This is an original study to quantify and grade defect healing in a large animal cranial bone substitute model. The study of various therapies to heal cranial defects requires an appropriate ?critical? animal model. An experimental animal model should be analogous and recognizable as an appropriate challenge to human physiology. In addition, the defect must fail to heal unless treated with the tissue engineering therapy under study. Sheep as a large animal model was chosen because of its ability to tolerate creation of large skull defects analogous to clinical scenario, and its biology of healing as a high order mammal would be closer human beings. There is no agreement on the critical size limits for cranial defects. Various sizes have been termed "critical" in publications utilizing sheep. These ranged from 20-22mm. This study will investigate whether a 20mm defect is adequate. Bilateral circular cranial defects of 10, 20 and 25mm diameters were created in 12 adult sheep. Based on guided tissue engineering principles, defect protection was utilized to prevent in-growth of fibroblasts and other connective tissue cells from the surroundings. As bone tissue regeneration strategies usually involve osteoconduction element, an animal model that considered the defect protection role of osteoconduction would be more appropriate. Repopulation and regeneration of the defect was maximized as an added challenge Bioresorbable polylactic acid co-polymer mesh (MacroPoreTM) and Titanium mesh (TiMeshTM) was used as defect protection. The cranial defects were harvested at 8 and 16 weeks. The end-point analysis included Faxitron X-ray images, DEXA (Dual Energy X-ray Absorptiometry), and histology. The defects were graded to assess their ability to eventually heal. 10mm defects fully healed at 16 weeks. There was new bone formation spanning the entire defect seen on histology. 25mm defects were spanned by thin fibrous tissue only. There was variability in the healing potential of 20mm defect. Based on presence of bone islands within the defect, half of the 20mm defects demonstrated ability to heal while the other half actually had new bone spanning the defects on histology. Critical size cranial defect in sheep for the study of bone graft substitute has to be larger than 25mm diameter. The model is then utilized to study the use of Pro Osteon and AGF compared with the gold standard of autologous bone graft.

Thesis (M.Orth.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 137-147) Also available in print.

Thesis (M.Orth.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves [102]-122) Also available in print.

Choline is indispensable for a number of fundamental processes in the body. Besides being the precursor of the acetylcholine, an important neurotransmitter, choline is found in the cell membrane structure combining with fatty acids, phosphate and glycerol. Its deficiency may result in nervous system disorders, fatty acid build up in the liver, along with increased cholesterol levels, high blood pressure and memory loss. Thus, rapid detection methods are required for the determination of choline in biological fluids. In this study a choline oxidase biosensor was constructed for the determination of choline. During construction of the biosensor, glucose oxidase was used as a model enzyme, before choline oxidase used. The Teflon (PTFE) membrane of the oxygen electrode was grafted with 2-hydroxyethyl methacrylate (HEMA, 15%, v/v) in the presence of ferrous ammonium sulphate (FAS, 0.1%, w/v) by gamma irradiation and ethyleneglycol dimethacrylate (EGDMA, 0.15 %, v/v) was used as a crosslinker in a series of membranes. HEMA-grafted membranes were activated with epichlorohydrin or glutaraldehyde to maintain covalent immobilization of enzyme. The enzyme activity was measured with an oxygen electrode unit based on oxygen consumption upon substrate addition. Membranes were characterized in terms of grafting conditions and mechanical properties. Membranes, gamma irradiated in a solution of HEMA (15%) and FAS (0.1%) for 24 h, were found to be suitable for use in the further studies. Mechanical test results revealed that HEMA grafting made Teflon membrane more flexible and the presence of EGDMA made the grafted membrane stiffer. During optimization stage, it was found that the immobilized enzyme amount was not sufficient to obtain enzyme activity. Thus, the membrane preparation stage was modified to obtain thinner membranes. The immobilized glucose oxidase and choline oxidase contents on thin HEMA grafted membranes were determined by Bradford and Lowry methods. The influence of EGDMA presence and the epichlorohydrin activation duration on enzyme activity studies revealed that the membrane should be prepared in the absence of EGDMA and 30 min activation duration is appropriate for epichlorohydrin coupling. The study on the influence of membrane activation procedures revealed that the membranes activated with glutaraldehyde had a higher specific activity than the membranes activated with epichlorohydrin. Upon stretching membrane on the electrode directly rather than placing in the sample unit, the response of the enzyme immobilized sensor improved with high specific activity. The optimum choline oxidase concentration was found to be 2 mg/mL considering the effect of immobilization concentration on enzyme activity. With the choline oxidase biosensor, the linear working range was determined as 0.052-0.348 mM, with a 40 &plusmn / 5 &micro / M minimum detection limit. The response of the sensor decreased linearly upon successive measurements.

No description available.

published_or_final_version / abstract / toc / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

This thesis describes the preparation of surface-attached monolayer tethers from electroreduction of aryldiazonium ions using a protection-deprotection strategy. Monolayers of ethynylphenyl, carboxyphenyl, aminophenyl and aminomethylphenyl were prepared. Glassy carbon (GC) and pyrolysed photoresist film (PPF) surfaces were modified electrochemically and characterised by redox probe voltammetry. The monolayer tethers were coupled with electro-active ferrocenyl (Fc) and nitrophenyl (NP) groups for the indirect electrochemical estimation of the surface concentration. Film thickness measurement was carried out using an atomic force microscopy (AFM) depth profiling technique. The surface concentration and film thickness measurement results were consistent with the formation of monolayer films after removal of the protecting groups. Preparation of mixed monolayers was studied using three different modification strategies: i) grafting from a solution containing two different protected aryldiazonium ions, ii) sequential grafting of two different protected aryldiazonium ions, and iii) grafting of protected aryldiazonium ions followed by removal of the protecting group and reaction of an amine or carboxylic acid derivative directly with the GC surface. The composition of the mixed layer prepared using the first method is difficult to control, whereas the possibility of multilayer formation cannot be discounted using the second method. Multilayer formation is unlikely using the third method. The electrocatalysis of oxygen reduction at mixed monolayer films was investigated briefly. The origin of the two reduction peaks frequently observed for electroreduction of aryldiazonium ions at carbon surfaces was studied. Electroreduction was carried out at GC and HOPG surfaces. The reduction peak at the more positive potential is surface sensitive, while the peak at the more negative potential is not. However, both reduction peaks lead to deposition of films and it is tentatively proposed that the more positive peak corresponds to reduction at a ‘clean’ GC electrode, and the more negative peak corresponds to reduction at the already grafted layer.

This is an original study to quantify and grade defect healing in a large animal cranial bone substitute model. The study of various therapies to heal cranial defects requires an appropriate ?critical? animal model. An experimental animal model should be analogous and recognizable as an appropriate challenge to human physiology. In addition, the defect must fail to heal unless treated with the tissue engineering therapy under study. Sheep as a large animal model was chosen because of its ability to tolerate creation of large skull defects analogous to clinical scenario, and its biology of healing as a high order mammal would be closer human beings. There is no agreement on the critical size limits for cranial defects. Various sizes have been termed "critical" in publications utilizing sheep. These ranged from 20-22mm. This study will investigate whether a 20mm defect is adequate. Bilateral circular cranial defects of 10, 20 and 25mm diameters were created in 12 adult sheep. Based on guided tissue engineering principles, defect protection was utilized to prevent in-growth of fibroblasts and other connective tissue cells from the surroundings. As bone tissue regeneration strategies usually involve osteoconduction element, an animal model that considered the defect protection role of osteoconduction would be more appropriate. Repopulation and regeneration of the defect was maximized as an added challenge Bioresorbable polylactic acid co-polymer mesh (MacroPoreTM) and Titanium mesh (TiMeshTM) was used as defect protection. The cranial defects were harvested at 8 and 16 weeks. The end-point analysis included Faxitron X-ray images, DEXA (Dual Energy X-ray Absorptiometry), and histology. The defects were graded to assess their ability to eventually heal. 10mm defects fully healed at 16 weeks. There was new bone formation spanning the entire defect seen on histology. 25mm defects were spanned by thin fibrous tissue only. There was variability in the healing potential of 20mm defect. Based on presence of bone islands within the defect, half of the 20mm defects demonstrated ability to heal while the other half actually had new bone spanning the defects on histology. Critical size cranial defect in sheep for the study of bone graft substitute has to be larger than 25mm diameter. The model is then utilized to study the use of Pro Osteon and AGF compared with the gold standard of autologous bone graft.

Bibliography: leaves 151-206. / xxvi, 206 leaves, [8] plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the hypothesis that HIV infected bone allografts can be sterilized with 25 kGy of gamma irradiation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Orthopaedic Surgery & Trauma, 1998?