souris ne présentent de modification ni de leurs paramètres hémodynamiques, ni de leurs diamètres aortiques. En revanche, environ 20 % des souris Col3a1+/G183S présentent des plaies au niveau du dos et des pattes. Ce nouveau modèle de souris est actuellement le seul à récapituler aussi fidèlement le phénotype des patients SEDv. Il devrait donc permettre de tester différentes approches thérapeutiques. / Vascular Ehlers-Danlos (vEDS) syndrome is a rare, inherited, autosomal dominant disease that results from mutations in the COL3A1 gene, encoding type III collagen. Patients are mostly affected by missense mutations probably acting through a dominant negative mechanism. A few patients present large deletions or nonsense mutations leading to a haploinsufficient mechanism. These mutations are supposed to lead to a defect in the synthesis and secretion of collagen type III, resulting in arterial wall fragility. Consequently, vEDS is mostly characterized by ruptures/dissections in arteries at a young age, which ultimately lead to premature death. While there is currently no surgical or therapeutic treatments available, a recent study reported the beneficial effect of the beta-blocker celiprolol, which prevents vascular complications in patients.To investigate the vascular phenotype of vEDS, a mouse model of this disease has been generated by the complete and ubiquitous inactivation of the COL3A1 gene. Col3a1-/- mice exhibit severe perinatal mortality and die prematurely from spontaneous vascular rupture. However, Col3a1+/- mice are viable and exhibit no obvious vascular phenotype. To determine the susceptibility of Col3a1+/- mice to develop vascular rupture/dissection, an experimental model of aneurysm induction was used, through the chronic infusion of Angiotensin II (Ang II). Our results showed that Ang II infusion led to severe premature mortality in Col3a1+/- compared to wild type. This fragility was characterized by the development of rupture/dissection in the ascending aorta. These lesions could be caused by the elevation of blood pressure and/or the activation of Ang II signaling pathways. We showed that treatment with a beta-blocker (propranolol) and an arterial vasodilator (hydralazine) reduced the mortality induced by Ang II in Col3a1+/- mice. These results suggest the beneficial effect of adding a preventive treatment inhibitor of Ang II to the beta-blocker treatment recommended in human pathology.Meanwhile, given that a majority of human vEDS cases is caused by missense mutations in the COL3A1 gene, we established a knock-in mouse model bearing a point mutation (Gly183Ser) found in vEDS patients. The preliminary characterization of this model showed that Col3a1+/G183S mice die spontaneously as early as 4 weeks of age from a dissection or rupture of the ascending aorta. However, these mice do not showed any changes of their hemodynamic parameters or aortic diameter. Furthermore, about 20 % of mouse Col3a1+/G183S display wounds in the back and legs. This new mouse model is currently the only that mimic more closely the human disease and could therefore be used to test different therapeutic strategies.