Earl Y. Cheng

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

PURPOSEnSmall 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.nnnMATERIALS AND METHODSnPrimary 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.nnnRESULTSnProgressive 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.nnnCONCLUSIONSnSmall 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.

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.

Snodgrass hypospadias repair with vascularized dartos flap: the perfect repair for virgin cases of hypospadias?

PURPOSEnSince its introduction, the Snodgrass hypospadias repair has been applied to virtually all forms of hypospadias repair. However, fistula rates have still been reported to be as high as 5% from large center, multiple surgeon studies and 16% from smaller center studies. We report on the use of the Snodgrass repair in conjunction with routine use of a vascularized dartos flap and 2-layer closure of the neourethra from multiple institutions.nnnMATERIALS AND METHODSnRecords of patients who underwent a primary 1-stage hypospadias repair with the Snodgrass technique in conjunction with vascularized dartos flap coverage were reviewed. Nearly identical surgical technique was used by all 6 surgeons in each case, which included a 2-layer closure of the neourethra, preservation of the well vascularized periurethral tissue and routine use of vascularized dartos flap coverage. A total of 514 cases were identified, including 414 with distal and 100 with midshaft or proximal hypospadias. Stents were used in 292 of the 514 repairs.nnnRESULTSnOf the 414 distal cases there were no fistulas and 1 case of meatal stenosis. Of the 100 proximal cases there were 3 fistulas and 1 case of meatal stenosis. The overall complication rate was less than 1% for all cases combined.nnnCONCLUSIONSnThis series represents the largest reported multi-institutional experience with the Snodgrass technique. When used in conjunction with vascularized dartos flap coverage, 2-layer closure of the neourethra and special attention to preservation of the periurethral vascular supply, this repair can be performed with a near 0 complication rate. We believe that this is the optimal repair for routine cases of hypospadias.

Urologic tissue engineering with small-intestinal submucosa: potential clinical applications

Abstract Small-intestinal submucosa (SIS) is a unique biomaterial that has been shown to induce tissue-specific regeneration in numerous organ systems. In the urinary tract, animal studies have demonstrated that SIS promotes functional bladder regeneration. Other preliminary studies have suggested that SIS may also be extremely useful for several other types of urologic surgery application where new tissue is needed or reinforcement of native structures is desired. This article reviews past and current work with SIS in the urinary tract and focuses on applications that will likely have future clinical utility.

MULTI-INSTITUTIONAL STUDY OF TESTICULAR, MICROLITHIASIS IN CHILDHOOD: A BENIGN OR PREMALIGNANT CONDITION?

PURPOSEnTesticular microlithiasis, a rare ultrasonographic diagnosis in children, has been shown to coexist in benign and malignant conditions. The natural history of incidentally discovered testicular microlithiasis has not been well defined in the pediatric population. The concern that testicular microlithiasis may be a premalignant condition has been raised. Reports indicate as much as a 45% association of germ cell tumors with testicular microlithiasis at the time of tumor diagnosis and there have been 4 cases of interval testicular tumor development associated with preexisting testicular microlithiasis. To address this issue we performed a multi-institutional study to evaluate children with incidentally diagnosed testicular microlithiasis.nnnMATERIALS AND METHODSnData on 26 patients with a mean age of 12.3 years at presentation with incidentally discovered testicular microlithiasis were collected from 7 institutions. Presenting scrotal conditions were reviewed. Two children with a previous testicular malignancy were excluded from study.nnnRESULTSnFollowup ranged from 1 month to 7 years (mean 27.6 months). Testicular biopsy and tumor marker (alpha-fetoprotein and beta-human chorionic gonadotropin) determinations were performed in 9 and 15 patients, respectively. To date no testicular tumor has developed during clinical followup.nnnCONCLUSIONSnOur multi-institutional study has not yet shown a trend toward the malignant degeneration of incidentally diagnosed testicular microlithiasis in children. However, we still advocate close surveillance of patients with testicular microlithiasis, such as yearly testicular ultrasound, physical examination, and judicious tumor marker determinations. We propose that a registry be started to follow prospectively patients with testicular microlithiasis to define its significance better.

CHARACTERIZATION OF CULTURED BLADDER SMOOTH MUSCLE CELLS: ASSESSMENT OF IN VITRO CONTRACTILITY

PURPOSEnThe contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle.nnnMATERIALS AND METHODSnHuman and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments.nnnRESULTSnHuman SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore.nnnCONCLUSIONSnIn vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.

The use of small intestinal submucosa as an off-the-shelf urethral sling material for pediatric urinary incontinence

PURPOSEnUse of autologous rectus fascia for urethral slings in the pediatric population has produced reliable and predictable results. However, the potential morbidity and complications associated with harvesting the autologous rectus fascia have driven efforts to find a reliable off-the-shelf material for urethral slings. Small intestinal submucosa is a collagen based material that has been shown to promote tissue specific regeneration in a variety of organs. We report the clinical experience at 4 institutions with small intestinal submucosa for urethral slings.nnnMATERIALS AND METHODSnA total of 20 patients 3 to 18 years old (mean age 8.7) received urethral slings using the commercially available form of small intestinal submucosa (STRATASIS, Cook Urologic Spencer, Indiana) via a sling suspension procedure from a suprapubic approach.nnnRESULTSnThe material was consistently uniform to work with and user-friendly. All 20 patients tolerated the procedure well with no intraoperative complications. Postoperative followup has ranged from 9 to 26 months (mean 13), and 14 (70%) patients are completely dry (85% in females and 43% in males). Of the 14 dry patients 13 are on intermittent catheterization and 1 female with epispadias voids spontaneously.nnnCONCLUSIONSnThis report is the largest and longest followup series using small intestinal submucosa as an off the shelf urethral sling material in children. These continence rates are equal to autologous fascia without additional morbidity of graft harvest.

Reversed seromuscular flaps in the urinary tract in dogs

Reversed seromuscular flaps of ileum and standard bowel replacement procedures were performed in 16 dogs to evaluate their potential to decrease the likelihood of recognized complications in cases of standard bowel replacement. Of 12 dogs augmentation cystoplasty was done in 6 and ureteral replacement was done in 6. In each group 4 procedures were performed using reversed seromuscular flap, while the remaining 2 were done in the conventional manner (controls). All flap animals had partial to full re-epithelialization with transitional cells but they also had gross and microscopic evidence of flap contraction at the end of 6 months. In the flap augmentation group intravesical pressure measured preoperatively at bladder volumes of 30 cc and 60 cc averaged 25.8 and 45.8 mm. Hg compared to sacrifice pressures of 56.7 and 80.8 mm. Hg. Monthly serum blood urea nitrogen measurements were lower in reversed seromuscular flap animals compared to controls. An additional 4 dogs were studied to help elucidate the etiology of graft contraction, of which 2 underwent reversed seromuscular flap enterocystoplasty with no mucosal stripping while 2 had a procedure exposing intact intestinal serosa to the lumen of the bladder and urine. All of these animals demonstrated good re-epithelialization of the serosal surface with transitional cells as well as little or no evidence of flap fibrosis or contraction. Our results demonstrate that the use of reversed seromuscular flaps in the urinary tract in dogs results in good re-epithelialization of the serosal surface with transitional cells but also flap contraction. This fibrosis and scarring process is largely due to the trauma of mucosal stripping and not urine contact.

Use of Small Intestinal Submucosa for Corporal Body Grafting in Cases of Severe Penile Curvature

PURPOSEnSmall intestinal submucosa is a unique biomaterial that has been found to promote tissue specific regeneration in the urinary tract. We present our experimental and clinical experience with small intestinal submucosa (SurgiSis, Cook Biotech, Spencer, Indiana) for pediatric corporal body reconstruction.nnnMATERIAL AND METHODSnA total of 20 Fischer rats underwent implantation of a 7 x 3 mm. small intestinal submucosa graft following excision of an ellipse of tunica albuginea and 14 control animals underwent tunical excision with reimplantation of this autologous segment. The animals were euthanized, and the penis was sectioned and histologically studied at intervals of 1, 2, 4, 6, 16 and 24 weeks. In 15 pediatric patients small intestinal submucosa was used for corporal body grafting. The grafting procedure was performed along the ventral (hypospadias cases) or dorsal (epispadias cases) surface of the corporal bodies. The tunica albuginea was incised full thickness at the point of maximal curvature down to the cavernosal tissue and the defect was filled with a single layer of small intestinal submucosa.nnnRESULTSnMeasurements of the animal small intestinal submucosa grafts did not reveal significant graft contraction through 6 months. There was no graft expansion or ballooning after pharmacological induction of an artificial erection. Histologically, marked inflammation at 1 week precipitously decreased to a normal appearing tunica albuginea at 3 and 6 months. In all clinical cases small intestinal submucosa was found to be technically easy to handle. Mean followup is 14 months (range 5 to 26). All patients have a straight phallus as documented by observation of spontaneous erections or artificial erection at the time of stage 2 hypospadias repair. No complications occurred.nnnCONCLUSIONSnSmall intestinal submucosa demonstrates tissue specific regeneration properties in the rat and human tunica albuginea. It is an off-the-shelf material that is safe, technically easy to use and readily available.

Urine levels of transforming growth factor-beta 1 in children with ureteropelvic junction obstruction.

OBJECTIVESnTo determine if there are measurable quantities of transforming growth factor-beta 1 (TGF-beta 1) in the urine of children with either normal or pathologic conditions of the urinary tract, specifically vesicoureteral reflux (VUR) and ureteropelvic junction obstruction (UPJO). We also sought to determine if the urine TGF-beta level could distinguish between renal obstruction and no obstruction.nnnMETHODSnPreoperative bladder urine from consecutive patients undergoing pyeloplasty (UPJO group; n = 13), ureteral reimplantation (VUR group; n = 11), or circumcision/orchiopexy (control group; n = 19) as well as urine from the renal pelvis of the UPJO group was collected. The urine level of TGF-beta 1 was measured using a quantitative sandwich enzyme immunoassay technique.nnnRESULTSnUrine level of TGF-beta 1 was detected in each group: control (26.6 +/- 6.3 pg/mL), reflux (22.1 +/- 9.6), UPJO-pelvic urine (82.4 +/- 19.3), UPJO-bladder urine (31.2 +/- 8.2). The urine TGF-beta 1 concentration in pelvic urine in the UPJO group was significantly higher than that in bladder urine in children in the UPJO group (p = 0.03). TGF-beta 1 concentrations were similar from the bladder of children in all three study groups (p = NS).nnnCONCLUSIONSnUrine TGF-beta 1 is detectable in children with normal and pathologic urinary tracts. The level of this urine marker is elevated in the renal pelvis of children with UPJO compared to the level in the bladder of either obstructed or nonobstructed upper urinary tracts.