"The effectiveness of exogenous cellular therapies has been limited by the ability to efficiently and locally deliver cells to a region of interest. We have developed biological sutures, formed from fibrin microthreads, to overcome these delivery issues and demonstrated increased cell engraftment compared to the current gold standard. However, the cell seeding efficiency onto the sutures is low and during implantation cells are subjected to shear forces as the sutures are pulled through the tissue. As a result, cells go unused after seeding and an uneven distribution of cells from the entry point to exit of the suture. By adding cell attachment and adhesion promoters and increasing culture time we proposed to overcome these issues. We have developed a shear loading method to evaluate the changes in cellular adhesion. Either poly-l-lysine or vitronectin was used to coat sutures. Uncoated control and coated sutures were then seeded with 100,000 human mesenchymal stem cells (hMSCs) for 24hrs or control sutures were seeded for 48hrs. An in vitro shear stress model was created by spinning seeded sutures with a centrifuge. Cell number per unit length prior to and post spinning were compared. To compare the effect of modifications on cell morphology cells were qualitatively assessed and nuclear alignment was evaluated as a robust measurement for overall cellular angle. Control sutures were found to have 6,821±739cells/cm prior to spinning, while sutures modified with poly-l-lysine resulted in 4,226±1,003cells/cm and vitronectin had 19,604±1,829cells/cm (p<0.05 vs. control and poly-l-lysine). 48hrs seeding resulted in a cell number to 4,417±2,266 cells/cm. Spinning resulted in relative decreases in cell number for control and coated sutures. Cells remained attached after sutures were spun after increased incubation time. Cells aligned along the long axis of individual microthreads; the alignment on control sutures was significantly different from all modifications. There was no difference in alignment between modifications, although they were significantly different compared to cells grown on topographically flat tissue culture plastic. These results demonstrated increased cell seeding efficiency and cell number for vitronectin coated biological sutures and increased cell adhesion following increased incubation time. The combination of these two modifications may lead to increased quantity and more evenly distributed cells delivered to diseased tissues by increasing initial cell number, increasing cell engraftment, and increased resistance to shear."

Thesis (M. D. S.)--University of Hong Kong, 2003. / Title proper from title frame. Also available in printed format.

Three samples of the biopolymer hyaluronic acid (HA) were characterized in the following manner: the molecular weights were obtained via multi-angle laser light scattering; the intrinsic viscosities were calculated through dilute solution viscometry, and the rheology of HA solutions was determined with constant rate rotational viscometry and dynamical mechanical testing. In addition, the highly debated role of hyaluronic acid in wound healing was examined by studying the effect that HA has upon thrombin-catalyzed fibrin clotting. Fibrin, in phosphate-buffered saline, was clotted both alone and after being incubated with HA. It was determined that the presence of hyaluronic acid resulted in a slower clotting process; in effect, HA acts as an anti-coagulant. Based upon the experimental evidence, it is proposed that this anti-coagulant phenomenon arises through a combination of two mechanisms: 1) specific binding between HA and fibrin, which acts to retard fibrin clotting through steric hindrance, and 2) the formation of an HA network which slows fibrin clotting by hindering free diffusion of fibrin and thrombin. Finally, creation of a synthetic replacement for synovial fluid was attempted using xanthan gum and locust bean gum in phosphate-buffered saline. The phenomenon of gum synergism was utilized in an effort to exert some degree of fine-tuning over the final rheological properties of the solution. This also would provide the side benefit of reducing the weight of gum required per unit volume. By mixing the solutions at different temperatures, it was possible to exploit the tendency of xanthan gum to uncoil at higher temperatures and therefore bind more strongly to locust bean gum. However, it was determined that no combination of gum concentrations and processing conditions resulted in a gum solution that adequately mimicked the rheology of a hyaluronan solution. / Graduation date: 2003

Thesis (Ph. D.)--University of Wisconsin, 1944. / Typescript. Includes bibliographical references.

No description available.

Abnormalities in the structure of fibrin fibers and blood platelets have been broadly studied and well correlated to functional abnormalities in the coagulation system due to disease, genetics or environmental factors. The importance of these and further ultrastructural investigations of fibrin fiber networks and platelets is therefore paramount in understanding iii the aetiology of haemorrhagic or thrombogenic tendencies and disorders. To study structural abnormalities and variations of fibrin fibers and platelets, electron microscopy is essential; unfortunately the use of electron microscopy necessitates several preparation steps to make a biological specimen stable enough to withstand the high vacuum environment of an electron microscope and also electron beam irradiation. The two most common procedures to accomplish this is chemical fixation and freeze fixation. Chemical fixation entails the chemical alteration of a specimen by means of the introduction of chemical bonds and cross-links that keep molecules and structures in place, followed by several rinsing and dehydration steps. Freeze fixation preserves biological specimens by the removal of thermal energy from the specimen at an extremely fast rate (> 105 In this study, methods to fixate fibrin fiber and platelet networks by freeze fixation was developed, optimised and subsequently compared to chemical fixation methods to ascertain the optimum preparation technique for transmission and scanning electron microscopy for ultrastructural studies of platelets and fibrin networks. K/s) allowing the water in a biological specimen to reach a super-cooled stabilized state (vitrification). The general consensus in the scientific community is that ultrastructural preservation by high pressure freeze fixation is superior to that of chemical fixation, although the facts are that different fixation methodologies have dissimilar chemical and physical interactions with different specimens and as a result different artefact introductions. Therefore the best possible specimen preparation method to ensure an accurate likeness of the fixated specimen to its in vivo condition needs to be ascertained and used. In this study, methods to fixate fibrin fiber and platelet networks by freeze fixation was developed, optimised and subsequently compared to chemical fixation methods to ascertain the optimum preparation technique for transmission and scanning electron microscopy for ultrastructural studies of platelets and fibrin networks. K/s) allowing the water in a biological specimen to reach a super-cooled stabilized state (vitrification). The general consensus in the scientific community is that ultrastructural preservation by high pressure freeze fixation is superior to that of chemical fixation, although the facts are that different fixation methodologies have dissimilar chemical and physical interactions with different specimens and as a result different artefact introductions. Therefore the best possible specimen preparation method to ensure an accurate likeness of the fixated specimen to its in vivo condition needs to be ascertained and used. Ultimately it was found that high pressure freezing coupled with freeze substitution is a superior method for fine structure preservation of fibrin fiber networks and platelets when utilizing transmission electron microscopy. Contrastingly for scanning electron microscopy ultrastructural studies it was found that chemical fixation is the more optimal method for the preparation of fibrin networks and platelets. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Anatomy / Unrestricted

The carboxy terminal of the γ-chain of human fibrinogen contains at least three biologically. important functional domains: (i) the fibrinogen γ-chain polymerisation centre, (ii) the platelet receptor domain and (iii) the site for staphyloccocal clumping. The nature of the site specificity of these interactions necessitates the existence of a preferred conformation for this region, the nature of which has yet to be clearly established. A novel zinc metalloproteinase isolated from puff adder venom (PAV protease) capable of specifically cleaving the di-γ-chain of transglutaminase (Factor XIIIa) catalysed crosslinked plasmin derived D-dimer into apparently symmetrical monomers has been described. The activity is fibrin specific and displays an unusual site specificity for the γ-carboxy terminal domains within the crosslink region. The activity was reported to be potentiated by zinc. The effect of zinc on the digestion of D-dimer by PAV protease was evaluated by SDS-PAGE and by a fluorimetric technique utilising a fluorescent dansylcadaverine conjugate of the substrate (f-D-dimer). A differential zinc binding study determined that the potentiation of activity by zinc was due to a zinc-substrate rather than a zinc-enzyme interaction. The binding constant for zinc to D-dimer was determined by Scatchard analysis of zinc titration data. The interaction of zinc and f-D-dimer was confirmed by fluorescence anisotropy determinations. The nature of the coordination capsule around the metal cation was determined by examining a cobalt-fibrin-D-dimer complex and characterising the difference visible absorption spectrum thereof. The donor ligands from the D-dimer fragment for the metal ion were determined as histidines by examining zinc(II) and cobalt(II) binding to diethylpyrocarbonate modified fibrin-D-dimer and hydroxylamine treated DEPC-fibrin-D-dimer. Through this study it has been established that the PAV protease cleavage of the di-γ-chain of the plasmin derived D-dimer fragment is potentiated by zinc(II) ions through the formation of a novel zinc determined conformation of fibrin-D-dimer. This presents the possibility of a fibrinspecific neo-epitope being manifested in the presence of zinc ions that could provide a means to determine fibrin degradation products more specifically. A model for the neo-epitope has been proposed.

"Recent evidence suggests that delivering human mesenchymal stem cells (hMSCs) to the infarcted heart reduces infarct size and improves ventricular performance. However, cell delivery systems have critical limitations such as inefficient cell retention and poor survival, and lack targeted localization. Our laboratories have recently developed a method to produce discrete fibrin microthreads that can be attached to a needle and delivered to a precise location within the heart wall. We hypothesize that fibrin microthreads will support hMSC proliferation, survival and retention of multipotency, and may therefore facilitate targeted hMSC delivery to injured tissues such as infarcted myocardium. To test this hypothesis, we bundled 100 μm diameter microthreads to provide grooves to encourage initial cell attachment. We seeded hMSCs onto the microthread bundles by applying 50,000 cells in 100 μL of media. The number of cells adhered to the microthreads was determined up to 5 days in culture. Cell density on the fibrin microthreads increased over time in culture, achieving an average density of 730 ± 101 cells/mm2. A LIVE/DEAD assay confirmed that the cells were viable and Ki-67 staining verified the increase in cell number over time was due to proliferation. Additionally, functional differentiation assays proved that the hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that delivering 1 to 4 cell seeded microthread bundles to the infarcted rat myocardium has the potential to produce a positive improvement in mechanical function and these microthreads support hMSC proliferation and survival. Additionally these findings suggest that cell-seeded microthreads may serve a platform technology to improve localized delivery of viable cells to infarcted myocardium to promote functional tissue regeneration. "

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the comunity health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109(DE3). / Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109(DE3).

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the comunity health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109(DE3). / Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109(DE3).