Characterisation of bovine pericardium and alternative treatments for its application as biomaterial

Abstract

Bovine pericardium is an extensively used biomaterial utilised in a wide range of biomedical devices such as bioprosthetic heart valves. The characterisation techniques employed for the analysis of bovine pericardium and similar natural soft tissue biomaterials, vary across both information sought, preparation of sample and the method itself. Natural biomaterials differ from traditional synthetic types in that additional steps are required during the production process to make them suitable for their application in vivo. These steps can include chemical fixation using glutaraldehyde to preserve stability, strength and prevent against enzymatic degradation. This thesis assessed the biomechanical, physical, chemical and cytotoxic methods available for the analysis of bovine pericardium. Biomechanical assessment of bovine pericardium is varied across the literature in both techniques and methods used. The thesis investigated uniaxial testing by focusing on two standard test parameters of strain rate and preconditioning number of cycles, to elucidate recommendations for the standardisation of a uniaxial method, while also measuring not so common parameters of low modulus and hysteresis. The results demonstrated that an extension rate of 10 mm/min and 5 preconditioning load-unload cycles as a reference point for the standardisation of a uniaxial testing method. Imaging analysis of the collagen structure post mechanical testing using scanning electron microscopy, displayed the crimped pattern present after the removal of the stress. This provided a quantitative assessment of bovine pericardium post mechanical testing, that can be applied to further the understanding of the behaviour of the tissue under stress. Glutaraldehyde is an extensively used sterilant and fixative for the crosslinking of natural soft tissue biomaterials like bovine pericardium. There is significant debate around the reaction mechanism of this crosslinker with natural biomaterials. This section explored the reaction mechanism using a derivative calorimetry technique, quasi-isothermal modulated differential scanning calorimetry, most commonly used for the analysis of polymorphic transformations in pharmaceuticals, to measure the rate of crosslinking between glutaraldehyde and bovine pericardium. The analysis showed that crosslinking reaction was completed after approximately 10 min and provided further evidence of the changing monomeric chemistry of glutaraldehyde. Additional characterisation of crosslinked bovine pericardium using Ninhydrin assay, proved to be a fast and convenient method to qualitatively demonstrate the crosslinked status of the tissue. Also, the structural assessment of the tissue using electron microscopy methods of scanning and transmission, and atomic force microscopy provided further insight into the directionality of collagen, ultrastructure analysis of cellular components and quantitative measurements of the D-banding pattern of collagen fibrils. The final phase of the thesis compared glutaraldehyde fixation with that of alternative treatments using genipin and decellularisation. A concentration of 3 mM of Genipin was recommended due to its increase in the thermal denaturation temperature (Td), producing mechanical properties similar to those of glutaraldehyde. The decellularisation protocol using sodium dodecyl sulfate produced a favourable cytotoxic evaluation compared to glutaraldehyde fixed bovine pericardium. While decellularisation used in combination with glutaraldehyde or genipin improved both its mechanical and cytotoxic properties. Together this data demonstrated that Genipin and the decellularisation protocol are viable alternatives for the treatment of bovine pericardium.