Bradley P. Kropp

Regenerative Urinary Bladder Augmentation Using Small Intestinal Submucosa: Urodynamic and Histopathologic Assessment in Long-term Canine Bladder augmentations

PURPOSE To evaluate small intestinal submucosa (SIS) as a possible bladder augmentation material. MATERIALS AND METHODS Nineteen male dogs underwent 35 to 45% partial cystectomy with immediate augmentation with SIS grafts. All dogs were evaluated pre- and postoperatively with blood chemistries, urine cultures, intravenous urograms, cystograms and cystometrograms. Postoperatively (1 to 15 months), bladders were examined with routine histology and image analysis. RESULTS All dogs survived their intended survival period without morbidity. All results were normal. Histologically, all 3 layers (mucosa, smooth muscle, serosa) of the normal bladder showed evidence of regeneration. CONCLUSIONS Small intestinal submucosa acts as a scaffold for bladder augmentation through regeneration and could be a potential option for bladder reconstruction.

Experimental assessment of small intestinal submucosa as a bladder wall substitute

OBJECTIVES This study determined the feasibility of promoting urinary bladder regeneration with porcine-derived small intestinal submucosa (SIS). METHODS Twenty-two Sprague-Dawley rats underwent partial cystectomy with immediate bladder augmentation with SIS. Bladders were harvested for histologic evaluation at 2, 4, 8, 12, 24, and 48 weeks. RESULTS Histologically at 2 weeks, there was infiltration of the graft material with viable host cells consisting of fibroblasts, macrophages, and blood vessels covered by complete mucosal urothelium comprised of transitional cells. During the next 10 weeks, collagen formation and maturation were noted, and by the end of 12 weeks, the SIS graft was comprised of a mature collagen matrix admixed with thinly scattered disorganized smooth muscle bundles and covered by normal urothelium. At 48 weeks, all three layers of the normal bladder (urothelium, smooth muscle, and serosa) were present and were grossly and microscopically indistinguishable from the normal rat urinary bladder. CONCLUSIONS This study further supports the concept of bladder regeneration and suggests that SIS may be a viable material for bladder augmentations.

Rabbit urethral regeneration using small intestinal submucosa onlay grafts

OBJECTIVES To determine if small intestinal submucosa (SIS) can evoke urethral regeneration. METHODS Twenty male white New Zealand rabbits were assigned to one of three experimental groups. Group 1 (n = 4) underwent simple urethrotomy and closure. Group 2 (n = 8), a second control group, underwent an onlay urethroplasty with a graft of full-thickness preputial skin from the host rabbit. Group 3 (n = 8) underwent an onlay urethroplasty with an SIS graft. RESULTS All eight SIS onlay grafts promoted regeneration of the normal rabbit epithelium supported by a well-vascularized collagen and smooth muscle backing. Preputial free onlay grafts maintained a keratinizing squamous cell epithelium with a poor supportive backing, which resulted in the formation of urethral diverticulum. CONCLUSIONS SIS onlay patch grafts for urethroplasty promote rabbit urethral regeneration.

Characterization of Small Intestinal Submucosa Regenerated Canine Detrusor: Assessment of Reinnervation, in Vitro Compliance and Contractility

PURPOSE We characterized small intestinal submucosa regenerated canine bladder. MATERIALS AND METHODS We subjected 15-month small intestinal submucosa regenerated canine bladder strips to in vitro muscle bath compliance, contractility testing and immunohistochemical staining. RESULTS Compliance studies demonstrated no significant difference between small intestinal submucosa regenerated and control bladders, which were 30-fold more compliant than native small intestinal submucosal graft material. Contractility studies demonstrated contractile responses and innervation similar to those of normal canine bladder. Afferent nerves were demonstrated through immunohistochemical techniques. CONCLUSIONS These characteristics further support the regenerative capacity of small intestinal submucosa and its potential use as a bladder augmentation material.

Detrusor Regeneration in the Rat Using Porcine Small Intestinal Submucosal Grafts: Functional Innervation and Receptor Expression

PURPOSE To evaluate functional characteristics of regenerated bladder induced by small intestinal submucosa (SIS). MATERIALS AND METHODS Strips from bladder regenerated from SIS and normal rat bladder were evaluated by in vitro muscle bath contractility studies. RESULTS The present results indicate that SIS-regenerated bladder 1) demonstrates contractile activity; 2) expresses muscarinic, purinergic and beta adrenergic receptors; and 3) exhibits functional cholinergic and purinergic innervation that is similar to the normal rat urinary bladder muscle. CONCLUSIONS These functional characteristics of SIS-regenerated tissue demonstrated in the present study further support use of SIS material as a bladder augmentation material.

COCULTURE OF BLADDER UROTHELIAL AND SMOOTH MUSCLE CELLS ON SMALL INTESTINAL SUBMUCOSA: POTENTIAL APPLICATIONS FOR TISSUE ENGINEERING TECHNOLOGY

PURPOSE Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.

Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystectomy model

To evaluate small intestinal submucosa (SIS), unseeded or seeded, as a possible augmentation material in a canine model of subtotal cystectomy.

Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder

To assess the potential use of bone marrow stromal cell (BMSC)‐seeded biodegradable scaffold for bladder regeneration in a canine model, by characterizing BMSCs and comparing them to bladder smooth muscle cells (SMCs) by immunohistochemistry, growth capability, and contractility.

Small-intestinal submucosa for bladder augmentation: a review of preclinical studies

Abstract The need to find an alternative to the use of bowel for urinary reconstruction has renewed research interests involving bladder regeneration. Historically, alloplastic and biodegradable materials have demonstrated bladder regeneration; however, high complication rates and unreliable regenerative results have prevented any of these materials from being used clinically. Small-intestinal submucosa (SIS) is an acellular, nonimmunogenic, biodegradable, xenogeneic, collagen-based material that is derived from the submucosa layer of porcine small intestine. SIS has demonstrated regenerative capacities in multiple organ systems, including the aorta, vena cava, ligaments, tendons, abdominal wall, and skin. SIS has also demonstrated long-term reliable regenerative results in the rat and canine bladder-augmentation models. This article reviews the preclinical studies involving the use of SIS for bladder augmentation.

Bladder Regeneration with Cell-Seeded Small Intestinal Submucosa

This study was performed to determine the regenerative properties of smooth muscle cells (SMCs) and urothelial cells (UCs) seeded on small intestinal submucosa (SIS), utilizing a nude mouse model. Human bladder SMCs and UCs were seeded on SIS in a layered coculture fashion. Cell-seeded SIS grafts (1 x 1 cm(2)) were maintained in a CO(2) incubator for 14 days and subsequently folded with the seeded cells facing the lumenal side and implanted subcutaneously into the flanks of nude mice (n = 20). Unseeded SIS grafts were implanted into the contralateral flanks of the mice to serve as controls. Grafts were harvested at 4, 8, and 12 weeks after implantation. By 12 weeks, layered urothelium with a central lumen was noted with early smooth muscle bundle formation peripherally. At each time point, the regenerated SMCs stained positive for alpha-smooth muscle actin, and the UCs stained positive for cytokeratin AE1/AE3. The control group demonstrated no evidence of organized bladder regeneration. This study demonstrates the potential for cell-seeded SIS to induce organized bladder regeneration in vivo. This also provides the basis for additional work utilizing seeded SIS grafts for bladder augmentation.