Optimisation and characterisation of cholecyst-derived extracellular matrix for abdominal wall reconstruction

Abstract

Polypropylene mesh was created by Usher in 1962 after he experienced unsatisfactory results from using autografts and allografts for hernia repairs. More than fifty years later, polypropylene is still the most popular hernia mesh in use today. More recently, biologic meshes were introduced for their ability to integrate and be replaced with natural host tissue, and ability to resist infection. From a humble, and perhaps doubtful beginnings, biologic meshes are now accorded a category of their own to meet the needs of both routine as well as the very complex reconstruction. However, over-enthusiastic use of these biologic meshes has resulted in implant stretching, abdominal bulging and hernia recurrence. The work described in this thesis focuses on the development of a crosslinked extracellular matrix for abdominal wall repair, with the aim of eliminating unfavourable early biomaterial degradation in vivo. This project centred on the characterisation and optimisation of cholecyst-derived extracellular matrix (CEM), a novel biomaterial discovered by this group. The key hypothesis of this work was that a slowly degradable, crosslinked CEM with optimised degradation profile would improve tissue response and clinical outcome in hernia repair, in particular, reducing implant area stretching and bulging. In the first phase of this work, CEM decellularisation and its composition was established, followed by development of crosslinking strategies. CEM has a loose randomly arranged, mesh-like fibrous matrix network on SEM. Glutaraldehyde crosslinking of CEM resulted in reduced susceptibility to collagenase enzyme degradation in vitro. The degree of crosslinking was characterised for physiochemical, mechanical and degradation studies, in addition to cell culture studies. However, glutaraldehyde-crosslinked CEM was associated with very poor cell attachment and proliferation, due to the well-known toxicity effects of glutaraldehyde. In the second phase, an alternative crosslinker using the carbodiimide system (EDC/NHS) was chosen as the molecule does not incorporate itself within crosslinks (zerolength crosslinker), is water soluble and does not leave any cytotoxic residues in the substrate. The extent of crosslinking was controllable and an optimised method established. Furthermore, cell culture studies showed that cell viability was maintained and cells proliferated on the EDCxCEM crosslinked scaffolds with normal cell morphology. Subcutaneous implantation study in rats was carried out to evaluate in vivo degradation profile and host response of EDCxCEM. XXIII The third phase of this work was aimed to develop an improved in vivo model for testing surgical meshes for abdominal wall repair. Modifications to current existing models were made to detect implant area changes more objectively, which is either shrinkage or stretching, seen with synthetic meshes and non-crosslinked biological meshes, respectively. Three model implants were used to confirm the utility of our rabbit model. In parallel, a stereological approached was utilised for quantitative analysis of histological parameters of the implant area. Using this approach, statistical differences such as volume fractions of residual implant, nuclei and blood vessels were used to provide supportive quantitative data for comparison. In the final phase, a prototype EDCxCEM implant was compared with commercially available polypropylene, four-layer small intestinal submucosa and glutaraldehydecrosslinked bovine pericardium in vivo. Clinical parameters including seroma rate, adhesion formation and macroscopic changes to implant area were compared. Furthermore, host tissue response parameters were evaluated with stereological quantification. Crosslinked EDCxCEM showed promise due to its improved resistance to implant area stretching and more favourable tissue response. Thus, supplementary crosslinking augments the in vivo degradation profile and supports new host tissue formation and maturation, before critical degradation of the scaffold.