Alan B. Retik

Tissue-engineered autologous bladders for patients needing cystoplasty

BACKGROUND Patients with end-stage bladder disease can be treated with cystoplasty using gastrointestinal segments. The presence of such segments in the urinary tract has been associated with many complications. We explored an alternative approach using autologous engineered bladder tissues for reconstruction. METHODS Seven patients with myelomeningocele, aged 4-19 years, with high-pressure or poorly compliant bladders, were identified as candidates for cystoplasty. A bladder biopsy was obtained from each patient. Urothelial and muscle cells were grown in culture, and seeded on a biodegradable bladder-shaped scaffold made of collagen, or a composite of collagen and polyglycolic acid. About 7 weeks after the biopsy, the autologous engineered bladder constructs were used for reconstruction and implanted either with or without an omental wrap. Serial urodynamics, cystograms, ultrasounds, bladder biopsies, and serum analyses were done. RESULTS Follow-up range was 22-61 months (mean 46 months). Post-operatively, the mean bladder leak point pressure decrease at capacity, and the volume and compliance increase was greatest in the composite engineered bladders with an omental wrap (56%, 1.58-fold, and 2.79-fold, respectively). Bowel function returned promptly after surgery. No metabolic consequences were noted, urinary calculi did not form, mucus production was normal, and renal function was preserved. The engineered bladder biopsies showed an adequate structural architecture and phenotype. CONCLUSIONS Engineered bladder tissues, created with autologous cells seeded on collagen-polyglycolic acid scaffolds, and wrapped in omentum after implantation, can be used in patients who need cystoplasty.

Endoscopic treatment of vesicoureteral reflux with a chondrocyte-alginate suspension

Injection of polytetrafluoroethylene (Teflon) or collagen has been used in the endoscopic treatment of vesicoureteral reflux. Although the principle of an endoscopic treatment is valid, there are concerns regarding the long-term safety and effectiveness of these substances. In search of a different injectable material we conducted experiments using chondrocytes in a biodegradable polymer solution for the treatment of vesicoureteral reflux in an animal model. Reflux was created in 4 mini-pigs and confirmed with a cystogram. Cartilage was obtained from the auricular surface of each animal. Chondrocytes were harvested and expanded in vitro. The cells were individually quantitated and concentrated to 40 million cells per cc. The cell suspensions were mixed with a sodium alginate and calcium sulfate solution. Each pig was injected unilaterally in the subureteral region with the autologous chondrocyte suspension. The opposite ureter served as an internal control in all animals. Cystograms showed resolution of reflux in the treated side and persistence of reflux in the opposite untreated side in each instance. Excretory urograms revealed no evidence of obstruction. Histological examination of the subureteral region demonstrated cartilage. Autologous chondrocytes can be readily harvested, expanded in vitro and injected cystoscopically. The cells survive and form a cartilage nidus that is nonantigenic. This system is able to correct reflux without any evidence of obstruction.

Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vitro

The use of autologous urothelium would be advantageous for urothelial replacement in many genitourinary reconstructive procedures. Urothelial tissue grafts might be created using isolated populations of transitional epithelium or tissue in concert with an appropriate synthetic substrate. We describe the results of experiments designed to determine the feasibility of using biodegradable polymers as delivery vehicles for the creation of new urothelial structures in vivo from dissociated cells. Primary cultures enriched in uroepithelial cells were obtained from New Zealand white rabbits using a new technique of cell harvest. Cells were seeded onto nonwoven meshes of polyglycolic acid polymers in culture and, after 1 to 4 days in vitro, the cell-polymer scaffolds were implanted into the mesentery, omentum or retroperitoneum of athymic mice. Polymers implanted without cells served as controls. Animals were sacrificed at 5, 10, 20 and 30 days after implantation and 75 implants were examined histologically. Ten days after implantation isolated single cell layers were seen lining the polymer fibers. At 20 and 30 days polymer degradation was evident and urothelial cells lined the polymer in continuous layers of 1 to 3-cell thickness. Anticytokeratin western blots demonstrated the presence of a urothelium-associated cytokeratin in cell-polymer implants recovered after 30 days. These results demonstrate that urothelial cells can be successfully harvested, survive in culture and attach to artificial biodegradable polymers. The urothelial-polymer scaffolds can be implanted into host animals and the implanted cells can achieve spatial orientation as the polymer undergoes biodegradation. These findings suggest that it may be possible to use autologous urothelium, reconfigured on a synthetic substrate, in reconstructive procedures involving the ureter, bladder and urethra.

Pediatric Robot Assisted Laparoscopic Dismembered Pyeloplasty: Comparison With a Cohort of Open Surgery

PURPOSE We assessed the usefulness of RALP in children and compared an age matched cohort undergoing OPN to RALP for safety, efficacy, operative time, blood loss, in-hospital narcotic use and LOS. MATERIALS AND METHODS We performed a retrospective case-control study from 2000 to 2004 of 33 patients undergoing RALP and 33 undergoing OPN. Average age of each group was not significantly different (RALP 7.8 years vs OPN 7.6 years, p = 0.75). Mean followup of RALP and OPN groups was 10 and 21 months, respectively. RESULTS Etiology of the obstruction was not significantly different. Mean operative time was significantly less for OPN (181 minutes vs 219 minutes for RALP, p = 0.031). As RALP experience increased, operative times improved and approached the OPN experience. RALP complications included 1 patient requiring reoperative surgery vs no complications in the OPN group (p = 0.15). Patients undergoing RALP had a mean LOS of 2.3 days compared to 3.5 days for OPN (p <0.001). Total narcotic requirements were significantly less in the RALP group (p = 0.001). All patients in the OPN and 31 in the RALP group had either resolution of hydronephrosis, improvement in drainage or relief of symptoms. CONCLUSIONS We documented the safety and efficacy of RALP in children. RALP showed advantages of decreased hospital stay, decreased narcotic use and operative times approaching those of open surgery. RALP is an option for pyeloplasty, and as robotic technology improves, this method of repair may become the minimally invasive treatment of choice.

Implantation in Vivo and Retrieval of Artificial Structures Consisting of Rabbit and Human Urothelium and Human Bladder Muscle

The harvest of human bladder muscle and urothelial cells with subsequent growth may be useful for tissue replacement in genitourinary reconstruction. We previously reported the development of a system for the harvest, delivery and growth of rabbit urothelium in vivo using biodegradable polymers. We have now expanded and adapted this system for the harvest and in vivo implantation of human bladder urothelial and muscle cells. Synthetic polymer fibers of polyglycolic acid can serve as a scaffold and a delivery vehicle for the implantation of rabbit uroepithelial cells into athymic host animals. The polymers, which slowly degrade in vivo, allow the urothelial cells to survive at the implant site. We demonstrate that polyglycolic acid polymers support the proliferation of rabbit urothelial cells in situ and can serve as a maleable substrate for the creation of new urological structures that replace the degrading polymer fibers. We also show that human urothelial cells and bladder muscle cells, when implanted on polyglycolic acid fibers, from new urological structures in vivo composed of both cell types. The human cell-polymer xenografts can be recovered from host animals at extended times after implantation. These data suggest that feasibility of using polyglycolic acid polymers as substrates for the creation of human urothelial and muscle grafts for genitourinary reconstruction.

Injectable Alginate Seeded with Chondrocytes as a Potential Treatment for Vesicoureteral Reflux

Injection of polytetrafluoroethylene (Teflon) or collagen has been used in the endoscopic treatment of vesicoureteral reflux. Although the principle of an endoscopic treatment is valid, there are concerns regarding the long-term safety and effectiveness of these substances. The goal of several investigators has been to find alternate implant materials that would be safe for human use. Toward this goal we conducted a study to determine the effect of chondrocytes using a biodegradable polymer solution as a template. Hyaline cartilage was obtained from the articular surfaces of calf shoulders and chondrocytes were harvested. Chondrocyte suspensions were concentrated to 20, 30 and 40 x 10(6) cells per cc and mixed with dry alginate powder (a biodegradable polymer) to form a gel. Twelve athymic mice were injected subcutaneously with a chondrocyte-alginate solution. Each mouse had 4 injection sites, consisting of control, 10, 15 and 20 x 10(6) chondrocyte cells (48 injection sites). Mice were sacrificed at 2, 4, 6 and 12 weeks after injection. Histological examination of the injection sites demonstrated evidence of cartilage formation in 34 of the 36 experimental injection sites. Gross examination of the injection sites with increasing time showed that the polymer gels were progressively replaced by cartilage. The ultimate size of the cartilage formed was related to the initial chondrocyte concentration injected, and appeared to be uniform and stable within each category. There was no evidence of cartilage formation in the 12 controls. Histological analyses of distant organs showed no evidence of cartilage or alginate gel migration, or granuloma formation. In conclusion, chondrocyte-alginate gel suspensions are injectable, appear to be nonmigratory and are able to conserve their volume. In addition, the use of autologous cartilage cells would preclude an immunological reaction. These preliminary studies indicate that autologous cartilage-polymer gel solutions may be potentially useful in the endoscopic treatment of reflux.

Phenotypic and Cytogenetic Characterization of Human Bladder Urothelia Expanded in Vitro

A simple method for the harvest of bladder cell types from surgical specimens was used to generate strains of normal human urothelial cells that could be reproducibly cultivated, passaged and extensively expanded in serum-free medium. Immunostaining of the bladder epithelial cells with broadly reacting anti-cytokeratin antibodies and with an anti-cytokeratin antibody specific to cytokeratin 7, a transitional cell marker, indicated that they expressed a stable epithelial phenotype with serial passage. Low levels of immunostaining for E-cadherin and low levels of E-cadherin messenger ribonucleic acid, as determined by Northern blot analysis, and strongly positive immunostaining with an anti-vimentin antibody indicated collectively that the uroepithelial cells express a nonbarrier-forming phenotype under these culture conditions. However, when the urothelial cells were implanted subcutaneously into athymic mice on biodegradable synthetic polymers, they formed multilayered structures, suggesting that they retain the capability to differentiate in a living host. The urothelial cells proliferated in an epidermal growth factor independent manner and expressed high levels of transforming growth factor-alpha and amphiregulin messanger ribonucleic acids, suggesting the possibility of autocrine regulation of growth by epidermal growth factor-like factors. Cytogenetic analysis indicated that urothelial cells cultured for 6 passages possessed a normal chromosomal complement. These results demonstrate that primary cultures of autologous human bladder epithelial cells can be extensively expanded in vitro and, consequently, might be used in cell transplantation strategies for genitourinary reconstruction.

URETHRAL STRICTURE REPAIR WITH AN OFF-THE-SHELF COLLAGEN MATRIX

ABSTRACTPurpose: In select patients with urethral strictures in whom genital skin is insufficient alternative tissues are needed for urethral reconstruction. We explored the feasibility of using a bladder submucosa collagen based inert matrix as a free graft substitute for urethral stricture repair.Materials and Methods: A total of 28 patients 22 to 61 years old with a diagnosis of urethral stricture underwent reconstructive surgery using a collagen based inert matrix for urethral repair. The inert collagen matrix was trimmed to size as needed for each patient and the neourethra was created by anastomosing the matrix in an onlay fashion to the urethral plate with continuous 6-zero absorbable sutures. The size of the created neourethra ranged from 1.5 to 16 cm. A voiding history, physical examination, retrograde urethrography, uroflowmetry and cystoscopic examinations were performed preoperatively and postoperatively. Random urethral biopsies were also performed.Results: After a 36 to 48-month followup (me...

Pediatric Laparoscopic Dismembered Pyeloplasty

We performed laparoscopic dismembered pyeloplasty in a boy with right ureteropelvic junction obstruction using 4 cannula sites, and a dismembering and reanastomosis technique identical to that used in open pyeloplasty. Interrupted sutures were placed and tied intracorporeally. A nephrostomy tube was placed under direct vision for drainage but no ureteral stent was used. Total operating time was 5 hours. The patient was discharged home 36 hours after the procedure. The nephrostomy tube was removed 10 days postoperatively after radiographic demonstration of patency and 24 hours of clamping without pain. Followup excretory urography at 6 weeks showed much less hydronephrosis and a widely patent anastomosis. Our case illustrates the technical features and feasibility of laparoscopic pyeloplasty in children, and should encourage further development of pediatric urological reconstructive laparoscopic techniques.

A NOVEL INERT COLLAGEN MATRIX FOR HYPOSPADIAS REPAIR

PURPOSE In select patients with hypospadias in whom genital skin is insufficient alternative tissues are needed for urethral reconstruction. Although skin and mucosal grafts may be used, they may increase hospitalization and morbidity. We explored the feasibility of using a bladder submucosal, collagen based inert matrix as a free graft substitute for urethral repair. MATERIALS AND METHODS Four patients with a history of hypospadias underwent repeat hypospadias repair using a collagen based inert matrix for urethral reconstruction. The inert collagen matrix was trimmed to size as needed for each patient. The neourethra was created by anastomosing the matrix in an onlay fashion to the urethral plate with continuous 6-zero polyglactin sutures. The created neourethra size ranged from 5 to 15 cm. RESULTS After a 22-month followup 3 of the 4 patients had a successful outcome in regard to cosmesis and function. One patient in whom a 15 cm. neourethra was created had a subglanular fistula. CONCLUSIONS The use of a collagen inert matrix appears to be beneficial in patients who have undergone previous hypospadias repair and who may lack sufficient genital skin for reconstruction.