Understanding roles of porcine small intestinal submucosa in urinary bladder regeneration: identification of variable regenerative characteristics of small intestinal submucosa

Abstract

Neuropathic bladders are the result from damages to the central or peripheral nervous system, and ultimately may require surgical reconstruction to increase bladder volumes and to reduce the risk of damages to the kidneys. Surgical reconstruction through bladder augmentation has traditionally been practiced using a segment of the ileum, colon, or stomach from the patient through enterocystoplasty. However, the use of gastrointestinal segments can lead to serious adverse consequences. Porcine small intestinal submucosa (SIS), a xenogeneic, acellular, biocompatable, biodegradable, and collagen-based bioscaffold is best known to encourage bladder regeneration without ex vivo cell seeding before implantation in various experimental and preclinical animal models. Although it has been demonstrated that SIS supports bladder cell growth in vitro, and SIS-regenerated bladders are histologically and functionally indistinguishable from normal functional tissues, clinical utilization of SIS for bladder augmentation has been hampered by inconsistent preclinical results. Several variables in SIS, such as the age of pigs, the region of the small intestine, and method of sterilization, can have different physical properties, biochemical characteristics, inflammatory cell infiltration, and regenerative capacity due to cellular responses in vitro and in vivo. These parameters are particularly important for bladder regeneration due to its specific biological function in urine storage. Clinical application of SIS for surgical bladder reconstruction may require graft materials to be prepared from a specific region of the small intestine, or to be further formulated or processed to provide uniform physical and biochemical properties for consistent, complete, and functional bladder regeneration.

Figures

FIG. 1.

Culture of human bladder cells on small intestinal submucosa (SIS). Human bladder cells were seeded on commercial SIS and cultured for a period of 7 days. (A) Masson's trichrome stained section of primary cultures of urothelial cells (brown) grown on the mucosal surface of SIS (blue) in vitro. (B) Masson's trichrome staining of primary cultures of human bladder smooth muscle cells (SMCs) (brown) grown on the mucosal side of SIS (blue). (C) Masson's trichrome staining of the sandwich technique: urothelial cells were grown on the mucosal side of SIS with bladder SMCs seeded on the opposite side (serosal side) of SIS. (D) Double immunohistochemical staining of cells grown in layered coculture on SIS: urothelial cells stain positive for cytokeratin marker AE1/AE3 (red) and grow in multiple layers with some early polarity on top of several layers of α-actin-positive SMCs (brown), which are beginning to penetrate the SIS. (E) Masson's trichrome staining of human microvascular endothelial cell (HMEC) (red) grown on the mucosal surface of SIS (blue) in cultures.

FIG. 2.

Histological evaluation of bladder regeneration in a rat bladder augmentation model. Immunohistochemical and Masson's trichrome staining of regenerating bladders was performed at day 56 postaugmentation (A). Detection of uroplakin-positive immunoreactivity specifically in superficial and deeper layers of urothelial cells. (B) Diffused and homogeneous ZO-1-positive immunoreactivity throughout the urothelium. (C) Development of smooth muscle bundles demonstrated by α-SMA-positive staining. (D) Capillary blood vessel formation shown by CD31-positive immunohistochemistry. (A) and (B) were adapted from Roth et al.

FIG. 3.

Dog bladder regeneration using e-beam sterilized and lyophilized SIS. Using the dog bladder augmentation model, regenerative bladders were harvested at 10 weeks following SIS augmentation. (A) Graft shrinkage and deep calcification (arrow) deposit on the surface using lyophilized SIS. (B) Graft shrinkage and long bone-like structure (arrow) using e-beam sterilized SIS. (C) Scar development and central calcification (arrow) augmented with lyophilized SIS. (D) Lack of muscle regeneration and transmural bone formation in the dog regenerative bladder augmented with lyophilized SIS.

FIG. 4.

Differential regenerative properties of SIS prepared from different segments of the small intestine. Bladder regeneration was followed in the dog bladder augmentation model following partial cystectomy. Bladders were harvested at 10 weeks following augmentation. Masson's trichrome staining was performed for histological evaluation. (A) Completed bladder regeneration was observed after augmentation distal SIS. (B) Histological presentations of urothelial and smooth muscle layers in distal SIS-augmented dog bladder. (C) Graft shrinkage and stone formation in bladder augmented with proximal SIS. (D) Histological features of proximal SIS-augmented bladder showing incomplete regeneration with lack of urothelium and smooth muscle development.

FIG. 5.

Structural and physical characteristics of SIS. (A) Under scanning electron microscopy, no significant differences were observed in the serosal sides of proximal and distal SIS, except in the distribution of fibers on the surface. The mucosal side of the SIS membranes showed less porous architecture relative to the corresponding serosal side. Relaxation behavior under different stages of ramp and hold tests for (B) SIS and (C) synthetic 50:50 poly D,L-lactic-co-glycolic acid membranes. Each stage was normalized to the origin by subtracting the values at the end of the previous stage. (B) and (C) were adapted from Mirani et al.

FIG. 6.

Differential inflammatory responses elicited by distal and proximal SIS. Using the rat bladder regeneration model, infiltrated inflammatory cells were distinguished by single-cell-type specific histological staining. (A–C) Proximal SIS recruits significantly higher numbers of eosinophils at early and late phases of bladder augmentation as compared to distal SIS. (D–F) Sustained recruitment of macrophages is observed in late phases of distal SIS-augmented bladder versus proximal SIS-augmented bladder (Fig. 6B). Arrows denote location where high-power inset images (×60) were taken. (A–F) were adapted from Ashley et al.