Bladder tissue regeneration using acellular bi-layer silk scaffolds in a large animal model of augmentation cystoplasty

Abstract

A cellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6×6cm2) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded non surgical control gains (144%) encountered due to animal growth. Similarly, elevations in bladder compliance were substantially higher in augmented animals from baseline (Group 1: 357%; Group 2: 147%) in comparison to controls (41%). Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,β-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair.

Human Urothelial Cells Isolation, In Vitro Expansion and Characterization for Evaluating Bio-Engineering Potentials

“…CK7, CK18, and CK19 are the markers expressed throughout all layers in normal urothelium [7] . Available literature towards urothelium basal layer characterization have shown that urothelium cells were expressing phenotypic marker CK5 and CK17, but they did not express phenotypic marker CK20, which is generally associated with differentiated superficial cells [7,8,9] . The urothelium or transitional epithelium of the urinary tract shows alterations in the expression and configuration of cytokeratin isotypes associated with stratification and differentiation.…”

“…Urothelial stem cells capable of regenerative potential were isolated from mice tissue, which was found to express sonic hedgehog and p63 proteins in the basal cell layers of the bladder urothelium [7,8] . A recent report has also shown p63 expression in porcine urothelial and human urothelial cells [9] .…”

Whyever bladder tissue engineering clinical applications still remain unusual even though many intriguing technological advances have been reached?

“…Though out of the true synthetic biomaterials field,"silk fibroin"made scaffolds, coated with ECM proteins, have been timely used, in animal model bladder tissue engineering, as templates for urothelial/smooth muscle cell seeding procedure, thus achieving a construct endowed with effective structural and functional proper such as particularly biodegradability, plasticity, compliance (42,43). In this regard, recent animal model studies have shown that the stretched electrospun silk fibroin matrix implantation can strongly promote bladder tissue regeneration with proper structure/function features, in comparison with BAM (44).…”

Silk scaffolds for musculoskeletal tissue engineering

“…To overcome the limitation, combined scaffolds have been developed. Combination fabrication is a method that fabricates scaffolds with two or more fabrication techniques, such as porous scaffold/ particles, 24 porous scaffold/membrane, 45 and hydrogel/ fibers. 46 Combination fabrication can better match the tissue regeneration requirement and broaden the use of SF materials.…”