published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (M.S.)--University of Missouri--Columbia, 2004. / "July 2004." Typescript. Includes bibliographical references (leaves 63-70). Also issued on the Internet. Also available on the Internet.

Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Includes bibliographical references (leaves 63-70). Also issued on the Internet. Also available on the Internet.

L’utilisation de greffon costal en chirurgie reconstructrice est connue et utilisée depuis longtemps et notamment en chirurgie maxillo-faciale. La greffe de cartilage costal fait partie de l’arsenal thérapeutique pour le service de Traumatologie, Orthopédie, Chirurgie Plastique et Reconstructrice du CHU de Besançon. Proposée initialement dans le traitement chirurgical de la rhizarthrose, les opérateurs de ce service, se sont progressivement appropriés cette technique et l’on utilisée pour d’autres indications traumatiques ou dégénératives pour réaliser une restauration cartilagineuse. Ainsi depuis 15 ans, en dehors de son utilisation en routine pour la rhizarthrose, ce greffon a été proposé dans l’arthrose péri-scaphoïdienne ou radio-carpienne mais aussi pour quelques cas de reconstruction post-traumatiques d’articulations digitales, de cals vicieux de radius, dans la maladie de kienböck avancée ou lors d’une perte de substance ostéo-cartilagineuse d’une tête humérale. Dans un premier temps, ce travail rapporte les différentes indications thérapeutiques et résultats cliniques de cette technique originale qui ont fait l’objet de publications scientifiques et notamment dans l’arthrose radio-scaphoïdienne débutante et dans un cas de reconstruction de tête humérale.Cette technique chirurgicale impose pour le prélévement du greffon ostéo-chondral costal, l’abord du thorax au niveau de la 8e côte en général. Les complications pleuro-pulmonaires sont très rares. La technique de prélèvement est simple mais doit être « démystifiée ». Dans ce but, le deuxième temps de ce travail propose de donner les repères topographiques et anatomiques nécessaires au prélèvement de ce greffon. Une étude anatomique complétée par une étude en radio imagerie a permis de situer la 8e jonction ostéo chondrale costale entre les processus épineux de la 11e vertèbre thoracique et la première vertèbre lombaire mais le plus souvent au niveau du processus épineux de la 12e vertèbre thoracique et au 2/3 de l’hémi périmètre thoraco-abdominal en partant du sillon médian du dos, quel que soit la morphologie thoracique, l’âge et le sexe du patient. Cette 8e jonction est de plus toujours située sous un seul muscle, le muscle oblique externe et protégée par un épais périchondre qui la protège des pédicules de l’espace inter costal, du fascia endo thoracique et de la plèvre pariétale.En chirurgie du membre thoracique, les implants de pyrocarbone utilisés dans l’arthrose radio carpienne et la rhizarthrose, avec les mêmes principes que les greffes ostéo chondrales costales, ne semblent pas donner pour le moment de résultats satisfaisants. Les cultures de chondrocytes autologues ou de cellules stromales mésenchymateuses avec des produits déjà commercialisés posent le problème de leur matrice qui doit à la fois contenir les cellules greffées, des facteurs de croissance et se substituer à la perte de substance cartilagineuse le temps de l’intégration. Enfin, au vue des résultats intéressants apportés par cette technique de greffe ostéo chondrale costale en chirurgie orthopédique du membre thoracique, il est envisagé d’éventuelles greffes ostéo cartilagineuses pour restaurer des pertes de substances cartilagineuse au membre pelvien que ce soit en situation d’urgence ou pour des situations dégénératives comme l’ostéo chondrite disséquante de genou ou au niveau de la cheville. Des tests biomécaniques doivent être réalisés pour vérifier la capacité du greffon ostéo chondral costal à supporter une mise en charge sur une articulation portante. / The use of costal graft in reconstructive surgery is known and used for a long time in maxillo-facial surgery. It is the same practice for the service of traumatologic, orthopédic, plastic and reconstructive surgery of the university hospital of Besançon. Firstly proposed in the surgical treatment of trapezo-metacarpal osteoarthritis, the operators of this service gradually appropriated this technique and used it for other traumatic or degenerative indications to restore a cartilage defect. Thus, since 15 years, this surgical technique has been proposed in trapezo-metacarpal osteoarthritis, peri-scaphoid or radio-carpal osteoarthritis, for some cases of post-traumatic fingers joint reconstruction, for malunion of distal radius, for advanced Kienbock disease and defect of osteo-cartilaginous substance of a humeral head. In a first time, this work reports the different therapeutic indications and clinical results of this original technique which have been the subject of scientific publications, especially in radio-scaphoid osteoarthritis and in a case of humeral head reconstruction.To obtain the osteo-chondral costal graft, this technique needs the surgical approach of the thorax and generally of the 8e rib. Pleuro-pulmonary complications are very uncommun. This surgical technique is simple but must be "demystified". Therefore, the second phase of this work proposes giving topographical and anatomical landmark necessary to obtain this graft. An anatomical study supplemented by a radio imaging study helped to situate the 8e osteo-chondral rib junction between the epineus process of the 11e thoracic vertebra and the first lumbar vertebra but more often at the 12e thoracic vertebra. In the thoraco-abdominal hemi perimeter, this junction was locate at the 2/3 starting from the median furrow of the back, whatever the thoracic morphology, age and sex of the patient. This 8e junction is more always located under the obliquus externus abdominis muscle and coated by a thick perichondrium which protects it from the inter costal pedicle, from the endo thoracic fascia and the parietal pleura. In surgery of upper limb, the pyrocarbon implants, used actually in radio carpal osteoarthritis and trapezo metacarpal osteoarthritis, with the same principles as the osteo chondral costal graft, do not appear to give satisfactory results. Autologous transplantation of chondrocytes or mesenchymal stromal cells cultures have problem of their matrix which must contain the grafted cells, growth factors and have to substitute for the cartilaginous defect. Finally, due to this interesting results brought by this costal osteo chondral grafting technique in orthopaedic surgery of the upper limb, we considered eventual cartilaginous transplants to restore articular defect to the lower member following a injury or a degenerative lesion such as dissecans osteo chondritis of knee or ankle. Biomechanical tests should be realised to verify the ability of the costal osteo chondral graft to support a load on a bearing joint.

Cartilage repair and/or replacement is necessary for many orthopaedic conditions including fissures from blunt trauma, autograft or allograft transplantation, and replacement of focal defects with biological or synthetic constructs. In cartilage repair, initial integration between the host tissue and repair site is desirable to allow for nutrient transport, molecular deposition to enhance fixation, and eventual stress transmission across the interface. It has been postulated that effective transport and crosslinking of newly synthesized collagen molecules across a repair site may be vital to the process of integrative repair, and recent experiments have correlated collagen deposition with the strength of such repair. Other investigations have shown that enzymatic degradation of the cartilage surface may enhance integrative repair and can increase bond strength of an adhesive to cartilage. This study explored a novel approach involving photochemical bonding of cartilage tissue samples through collagen crosslinking as a means to achieve rapid and effective initial fixation, with the goal of enhancing biological integration. Photosensitized collagen gels were first analyzed via FTIR to determine the crosslinking effects with respect to collagen type and photochemical mechanism. Using the photogellation FTIR results as a parametric guide, in vitro mechanical testing of photochemically bonded meniscal fibrocartilage and hyaline articular cartilage tissues was performed using a modified single-lap shear test. Finally, the cellular viability and bond stability of a photochemically bonded cartilage interface was evaluated over seven days of in vitro culture, where the bond strength was assessed by pushout of cores from annular defects. Results of this study have demonstrated the potential of combining enzymatic surface modification with photodynamic techniques to directly bond cartilage tissues for initial fixation.

Articular cartilage provides a low-friction surface during normal joint motion and distributes forces to the underlying bone. The extracellular matrix (ECM) composition of healthy cartilage has previously been shown to be an excellent predictor of its mechanical properties. Changes in ECM composition and loss of mechanical function are known to occur with degenerative conditions such as osteoarthritis. Identifying differences in the composition-function relationships of cartilage under different anabolic, catabolic, and homeostatic conditions may thus be a useful approach for identifying factors (e.g. ECM content, distribution, and structure) which are critical to mechanical function. In addition, diagnostic tools capable of monitoring changes in the cartilage ECM may increase our understanding of the effects of ECM changes on composition-functions relationships. The goals of this work were to investigate composition-function relationships in healthy, degraded, and engineered cartilage and to develop a microcomputed tomography-based approach to analyze changes in matrix composition and morphology in articular cartilage. In healthy explants, compressive and shear properties were dependent on both sGAG and collagen content. In contrast, the compressive properties of IL-1stimulated cartilage were dependent on sGAG but not collagen content. To assess changes in sGAG content, EPIC-microcomputed tomography, a 3D contrast-enhanced microcomputed tomography technique was developed. EPIC-microcomputed tomography attenuation was found to be an excellent predictor of sGAG content in IL-1-stimulated cartilage explants and engineered cartilage. In addition, analytical approaches were developed to use EPIC-microcomputed tomography for the in situ analysis of cartilage morphology. EPIC-microcomputed tomography was also used to analyze spatial differences in sGAG accumulation in bilayer engineered cartilage for comparison with the local strain profile. This work underscores the significance of ECM composition and structure in regulating cartilage mechanical properties and validates the use of EPIC-microcomputed tomography as a diagnostic for monitoring sGAG content and potentially for assessing mechanical function in models of degeneration and regeneration.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on April 24, 2010). Includes bibliographical references.

The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy