Abstract
Valve disease is on the rise. Despite its prevalence, current treatment options for valve replacement are limited: mechanical valves necessitate lifelong anticoagulation and bioprosthetic valves tend to degrade in as little as 10 years. The need exists for a valve replacement option that is haemodynamically stable and can withstand degradation.
Tissue engineering may hold the answer. This is a branch of science that aims to recreate functional human tissue. A tissue engineered solution needs three components for success: a matrix, a cell population, and a collection of signallers. This thesis will address the three components of the ‘tissue engineering triad’ as they relate to heart valves.
First, I generated a decellularised matrix from bovine pericardium using a detergent-enzyme protocol. I found that the combination of two detergents, sodium deoxycholate and Triton X100, followed by enzymatic digestion with a non-specific nuclease is able to reduce DNA content to below 50ng/mg—a level deemed acceptable by the literature—while preserving the tissue’s native microarchitecture.
Great care must be taken to ensure matrices are non-immunogenic, and so I have reviewed the current literature and developed a subcutaneous rat model in vivo immunogenicity assessment. I then use this model to investigate the immunogenicity of the decellularised matrix, and compared it to the ‘industry standard’ glutaraldehyde fixed pericardium. I saw that the graft avoided an immune-mediated rejection and calcification typical of glutaraldehyde fixed tissue. I also saw an improvement in macrophage polarity in the decellularised grafts compared to the industry standard.
Next, I developed an isolation protocol for valvular interstitial cells, and identify growth factors that nudge them towards a desirable phenotype. I found that fibroblast growth factor 2 can reverse the myofibroblastic activation common to diseased valves, and reduce the calcific potential to healthy levels. I found that nitric oxide had no effect.
Finally, the three strands are woven together: valvular interstitial cells are seeded onto decellularised grafts that have been modified with growth factors to aid their differentiation. I saw beneficial changes in gene expression in valve cells seeded onto the matrix. This modified graft is also assessed in the subcutaneous rat model where it outperformed its unmodified counterpart in remodelling-associated gene expression.