For patients with end-stage bladder disease for which other treatment options have failed, the patient is treated surgically with either urinary diversion or bladder augmentation. Enterocystoplasty is the most common of these options, and it involves augmenting the bladder using a section of the patient's own intestine. However, there are several problems associated with the use of intestine for augmenting the bladder, and therefore an alternative augmentation material may be of benefit to patients. Numerous approaches have been used to develop tissue-engineered scaffolds capable of successfully augmenting bladders. Some of these approaches have involved the use of acellular tissue-derived materials, whereby the tissues are decellularised in order to remove immunogenic material and therefore prevent an immune reaction when transplanted allogeneically or xenogeneically. Decellularisation protocols typically involve a variety of chemical and physical processes which remove cells and other immunogenic material from the tissues.
A protocol was previously developed to decellularise full-thickness porcine bladders. This material may have utility in bladder augmentation. The process involved distending the bladders with, and placing them in, a series of solutions. It was demonstrated that distending the whole organs was a necessary step in the decellularisation process. It was thought that this procedure applied biaxial strain to the wall of the tissue and reduced its thickness sufficiently for the solutions to penetrate the entire wall of the tissue. However, the method of distending whole bladders was not compatible with a scalable manufacturing process, and therefore the biomaterial was not able to be developed further. The overall aim of this project was to develop a novel method of manipulating bladder tissue which would enable bladder tissue to be decellularised in a way which would be compatible with a commercial manufacturing process.
The original bladder decellularisation process used 500 ml of solutions to distend bladders. Preliminary experiments demonstrated that this volume was not always adequate to decellularise larger bladders. Filling experiments were performed to find relationships between bladder size and bladder capacity. A relationship between bladder capacity and bladder width*length was found to have a high correlation. Bladders were decellularised when filled to capacities calculated using the relationship. No signs of cellular material were observed in histological sections of these bladders, and DNA quantification indicated a removal of more than 99% of the DNA relative to native tissue.
In order to determine the state of mechanical deformation of bladders during decellularisation, markers were placed on the surface of twelve bladders which were immersed in isotonic solution and slowly filled. Images taken of the bladders and markers during filling were used to calculate the strain of the tissues during the tests. The previously found relationships for bladder capacity were used to calculate the capacity of these bladders. The stress, strain and thickness of the bladders were calculated at the point the bladders were filled to their respective capacities. These strains were invariant with bladder capacity, and were equal to 2.0 and 1.4 in the circumferential and longitudinal directions respectively. Applying these strains to three bladders during decellularisation appeared to result in a complete removal of cellular material.
It was thought that applying the required strains to bladder tissue deformed in a flat sheet configuration would be compatible with a manufacturing process. In order to apply biaxial strain to this highly compliant material, it was recognised that it would be appropriate to deform it using discrete points, placed along the edges of the tissue. The stretching of flat sheets of bladder using this method was modelled using finite element modelling to find an optimal stretching regime. The models demonstrated that deforming the tissue using five discrete points along each edge of the material would be adequate to ensure that the required strains would be applied to the tissue for decellularisation to occur.
So that flat sheets of bladder could be decellularised, a piece of equipment was designed to hold pieces of bladder in the state of deformation which was previously modelled. The equipment took the form of a 3D-printed frame. A procedure was developed to stretch bladder tissue onto the frame. To test the hypothesis that bladder tissue could be decellularised in a flat sheet configuration, six bladders were stretched onto the frames and subject to the decellularisation process. Histological sections taken from decellularised bladder samples demonstrated a complete removal of cellular material, and a DNA extraction and quantification assay demonstrated that 99% of the DNA had been removed relative to the native controls.
Bladders decellularised using the original process were transported in transport medium and processed within 4 h of bladder collection. A manufacturing process would require the tissue to be stored before processing. It was also recognised that it may not be necessary to transport bladders---which are destined for decellularisation---in transport medium, which was developed in order to maintain viable urothelial cells. To test the effects of freezing and transportation without transport medium, bladders were collected from the abattoir, transported without transport medium and subject to either one (six bladders) or two (six bladders) freeze-thaw cycles. Twelve fresh bladders were transported with transport medium. Bladders were immersed in solution and mechanically tested by distension, and their stress--strain curves calculated. There was no statistical difference between the toe region modulus and the transition stress of fresh, once-frozen and twice frozen bladders. There was a small but significant increase in the linear region modulus and transition stress of fresh bladders compared to the once- and twice-frozen bladders. No significant differences were found between once-frozen and twice-frozen bladders. To determine the effect of this revised transportation regime on bladder decellularisation, six bladders were transported without transport medium, subject to two freeze-thaw cycles and subject to the decellularisation process. Samples taken from these bladders for histological analysis and DNA quantification exhibited a complete removal of cellular material.
In conclusion, this study demonstrated that applying suitable strains to flat sheets of bladder tissue was a viable method of deforming bladder tissue in order for it to be decellularised. Freezing the tissue up to two times before decellularisation resulted in some small but significant changes to the mechanical properties of the tissue, but did not affect the efficacy of the decellularisation process. It therefore may now be feasible to commercially produce decellularised full-thickness porcine bladder tissue.
