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dc.contributor.advisorMeiring, S. M.
dc.contributor.advisorSmit, F.
dc.contributor.authorKhemisi, Mmakgabu Martha
dc.date.accessioned2015-10-28T14:26:00Z
dc.date.available2015-10-28T14:26:00Z
dc.date.issued2015-02
dc.identifier.urihttp://hdl.handle.net/11660/1478
dc.description.abstractEnglish: The use of decellularized biological scaffolds for the reconstruction of small-diameter (< 6 mm) vascular grafts has been the focus of tissue engineering studies. These biological materials constructed through decellularization processes are thought to be ideal graft materials for replacement of diseased vessels. However thrombogenecity is a major cause of obstruction in these vascular constructs and result in early graft occlusion. Seeding of the decellularized vascular constructs with endothelial cells (ECs) is an attractive proposition as the endothelial layer incorporates many of the anti-thrombogenic properties of blood vessels. The aim of this study was to determine if we could successfully re-endothelialize decellularized baboon arteries, thereafter perfuse the newly engineered arteries with whole blood to investigate the possibility of thrombus formation. First, the histology of the decellularized baboon arteries were compared to normal arteries in order to assess the efficiency of the decellularization process. Collagen and elastin fibres were also stained to determine whether the remaining extracellular matrix scaffold was preserved after decellularization. Human umbilical vein endothelial cells (HUVECs) were then cultured under optimal conditions. The viability and proliferation rate of the cultured ECs were assessed using the MTT cell viability and proliferation assay. The cultured endothelial cells were then used to seed the luminal surfaces of decellularized baboon arteries. The confluent endothelial monolayer of the seeded decellularized arteries were then assessed using scanning electron microscopy (SEM) after 7 days. The seeded cells were then detached from the graft surfaces of small sections and tested for viability (metabolic activity and proliferation) using the MTT assay again. Afterwards, normal, decellularized and seeded decellularized arteries were perfused for 2 hours with baboon whole blood collected in 3.2% sodium citrate tubes. Thereafter, small artery sections were examined with SEM to determine whether thrombi was formed on the luminal surfaces of all arteries. Histology examinations showed that the decellularized arteries were not completely cell free. Nuclear and cellular remnants were still retained within the scaffold materials. Histology also revealed that the femoral arteries had retained more nuclear and cellular materials than the carotid and radial arteries which showed much less nuclear material remaining. However, the ECM of the decellularized arteries was preserved after the decllularization processes. Enough viable ECs were obtained in culture to seed three decellularized baboon arteries. After 7 days post seeding, a confluent endothelial monolayer was observed on the luminal surfaces of the decellularized scaffolds using SEM. The perfused normal artery and the seeded decellularized arteries showed no possible thrombus formation on their luminal surfaces. The decellularized arteries however showed wide-spread platelet adhesion and activation on the surface of the ECM. In conclusion, decellularization of arteries was not successfully and proved to be dependent on the thickness of the tissues used. However, the decellularization process produced morphologically preserved extracellular matrix. The endothelialization process was successful since the endothelialization of decellularized vascular grafts does prevent thrombus formation on artery surfaces after perfusion with whole blood, while a decellularized scaffold does promote thrombus formation.en_ZA
dc.description.abstractAfrikaans: Die gebruik van gedeselluleerde biologiese stutte om klein-deursnee (<6mm) vaskulêre oorplantings op te bou, is die hooffokus van weefsel-ingenieurswese. Hierdie biologiese materiale wat deur desellulering opgebou word, word as die ideale oorplantingsmateriale beskou om beskadigde weefsels te vervang. Maar trombusvorming is ‘n groot oorsaak van die verstopping van hierdie vaskulêre stutte. Deur die gedeselluleerde vaskulêre stut met endoteelselle te besaai, is ‘n aantreklike opsie omdat die endoteellaag teen-trombogeniese eienskappe van bloedvate bevat. Die doel van hierdie studie was om vas te stel of ons gedeselluleerde bobbejaanarteries weer kan her-endoteliseer. Daarna het ons die nuwe opgeboude arteries met heelbloed geperfuseer om the bepaal of trombi daarop vorm. Eers het ons die histologie van die gedeselluleerde bobbejaanarteries vergelyk met dié van normale arteries om vas te stel of die deselluleringsproses suksesvol vas. Ons het kollageen- en elastienvesels ook gekleur om te bepaal of die ekstrasellulêre matriks stut behoue gebly het na die deselluleringsproses. Menslike naelstring endoteelselle (HUVEC) is onder optimale kondisies gekultuur. Die oorlewings- en vermeerderingstempo van die selle in kutuur is bepaal met die MTT-seloorlewingstoets. Die kultuurselle is dan gebruik om die luminale oppervlaktes van die gedeselluleerde bobbejaanarteries te besaai. Die aaneenlopende endoteel enkellaag wat op die besaaide gedeselluleerde arteries gevorm het, was dan bestudeer deur skandeerelektronmikroskopie (SEM) na 7 dae. Die besaaide selle is dan van die stut los gemaak en getoets vir oorlewing en vermeerdering deur weereens die MTT-toets te gebruik. Daarna is ‘n normale, gedeselluleerde en ‘n besaaide arterie met bobbejaan-heelbloed in 3.2% natriumsitraat vir 2 ure lank geperfuseer. Klein deeltjies van die arterie is dan d.m.v. SEM ondersoek om te bepaal of trombi op die luminale oppervlaktes gevorm het. Histologiese ondersoeke het aangetoon dat die gedeselluleerde arteries nie heeltemal van selle bevry was nie. Kerne en seloorblyfles was nog in die stutte sigbaar. Hierdie ondersoeke het ook aangedui dat die femorale arteries heelwat meer kerne en seloorblyfsels bevat. Nogtans het die ekstrasellulêre matriks (ECM) van die gedeselluleerde arteries behoue gebly na die deselluleringsproses. Genoegsaam oorlewende endoteelselle kon gekultuur word om die gedeselluleerde bobbejaanarteries te besaai. Na 7 dae van saaiing kon ons ‘n aaneenlopende monolaag van endoteelselle op die luminale oppervlakte van die gedeselluleerde stut deur middel van SEM sien. Die geperfuseerde normale en besaaide arteries het geen trombusvorming op hulle luminale oppervlaktes getoon nie. Die gedeselluleerde arteries het egter wydverspreide plaatjie klewing en aktivering op die oppervlak van die ECM getoon. Om af te sluit, die desellulering van arteries was nie suksesvol nie en blyk afhanklik van die dikte van weefsel te wees. Nogtans het die deselluleringsproses ‘n ECM geproduseer waarvan die morfologie behoue gebly het. Die endoteliseringsproses van die vaskulêre oorplantings het wel trombusforming op die arteriële oppervlakte verhoed na perfusie met heelbloed waar ‘n gedeselluleerde stut trombusforming aangehelp het.af
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectDecellularizationen_ZA
dc.subjectTissue engineeringen_ZA
dc.subjectExtracellular matrixen_ZA
dc.subjectRe-endothelialisationen_ZA
dc.subjectSeedingen_ZA
dc.subjectThrombogenecityen_ZA
dc.subjectEndothelial seedingen_ZA
dc.subjectVascular graftsen_ZA
dc.subjectBlood-vesselsen_ZA
dc.subjectArteriesen_ZA
dc.subjectMedicine, Experimentalen_ZA
dc.subjectBaboons as laboratory animalsen_ZA
dc.subjectDissertation (M.Med.Sc. (Haematology))--University of the Free State, 2015en_ZA
dc.titleThrombogenecity of decellularized and re-endothelialized baboon arteriesen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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