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Title: Development of methods for assessment of the effects of sterilisation on acellular vascular grafts
Author: Milton-Barker, Jake Christopher
ISNI:       0000 0004 5915 9614
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2015
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Cardiovascular disease is the highest cause of morbidity worldwide, encompassing blood vessel disorders such as coronary artery and peripheral arterial disease. For small diameter applications (less than 6 mm) a tissue engineered commercial product is yet to be produced. Previously, a process for the production of acellular porcine carotid arteries was developed which showed excellent potential for clinical translation as vascular bypass grafts. Medical products, however, need to be sterilised to a sterility assurance level of 10-6 before they can be used clinically. The aim of this thesis was firstly to produce acellular vascular grafts from porcine carotid arteries using previously developed decellularisation technology. Secondly, to develop robust methods capable of evaluating the effects of different sterilisation methods on the biological and mechanical characteristics of acellular vascular grafts and finally to determine the compatibility of the main stream industrially available sterilisation processes (Gamma and E-Beam irradiation and ethylene oxide treatment) with acellular vascular grafts. Decellularisation of porcine carotid arteries was evaluated by determination of DNA content and histology. Biocompatibility was assessed using contact cytotoxicity. Acellular arteries were then subjected to 30 kGy (25 kGy min) E-Beam or Gamma irradiation or ethylene oxide treatment. The effects of sterilisation were determined using histology, immunohistochemistry, second harmonic generation multiphoton imaging, differential scanning calorimetry, denatured collagen content and determination of mechanical properties compared to non-sterilised acellular arteries. Mechanical properties were assessed using uniaxial tensile testing at a low strain rate to failure and burst pressure and compliance testing Histologically, the architecture of the arteries was retained post decellularisation and DNA content was reduced by greater than 95 %. The arteries were not cytotoxic. Stress strain mechanics were also retained but compliance testing showed a significant reduction. Post sterilisation with both Gamma irradiation and E-beam irradiation, there was a significantly increased stiffness of the elastin and collagen modulus of the acellular arteries. Ethylene oxide treatment significantly increased the elastin and collagen modulus. E-Beam irradiation and ethylene oxide treatment reduced compliance excessively. Histology showed the architecture of gamma and E-beam sterilised arteries to be consistent with acellular arteries and that ethylene oxide treated arteries exhibited some layer separation. Multiphoton imaging showed damage to all sterilised samples with Gamma sterilised acellular arteries the least affected. There were no significant differences in denaturation temperature. These results were in partial agreement with previously reported data on the effects of Gamma and E-beam sterilisation on tendons. Gamma, E-beam [30 kGy] irradiation and ethylene oxide treatment caused detrimental effects to the mechanical properties of acellular porcine carotid arteries. E-beam irradiation and ethylene oxide treatment caused severe reduction in the compliance of acellular porcine carotid arteries. Compliance mismatch is a known failure mechanism of vascular grafts, therefore E-Beam irradiation and ethylene oxide treatment were not compatible with acellular vascular grafts. Gamma irradiation caused the least damaging effects and is the most likely candidate of the three that could potentially be optimised for sterilising acellular vascular grafts.
Supervisor: Ingham, Eileen ; Fisher, John ; Stacy-Paul, Wilshaw Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available