Urethroplasty performed with an autologous urothelium-vegetated collagen fleece to treat urethral stricture in the minipig model.

INTRODUCTION AND OBJECTIVE: Tissue-engineered materials in urethral reconstructive surgeries are a promising field for innovative therapy. Collagen matrices increase stability of cell-based implants and can promote viability and proliferation of urothelial cells. In this study, a collagen type I-based cell carrier (CCC) with stratified multi-layer autologous urothelium was used for urethroplasty after induction of urethral stricture in eight minipigs. MATERIALS AND METHODS: Minipigs underwent surgical procedures to induce urethral stricture by thermocoagulation. Simultaneously, bladder tissue was harvested. Urothelial cells were expanded, labeled with PKH26 and seeded onto CCC in high density. 3 weeks after strictures were induced and verified by urethrography, minipigs underwent urethroplasty using the seeded CCC. Two animals were euthanized after 1, 2, 4, and 24 weeks. Urethras were histologically examined for integration and survival of seeded CCC. In vivo phenotype of multi-layered urothelium matrix constructs was characterized via immunofluorescence staining with pancytokeratin, CK20, p63, E-cadherin and ZO-1. RESULTS: Seeded CCCs showed excellent stability and suturability after manipulation and application. Transplanted cells were detected using positive PKH26 fluorescence up to 6 months after labeling. Urothelium matrix implants integrated well into the host tissue without sign of inflammation. Animals showed no sign of rejection or stricture recurrence (urethrography) at any time during experimental period. Immunofluorescence analysis confirmed epithelial phenotype, junction formation and differentiation after 2 weeks. CONCLUSION: CCC can be suitable for urologic reconstructive surgeries and represents a promising option for clinical application. Longer follow-up results are required to exclude re-occurrence of stricture reformation.

Ureteral reconstruction using autologous tubular grafts for the management of ureteral strictures and defects: an experimental study.

OBJECTIVE: To investigate whether the peritoneal cavity could function as a bioreactor to produce autologous tubular grafts for ureteral reconstruction in beagles. MATERIALS AND METHODS: 8-Fr Silastic tubes were implanted into the peritoneal cavities of 6 female beagles. At 3 weeks, the tubes were harvested and the tubular tissue covering the tubes was gently everted. A segment 3 cm in length of the right mid-ureter, involving two thirds of its diameter, was removed parallel to the ureteral axis, leaving a third of the ureteral wall. A 5-Fr double-J stent was inserted into the ureter through the created defect, and two thirds of the graft were anastomosed to both edges of the ureteral defect. One third of the graft was overlapped with the retained normal ureter and anastomosed to the external surface of the lumens. Thus, the graft was partly encapsulated by the remainder of ureteral wall. The stent was maintained for 6 weeks and removed. Excretory urography was performed at 8 (n = 3) and 12 weeks (n = 3), postoperatively. Meanwhile, the neoureter was harvested and analyzed. The left ureter served as the control and a simple intubated ureterotomy was performed. RESULTS: Histological analysis of the tubular tissue demonstrated transversely arranged myofibroblasts and an outer layer of mesothelium. The tissue was easily everted and transplanted as a ureteral graft. Eight weeks postoperatively, the neoureter demonstrated normal ureteral architecture, composed of multilayers of urothelium surrounded by smooth muscle bundles, which became increasingly organized with time. Excretory urography indicated no stenosis or hydronephrosis. CONCLUSIONS: These results show that autologous tubular tissue grown within the recipients' peritoneal cavity can be used for ureteral reconstruction in the beagle model.

[Tissue equivalent for the closure of extended urethral defects].

The aim of the present work was to develop a method for culturing epidermal keratinocytes to be used in a tissue equivalent for the closure of extended urethral defects. The experiment was carried out using 15 rabbits. Skin biopsies were obtained from the inner surface of the ear. The tissue equivalent consisted of collagen gel with embedded fibroblasts and epidermal keratinocytes grown on its surface; lavsan-mesh endoprosthesis served as the framework. Prefabrication of the neourethral plate was performed on the superficial fascia of m. rectus abdominis. The neourethral tube was formed after engraftment which was complete in all 15 animals. A histological study revealed morphological similarity of the neourethral tube thus engineered and the normal urethra.

Endodermal origin of bladder trigone inferred from mesenchymal-epithelial interaction.

PURPOSE: In the classic view of bladder development the trigone originates from the mesoderm derived wolffian ducts while the remainder of the bladder originates from the endoderm derived urogenital sinus. Recent molecular developmental studies have questioned the veracity of this received wisdom, suggesting an endodermal origin for the trigone. To shed further light on this issue we observed mesenchymal-epithelial interactions between trigone epithelium and fetal urogenital sinus mesenchyma to infer the trigonal germ layer of origin. MATERIALS AND METHODS: Mouse trigone epithelium was recombined with fetal rat urogenital sinus mesenchyma in tissue recombinant grafts that were placed beneath the renal capsule of athymic mouse hosts. Grafts were harvested at 4 weeks. Control grafts with bladder dome and ureteral epithelium were also examined. Tissues were evaluated with hematoxylin and eosin, and Hoechst dye 33258 to confirm cell species origin. Immunohistochemistry was done with androgen receptor, broad spectrum uroplakin, dorsolateral prostate secretions and seminal vesicle secretions to differentiate prostatic and seminal vesicle differentiation. RESULTS: Grafts of mouse trigone epithelium with fetal rat urogenital sinus mesenchyma yielded epithelial tissue that stained for dorsolateral prostate secretions but not for seminal vesicle secretions. Control grafts of bladder dome epithelium yielded the expected endodermal prostate differentiation. Control grafts of ureteral epithelium yielded the expected mesodermal seminal vesicle differentiation. CONCLUSIONS: The consistent finding of prostatic epithelium in tissue recombinants of trigone epithelium and fetal urogenital sinus mesenchyma reinforces the hypothesis that the trigone is derived from the endoderm and not from the mesoderm, as commonly accepted.

Construction of ureteral grafts by seeding urothelial cells and bone marrow mesenchymal stem cells into polycaprolactone-lecithin electrospun fibers.

The aim of the present study was to investigated the construction of polycaprolactone-lecithin (PCL-L) electrospun fibers as a novel scaffold material for a tissue-engineered ureter. The effect of bone marrow mesenchymal stem cells (BM-MSCs) on the neovascularization of the scaffolds and the viability of planted urothelial cells (UCs) on PCL-L were also studied. UCs were obtained from New Zealand rabbit bladders, cultured and then seeded onto the lumen of the tubular scaffolds before being subcutaneously transplanted into the space of nude mice. The cultured UCs showed vacuolar degeneration after 7 days of transplantation and they gradually degraded thereafter. To facilitate the regeneration of the tissue-engineered ureter and the survival of UCs in the implant, MSCs were seeded into the tubular grafts by rolling up the nanofibrous membrane, followed by the seeding of UCs. This facilitated the survival of the UCs, which formed several cellular layers after 30 days. The mean microvessel density was significantly increased in tissues seeded with MSCs. Cell-tracking experiments revealed that the transplanted MSCs did not integrate directly into capillaries for angiogenesis. Our results demonstrated that the PCL-L electrospun fibrous scaffold has a high potential for a tissue-engineered ureter especially when seeded with BM-MSCs, which enhanced angiogenesis.

Efficacy of a Urinary Bladder Matrix for Treating Wound Dehiscence With Hardware Exposure in a Patient With Rheumatoid Arthritis.

OBJECTIVE: This case report explores an effective treatment modality in a medically complicated patient, with considerable wound dehiscence refractory to treatment with negative pressure wound therapy (NPWT). CASE REPORT: A 35-year-old woman with a past medical history of hypothyroidism, osteoporosis, and rheumatoid arthritis treated with tumor necrosis factor (TNF) alpha inhibitors and disease-modifying antirheumatic drugs presented to the clinic following right great toe arthrodesis, metatarsal neck osteotomies, extensor tendon lengthening, and capsulotomy of the second, third, fourth, and fifth toes 2 weeks prior, with wound dehiscence of the right great toe and subsequent exposure of surgical hardware, complicated by infection. At the 2-week postop, a urinary bladder matrix was placed on the wound following failed NPWT, which was in place for 10 days. At the 3-month follow-up, the wound was closed and without any drainage. Patient reported a significant reduction in pain (visual analogue scale: 3) with adherence to weight-bearing restrictions. CONCLUSIONS: Wound healing was accomplished without removal of the exposed deep hardware in a patient with comorbidities and post-surgical wound dehiscence.

Determinants of success and failure of seromuscular colocystoplasty lined with urothelium.

PURPOSE: Seromuscular colocystoplasty lined with urothelium is a method of bladder augmentation that avoids incorporating intestinal mucosa into the urinary tract. Others have reported a repeat augmentation rate of 23%. We analyzed the results in 20 patients who underwent the procedure, as performed by one of us (RG), at 3 institutions. MATERIALS AND METHODS: After receiving institutional review board approval we retrospectively reviewed the charts of all patients operated on since 1998. Preoperative and postoperative bladder capacity at 30 cm H(2)O, expressed as the percent of expected capacity for age using the equation, bladder capacity in ml = (age +1) x 30, as well as prior, concomitant and subsequent bladder or bladder neck procedures, continence and the need for repeat augmentation were recorded. RESULTS: There were 20 patients, including 7 females, with a mean age at surgery of 9 years and a mean followup of 53 months. All patients had neurogenic bladder dysfunction. An artificial urinary sphincter was implanted at the time of seromuscular colocystoplasty in 10 patients, preoperatively in 6 and postoperatively in 1. A sling was used in 3 females. Patients were divided into 2 groups. The 15 group 1 patients underwent no concomitant procedure in the bladder and the 5 in group 2 underwent creation of a continent channel at seromuscular colocystoplasty. There were no failures of augmentation in group 1, in which bladder capacity increased from 60% of that expected for age to 100%. All patients were continent. Three of the 5 patients in group 2 required repeat augmentation. CONCLUSIONS: Seromuscular colocystoplasty lined with urothelium has proved to be an effective method to augment the bladder in patients who have an artificial urinary sphincter or who undergo simultaneous artificial urinary sphincter implantation. We do not recommend constructing a continent catheterizable channel at the time of seromuscular colocystoplasty lined with urothelium.

Tissue engineering in urinary bladder: morphological and functional characterization.

INTRODUCTION: In the last years, tissue engineering has attracted lots of researchers, in urology too. This is due to the possibility to use this technique in several pathologies' therapies, which generally require reconstructive surgical solutions. Our work's aim is to evaluate morphological and functional aspects of cultivated urothelial and detrusorial tissues, both in "monolayer growth" and on scaffolds, in order to understand the chance of using them in reconstructive surgery. MATERIALS AND METHODS: Tissue cultures of detrusorial and urothelial cells have been obtained from animals (pigs, rabbits) and men. The urothelial nature of obtained cells has been demonstrated by traditional histological observation (Hematoxylin - Eosin), by immuno-fluorescence assay (specific for cyto-keratins antibodies), by immuno-histo-chemistry techniques (using specific cyto-keratins 7, 17, and 20 antibodies). Detrusorial tissue has been studied by using antibodies specific for alpha-actin. RESULTS: Urothelial and smooth muscle cells, when isolated and expanded in vitro, keep the typical characteristics of original tissue, as showed by classical histological observation (H-E), immuno-histo-chemistry and immuno-fluorescence assays. These results were positive both in monolayer colonies and on scaffolds. In vitro results are encouraging and they demonstrate that it is possible to obtain in vitro vesical tissue that could have analogous characteristics to the original organ; even though clinical utilisation of this technique must be more investigated, both in vitro and in vivo.

Intravesical Administration of Xenogeneic Porcine Urothelial Cells Attenuates Cyclophosphamide-Induced Cystitis in Mice.

The urothelium of the bladder, renal pelvis, ureter and urethra is maintained through the regulated proliferation and differentiation of urothelial stem and progenitor cells. These cells provide a rich source of a novel urothelial cell therapy approach that could be used to protect, regenerate, repair and restore a damaged urothelium. Urothelial injury caused by physical, chemical and microbial stress is the pathological basis of cystitis (bladder inflammation). The loss of urothelial integrity triggers a series of inflammatory events, resulting in pain and hematuria such as hemorrhage cystitis and interstitial cystitis. Here we investigate a novel cell therapy strategy to treat cystitis by protecting the urothelium from detrimental stresses through intravesically instilling porcine urothelial cells (PUCs) into the bladder. Using a chemical-induced urothelial injury mouse model of cyclophosphamide (CPP)-induced hemorrhagic cystitis, we determined how the intravesical instillation of PUCs could protect the urothelium from toxic attack from CPP metabolites. We show that intravesical PUC instillation protected the bladder from toxic chemical attack in mice receiving CPP with reduced inflammation and edema. Compared with the vehicle control mice, the proliferative response to chemical injury and apoptotic cells within the bladder tissues were reduced by intravesical PUC treatment. Furthermore, the urothelium integrity was maintained in the intravesical PUC-treated group. After xenogeneic PUCs were introduced and adhered to the mouse urothelium, immunological rejection responses were observed with increased neutrophil infiltration in the lamina propria and higher immune-related gene expression. Our findings provide an innovative and promising intravesical PUC cell therapy for cystitis with urothelial injury by protecting the urothelium from noxious agents.

Direct conversion of fibroblasts into urothelial cells that may be recruited to regenerating mucosa of injured urinary bladder.

Urothelial cells play essential roles in protection of urine exudation and bacterial invasion at the urothelial mucosa, so that defect or damage of urothelial cells associated with urinary tract diseases may cause serious problems. If a sufficient number of functional urothelial cells are prepared in culture and transplanted into the damaged urothelial lesions, such technology may provide beneficial effects to patients with diseases of the urinary tract. Here we found that human adult dermal fibroblasts were converted into urothelial cells by transducing genes for four transcription factors, FOXA1, TP63, MYCL and KLF4 (FTLK). The directly converted urothelial cells (dUCs) formed cobblestone-like colonies and expressed urothelium-specific markers. dUCs were successfully expanded and enriched after serial passages using a specific medium that we optimized for the cells. The passaged dUCs showed similar genome-wide gene expression profiles to normal urothelial cells and had a barrier function. The FTLK-transduced fibroblasts were also converted into urothelial cells in vivo and recruited to the regenerating urothelial tissue after they were transplanted into the bladder of mice with interstitial cystitis. Our technology may provide a promising solution for a number of patients with urinary tract disorders.

Tissue engineering of urethra using human vascular endothelial growth factor gene-modified bladder urothelial cells.

Acquired or congenital abnormalities may lead to urethral damage or loss, often requiring surgical reconstruction. Urethrocutaneous fistula and strictures are common complications, due to inadequate blood supply. Thus, adequate blood supply is a key factor for successful urethral tissue reconstruction. In this study, urethral grafts were prepared by seeding rabbit bladder urothelial cells (UCs) modified with human vascular endothelial growth factor (VEGF(165)) gene in the decellularized artery matrix. A retroviral pMSCV-VEGF(165)-GFP vector was cloned by insertion of VEGF open reading frame into the vector pMSCV-GFP (murine stem cell virus [MSCV]; green fluorescent protein [GFP]). Retrovirus was generated using package cell line 293T. Rabbit UCs were expanded ex vivo and modified with either MSCV-VEGF(165)-GFP or control MSCV-GFP retrovirus. Transduction efficiency was analyzed by fluorescence-activated cell sorting. The expression of VEGF(165) was examined by immunofluorescence, reverse transcript-polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA). Decellularized rabbit artery matrix was seeded with genetically modified UCs and was subsequently cultured for 1 week prior to subcutaneous implantation into nude mice. Four weeks after implantation, the implants were harvested and analyzed by fluorescence microscopy, and by histologic and immunohistochemical staining. Ex vivo transduction efficiency of UCs was greater than 50% when concentrated retrovirus was used. The modified cells expressed both VEGF and GFP protein. Furthermore, the VEGF-modified UCs secreted VEGF in a time-dependent manner. Scanning electron microscopy and histochemical analysis of cross sections of the cultured urethral grafts showed that the seeded cells were attached and proliferated on the luminal surface of the decellularized artery matrix. In the subcutaneously implanted vessels, VEGF-modified cells significantly enhanced neovascularization and the formation of a urethral layer compared to GFP-modified cells. These results indicate that VEGF gene therapy may be a suitable approach to increase the blood supply in tissue engineering for treatment of urethral damage or loss.

Tissue engineered venous matrices for potential applications in the urogenital tract.

Tissue engineering is lacking inexpensive, easily applicable techniques for tissue replacement. We investigated the potential use of native veins for tissue-engineering applications in the urological field. Forty-eight porcine veins, half seeded with urothelial cells and half unseeded, were kept in vitro for 7 days. Four seeded and four unseeded scaffolds were analyzed after 3 and 7 days. The remaining 32 veins were implanted subcutaneously into 16 athymic mice. Four athymic mice were sacrificed after 2, 4, 8, and 12 weeks. Histochemistry, immunohistochemistry (anti-pancytokeratin AE1/AE3, anti-desmin), western blot analyses (CD31), and scanning electron microscopy were performed in the retrieved specimens. The histochemistry of the seeded matrices showed the presence of urothelial cells in vitro and in vivo. After 12 weeks, a multilayer of urothelial cells was present in the hemotoxylin and eosin staining, positive for anti-pancytokeratin AE1/AE3. The western blot analyses showed vascularization of the veins in vivo. The results of scanning electron microscopy revealed a cellular layer on the veins. Native venous matrices may be used as tissue-engineered constructs for reconstructing the urinary tract. The clinical relevance of this approach must be proven in a large-animal model.

De novo reconstitution of a functional mammalian urinary bladder by tissue engineering.

Human organ replacement is limited by a donor shortage, problems with tissue compatibility, and rejection. Creation of an organ with autologous tissue would be advantageous. In this study, transplantable urinary bladder neo-organs were reproducibly created in vitro from urothelial and smooth muscle cells grown in culture from canine native bladder biopsies and seeded onto preformed bladder-shaped polymers. The native bladders were subsequently excised from canine donors and replaced with the tissue-engineered neo-organs. In functional evaluations for up to 11 months, the bladder neo-organs demonstrated a normal capacity to retain urine, normal elastic properties, and histologic architecture. This study demonstrates, for the first time, that successful reconstitution of an autonomous hollow organ is possible using tissue-engineering methods.

Urethral Reconstruction Using Mesothelial Cell-Seeded Autogenous Granulation Tissue Tube: An Experimental Study in Male Rabbits.

Objective. This study was to evaluate the utility of the compound graft for tubularized urethroplasty by seeding mesothelial cells onto autogenous granulation tissue. Methods. Silastic tubes were implanted subcutaneously in 18 male rabbits, of which nine underwent omentum biopsies simultaneously for in vitro expansion of mesothelial cells. The granulation tissue covering the tubes was harvested 2 weeks after operation. Mesothelial cells were seeded onto and cocultured with the tissue for 7 days. A pendulous urethral segment of 1.5 cm was totally excised. Urethroplasty was performed with mesothelial cell-seeded tissue tubes in an end-to-end fashion in nine rabbits and with unseeded grafts in others as controls. Serial urethrograms were performed at 1, 2, and 6 months postoperatively. Meanwhile, the neourethra was harvested and analyzed grossly and histologically. Results. Urethrograms showed cell-seeded grafts maintained wide at each time point, while strictures formation was found in unseeded grafts. Histologically, layers of urothelium surrounded by increasingly organized smooth muscles were observed in seeded grafts. In contrast, myofibroblasts accumulation and extensive scarring occurred in unseeded grafts. Conclusions. Mesothelial cell-seeded granulation tissue tube can be successfully used for tubularized urethroplasty in male rabbits.

Right Atrial Tumor Resection and Reconstruction with Use of an Acellular Porcine Bladder Membrane.

Malignant cardiac tumors typically have a grave prognosis; their resection with negative margins is optimal. We present the case of a 21-year-old woman in whom we surgically resected a primary cardiac sarcoma and reconstructed the right atrium with use of a porcine urinary bladder membrane-the MatriStem(®) Surgical Matrix PSMX. The patient recovered uneventfully. Six months postoperatively, the right atrial wall had retained its integrity. In addition to our patient's case, we discuss the benefits of using the MatriStem membrane in cardiac reconstruction.

Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments for bladder augmentation: in vivo, long term, and functional pilot study.

BACKGROUND: Bladder augmentation technique has changed over the years and the current practice has significant adverse health effects and long-term sequelae. Previously, we reported a novel cell transfer technology for covering demucosalized colonic segments with bladder urothelium and smooth muscle cells through an aerosol spraying of these cells and a fibrin glue mixture. OBJECTIVE: To determine the long-term durability and functional characteristics of demucosalized segments of colon repopulated with urothelial cells in the bladder of swine for use in augmentation cystoplasty. STUDY DESIGN: Nine swine were divided into three groups. The first group (control) underwent standard colocystoplasty; the second group underwent colocystoplasty with colonic demucosalization and aerosol application of fibrin glue and urothelial cell mixture; in the third group detrusor cells were added to the mixture described in group two. The animals were kept for 6 months. Absorptive and secretory function was assessed. Bladders were harvested for histological and immunohistochemical evaluation. RESULTS: All animals but one in the experimental groups showed confluent urothelial coverage of the colonic segment in the bladder without any evidence of fibrosis, inflammation, or regrowth of colonic epithelial cells. Ten percent of the instilled water in the bladder was absorbed within an hour in the control group, but none in experimental groups(p = 0.02). The total urine sediment and protein contents were higher in the control group compared with experimental groups (p < 0.05). DISCUSSION: Both study groups developed a uniform urothelial lining. Histologically, the group with smooth muscle had an added layer of submucosal smooth muscle. Six months after bladder augmentation the new lining was durable. We were also able to demonstrate that the reconstituted augmented segments secrete and absorb significantly less than the control colocystoplasty group. We used a non-validated simple method to evaluate permeability of the new urothelial lining to water. To determine if the aerosol transfer of bladder cells would have behaved differently in the neurogenic bladder population, this experiment should have been performed in animals with neuropathic bladders. CONCLUSION: Aerosol spraying of single cell suspension of urothelial and muscular cells with fibrin glue resulted in coverage of the demucosalized intestinal segment with a uniform urothelial layer. This new lining segment was durable without regrowth of colonic mucosa after 6 months. The new reconstituted segment absorbs and secretes significantly less than control colocystoplasty.

Capsule induction technique in a rat model for bladder wall replacement: an overview.

The search for a reliable technique for functional genitourinary tissue replacement remains a challenging task. The most recent advances in cell biology and tissue engineering have utilized various avascular and acellular collagen scaffolds with or without seeded cells. These techniques, however, are frequently complicated by tissue necrosis, contracture and resorption due to limited vascularization. We employed a new three-stage, evolving animal model with stage I optimizing the culture delivery vehicle, stage II employing a seeded vascularized capsule flap, and stage III adding a contractile matrix in the form of pedicled gracilis muscle prelaminated with autologous, in vitro-expanded urothelial cells to reconstruct an entire supratrigonal bladder-wall defect in rats.Specimens stained with hematoxylin and eosin (H&E), alpha(1)-actin staining, and a specific immunohistochemical staining (AE(1)&AE(3)-anticytoceratin monoclonal antibody stain) showed a continuous, multilayered, functioning urothelial lining along the transposed prelaminated gracilis flap in the animals of the final-stage experiment. Successful urinary reconstruction requires a contractile neoreservoir resistant to resorption over time and a stable, protective urothelial lining. We demonstrated that a gracilis muscle flap can be seeded with autologous cultured urothelial cells suspended in fibrin glue. This prelaminated flap can be safely transposed onto its pedicle and become successfully integrated into the remaining bladder wall, demonstrating urothelial lining and the potential to contract. Further studies in larger animals with urodynamic assessment is warranted to determine if this type of bladder-wall replacement technique is suitable for urinary reconstruction in humans.

Transplantable urothelial cell sheets harvested noninvasively from temperature-responsive culture surfaces by reducing temperature.

Augmentation cystoplasty using gastrointestinal flaps may induce severe complications such as lithiasis, urinary tract infection, and electrolyte imbalance. The use of viable, contiguous urothelial cell sheets cultured in vitro should enable us to avoid these complications. Transplantable urothelial cell sheets were obtained by utilizing a temperature-responsive cell culture method, and then examined by immunostaining and electron microscopy. Canine urothelium was produced on the surfaces of temperature-responsive culture dishes covalently bonded with the thermally sensitive polymer, poly(N-isopropylacrylamide). Stratified urothelial cell sheets were cultured and then harvested intact without enzymatic treatment from these dishes by reducing the temperature. Histological structure and cell-to-cell junctions were compared between these urothelial cell sheets and those harvested with dispase. All urothelial cell sheets were harvested from the bonded surfaces by reducing the culture temperature without the need for dispase. Electron microscopy revealed well-developed microridge, microvilli, and cell junction complexes. Conversely, these same cell features were destroyed by dispase treatment. Immunoblotting revealed that dispase fragmented occludin, whereas it remained unchanged in the intact urothelial cell sheets. Novel urothelial cell sheets obtained by culture on temperature-responsive culture surfaces were successfully harvested much less destructively than with dispase. This technology should prove useful in urinary tract tissue engineering in the near future.