Currently, autologous gastrointestinal segments are utilized as the primary option for bladder reconstructive procedures despite their inherent morbidity and significant complication rate. Biomaterials derived from Bombyx mori silk fibroin represent attractive alternatives for bladder tissue engineering given their mechanical robustness, processing plasticity, and biodegradability. Our previous results have shown that silk scaffolds were capable of supporting tissue regeneration and voiding function in a murine model of bladder augmentation. In this study, we hypothesized that acellular silk matrices would effectively mediate tissue regeneration in a large animal model of bladder augmentation.
Scaffolds (6x6cm2) were generated from aqueous solutions of 6% silk fibroin by a previously reported solvent casting/sodium chloride-leaching process. Scanning electron microscopy (SEM) and tensile testing were performed to ascertain structural and mechanical properties of scaffolds prior to implantation. Matrices were anastomosed to the bladder dome of non-diseased Yorkshire pigs (N=2) either through open or robot-assisted augmentation cystoplasty and maintained for 3 months. Cystometric analysis was used to determine bladder capacity both pre-operatively and 3 months post-op. Following euthanasia, histological (H&E and Masson’s trichrome) and immunohistochemical (IHC) analyses of smooth muscle contractile protein expression (α-actin and SM22α) and urothelial-associated markers (cytokeratins and uroplakins) were assessed at the periphery and center of the original implantation site as well as a nonsurgical control region.
SEM characterization of silk matrices demonstrated the formation of a bi-layer structure consisting of an internal porous network (pore size ~300μm) buttressed on one side by a thin layer of amorphous silk (~50μm thick) which resulted in surface pore occlusion. Tensile testing of silk scaffolds revealed an average ultimate tensile strength of 430kPa, tensile modulus of 70kPa, and elongation to failure of 28%. Following 1 week of initial catheterization, animals were capable of voluntary voiding throughout the entire implantation period. Cystometric analyses of augmented bladders at 3 months post-op revealed substantial increases (>2-fold) in organ capacity in comparison to pre-operative values and weight-matched unoperated controls. Histological and IHC evaluations of both the periphery and central regions of the regenerated tissues demonstrated robust smooth muscle bundle formation displaying α-actin and SM22α expression as well as the presence of a multi-layered urothelium exhibiting both prominent uroplakin and cytokeratin positivity similar to control regions. In addition, substantial degradation of the silk matrix was noted with only discrete scaffold remnants present within the interior of the implantation site with no areas of fibrosis or stone formation observed.
Acellular bi-layer silk scaffolds represent an effective biomaterial system for mediating bladder tissue regeneration and functional outcomes in a large animal model and may offer advantages over conventional gastrointestinal segments and previously described cellularized biomaterials for augmentation cystoplasty.