Tissue engineering of genitourinary organs.
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Animals↗
Biomedical subjects
J J Yoo
Publications and source records attributed to J J Yoo.
Tissue engineered stents created from chondrocytes.
PURPOSE: Trauma, operations or instrumentation of the urethra or ureter may lead to stricture disease. The use of a natural urethral stent made of autologous tissue would be advantageous due to its biocompatibility. In this study we investigated the feasibility of engineering cartilage stents in vitro and in vivo. MATERIALS AND METHODS: We fabricated 40 cylinders 10 mm. long with an inner and outer diameter of 5 and 9 mm., respectively, from polyglycolic acid mesh coated with 50:50 polylactic-co-glycolic acid. Chondrocytes isolated from bovine shoulders were seeded onto the tubular polymer scaffolds at a seeding density of 60 x 106 cells per ml. Scanning electron microscopy was performed to determine the even distribution of chondrocytes throughout the polymer scaffolds. We implanted 20 cylinders under the skin of nude mice and 20 were cultured in stirred bio-reactors. Cytological characteristics, collagen content and mechanical durability were evaluated 4 and 10 weeks after cell seeding. RESULTS: Gross examination of the engineered stents showed the solid, glistening appearance of cartilaginous tissue. Cytological analyses with hematoxylin and eosin, trichrome, alcian blue and safranin O confirmed cartilage, and the deposition of collagen and glycosaminoglycan in each group. Increased deposition of collagen and glycosaminoglycan was observed in the stents created in vivo. Biomechanical testing demonstrated that the cartilaginous cylinders in each group were readily elastic and withstood high degrees of pressure. CONCLUSIONS: This study demonstrates the feasibility of creating cartilaginous stents in vitro and in vivo using chondrocyte seeded polymer matrices. This technology may be useful clinically for stricture disease in the genitourinary tract.
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Tissue-engineering applications for phallic reconstruction.
Pathologic penile conditions often require reconstructive surgery. Due to the limited amount of autologous tissues available for reconstruction, other tissue substitutes have been used. Phallic reconstruction using engineered autologous genital tissue, i.e., tissue derived from the patient's own cells, may be preferable. In this article we describe tissue-engineering approaches that may be applicable to genital reconstruction.
Biocompatible Materials↗
Experimental and clinical experience using tissue regeneration for urethral reconstruction.
Various urethral conditions often require additional tissue for reconstruction. Although several innovative tissues have been proposed for possible use as free grafts for urethral repair, all have specific advantages and disadvantages. The use of these tissues may be associated with additional procedures for graft retrieval, prolonged hospitalization, and donor-site morbidity. For these reasons, alternate materials have been sought for urethral repair. Our laboratory has developed an acellular collagen matrix that has shown adequate urothelial-cell epithelialization and urethral-tissue regeneration both experimentally and clinically. After a 3-year follow-up period, all patients who have had their urethras reconstructed with the acellular matrix are doing well, showing no clinical change from their immediate postoperative results. Other acellular materials may soon be tried clinically. Long-term studies need to be conducted before any of these materials can be accepted for routine use in urethral reconstructive procedures.
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Renal therapy using tissue-engineered constructs and gene delivery.
Currently available renal replacement therapies are not optimal for most patients. In addition to the inherent shortage of transplant organs, significant complications are associated with renal transplantation and immunosuppressive therapy. Dialysis neglects the resorptive, homeostatic, metabolic, and endocrinologic functions of the kidney and only partially replaces its filtration properties, resulting in morbidity and mortality. Application of tissue-engineering techniques may improve many aspects of renal function replacement. Identification of the growth factors capable of directing tissue development and of the technique to be used for their delivery would aid in the engineering of human tissue. The combination of tissue-engineering strategies with gene therapy might allow the transfection of diseased tissues with designated cDNA to eliminate inherent or acquired defects. Devices that have been targeted at replacing a single aspect of renal function, in addition to three-dimensional renal units that are capable of excreting urine-like solutes, have been used experimentally. Combination of these strategies may allow the formation of tissue-engineered kidneys in the future.
Biocompatible Materials↗
Defunctionalized bladders: effects before and after refunctionalization in an animal model.
PURPOSE: Bladder behavior after refunctionalization is usually unpredictable. We comparatively analyze various aspects of bladder defunctionalization and subsequent refunctionalization using an animal model. MATERIALS AND METHODS: A total of 18 rabbits were divided equally into 3 groups. Animals in group 1 underwent 2 successive surgical procedures, including bladder division and reattachment. Bladder division was performed by hemisecting the bladder from dome to trigone into a functioning and nonfunctioning chamber. Bladder reattachment was achieved by reanastomosing both hemibladders. Group 2 animals underwent sham operations, and group 3 animals were age matched normal controls. Serial urodynamic studies and fluoroscopic cystograms were performed in all animals. Gross, histochemical (hematoxylin and eosin, Masson's trichrome and Sirius red) and immunocytochemical (alpha-actin, collagen I and III) analyses, collagen content determination and organ bath studies were performed. RESULTS: The defunctionalized hemibladders demonstrated lower wet weight, capacity and compliance compared to the functional contralateral and normal control bladders. Refunctionalization of the bladders resulted in a progressive recovery of capacity and compliance with time. The bladder contractile response and connective tissue-to-muscle ratio were abnormal in the defunctionalized segments but normalized after bladder refunctionalization. CONCLUSIONS: Defunctionalization results in remarkable alterations in bladder growth, capacity, compliance and distribution of connective tissue. However, these bladders demonstrate an innate capacity to recover from these alterations following refunctionalization.
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Tissue engineering applications in the genitourinary tract system.
The concept of cell transplantation using tissue engineering techniques has provided numerous possibilities in the area of urologic tissue reconstruction. Tissue engineering applications in the genitourinary tract system have been investigated in almost every tissue in order to improve, restore and replace existing tissue function. Although most reconstructive efforts still remain in the experimental stage, several technologies have been transferred to the bedside with satisfactory outcome. In this article, we describe tissue engineering approaches attempted in the genitourinary system for reconstruction.
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Reconstitution of human corporal smooth muscle and endothelial cells in vivo.
PURPOSE: The availability of autologous erectile tissue composed of corporal smooth muscle and endothelial cells would be beneficial in patients undergoing penile reconstruction. We previously showed that cultured cavernous cells seeded on polymer scaffolds form corporal muscle when implanted in vivo. However, to reconstruct corporal tissue endothelial and corporal muscle cells are necessary. In this study we investigated the possibility of developing tissue composed of corporal cells in vivo by combining smooth muscle and endothelial cells. MATERIALS AND METHODS: Human corporal smooth muscle and endothelial cells were seeded on biodegradable polyglycolic acid polymer scaffolds at concentrations of 20 x 10(6) and 10 x 10(6) cells per cm3, respectively. A total of 60 polymer scaffolds seeded with cells and 20 control polymers without cells were implanted in the subcutaneous space of 20 athymic mice. Mice were sacrificed 1, 3, 5, 7, 14, 21, 28 and 42 days, respectively, after implantation. Immunocytochemical and histochemical analyses were performed with antifactor VIII, antipancytokeratins and anti-alpha actin antibodies. RESULTS: Histologically the retrieved polymers seeded with corporal smooth muscle and endothelial cells showed the formation of multilayered smooth muscle strips adjacent to endothelial cells 7 days after implantation. Increased organization of the smooth muscle tissue and accumulation of endothelium lining the luminal structures were evident by 14 days. A well organized tissue construct was noted 28 and 42 days after implantation. There was no evidence of tissue formation in controls. Immunocytochemical analysis using antifactor VIII to identify native vasculature only and antipancytokeratins to identify ECV 304 endothelial cells only distinguished the origin of the vascular structures in each construct. Anti-alpha-actin confirmed the smooth muscle phenotype. CONCLUSIONS: Human corporal smooth muscle and endothelial cells seeded on biodegradable polymer scaffolds formed vascularized corpus cavernosum muscle when implanted in vivo. To our knowledge this is the first demonstration in tissue engineering in which capillary formation was facilitated by the addition of endothelial cells in composite tissue in vivo.
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Direct in vivo gene transfer to urological organs.
PURPOSE: Patients with urological disorders may benefit from gene based therapy. We investigated the feasibility of delivering exogenous genes into urological tissues in vivo using direct in vivo electrotransfection. MATERIALS AND METHODS: Gene transfer to rat kidneys, testes and bladders was accomplished via direct local injection of pGL3/luciferase and beta-galactosidase reporter gene constructs, followed by an electrical pulse ranging from 55 to 115 msec at 100 V. Direct injection of deoxyribonucleic acid without an electrical pulse served as the control. The transfected and nontransfected organs were retrieved and analyzed by luciferase activity assay, histochemical and immunocytochemical staining for beta-galactosidase, and reverse transcription polymerase chain reaction with primers specific for beta-galactosidase messenger ribonucleic acid. RESULTS: There was significant luciferase activity 1, 3 and 5 days after direct in vivo electrotransfection in kidneys and testes, and after 3, 5, 7 and 10 days in bladders. Positive beta-galactosidase enzyme activity and beta-galactosidase immunoreactivity were observed in the transfected renal tubular cells, testicular interstitial and germ cells, and uroepithelial bladder layer. Reverse transcription-polymerase chain reaction products of the transfected organs were noted, indicating the successful transcription of messenger ribonucleic acid. CONCLUSIONS: This study demonstrates that direct in vivo electrotransfection is a feasible method of transient gene delivery into intact urological organs. Its apparent safety and relative simplicity suggest that direct in vivo electrotransfection may be useful clinically.
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Autologous engineered cartilage rods for penile reconstruction.
PURPOSE: Conditions such as inadequate and ambiguous genitalia that are caused by rudimentary penis, severe hypospadias or traumatic injury require surgical intervention. Although silicone penile prostheses are an accepted treatment modality, biocompatibility issues may be a problem in select cases. We previously demonstrated that rods composed of cartilage could be created using chondrocytes seeded on biodegradable polymer scaffolds. We showed that the cartilage rods engineered ex situ were readily elastic and withstood high degrees of pressure. We investigated the feasibility of applying the engineered cartilage rods in situ in an animal model. MATERIALS AND METHODS: Autologous chondrocytes harvested from rabbit ears were grown and expanded in culture. Cells were seeded onto biodegradable poly-L-lactic acid coated polyglycolic acid polymer rods at a concentration of 50 x 10(6) chondrocytes per cm3. A total of 18 chondrocyte polymer scaffolds were implanted into the corporal spaces in 10 rabbits. As controls, 1 corpus in each of 2 rabbits was not implanted. The animals were sacrificed 1, 2, 3 or 6 months after implantation. Histological analysis was performed using hematoxylin and eosin, aldehyde fuschin-alcian blue and toluidine blue staining. RESULTS: All animals tolerated the implants for the duration of the study without any complications. Gross examination after retrieval at 1 month showed well formed, milky white cartilage structures within the corpora. All polymers were fully degraded by 2 months. There was no evidence of erosion or infection at any of the implant sites. Histological analysis using alcian blue and toluidine blue staining revealed mature and well formed chondrocytes in the retrieved implants. CONCLUSIONS: Autologous chondrocytes seeded on preformed biodegradable polymer structures form cartilage structures within the rabbit corpus cavernosum. This technology appears to be useful for creating autologous penile prostheses.
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Acellular collagen matrix as a possible "off the shelf" biomaterial for urethral repair.
OBJECTIVES: To determine whether a naturally derived collagen-based tissue substitute developed for urethral reconstruction would be suitable for urethral repair in an animal model. Several urethral conditions often require nongenital tissues for reconstruction, such as skin grafts or mucosal grafts from the bladder or buccal regions. However, the use of these tissues for urethroplasty may be associated with additional procedures for graft retrieval, prolonged hospitalization, and morbidity. METHODS: A ventral urethral defect was created in 10 male rabbits. The acellular collagen matrix, obtained and processed from porcine bladder submucosa, was trimmed and used to replace the urethral defect in an onlay fashion. Serial urethrography was performed pre- and postoperatively at 0.5, 1, 2, 3, and 6 months. The animals were sacrificed 0.5, 1, 2, 3, and 6 months after surgery. The retrieved implants were analyzed grossly, histologically, and with immunocytochemistry. RESULTS: All animals survived until being sacrificed without any noticeable voiding dysfunction. Serial urethrograms confirmed the maintenance of a wide urethral caliber without any signs of strictures. Gross examination at retrieval showed normally appearing tissue without any evidence of fibrosis. Histologically, the implanted matrices contained host cell infiltration and generous angiogenesis by 2 weeks after surgery. The presence of a confluent transitional cell layer was confirmed by immunocytochemical analyses using pancytokeratin antibodies. Anti-alpha actin antibodies demonstrated the migration of unorganized muscle fiber bundles 2 months after implantation and organized muscle bundles 6 months after implantation. CONCLUSIONS: The acellular collagen matrix appears to be a useful material for urethral repair in the rabbit. The matrix can be processed easily, has good characteristics for tissue handling and urethral function, and has the advantage of being an "off the shelf" material.
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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.
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Progressive dilation for bladder tissue expansion.
PURPOSE: The use of gastrointestinal tissue for augmentation cystoplasty is associated with numerous complications. We previously reported the development of a system in which ureters were progressively dilated and used for ureterocystoplasty. We have now applied a similar system for the progressive expansion of native bladder tissue. We investigated whether the expanded bladder tissue retained normal functional and phenotypic characteristics. MATERIALS AND METHODS: Urodynamic studies were performed in 5 beagle dogs and the bladder was divided horizontally into a superior bladder neo-reservoir, and an intact smaller bladder inferiorly with both ureters left intact and draining. A silicone catheter was threaded into the newly formed, superiorly located neo-reservoir, and connected to an injection port which was secured subcutaneously. A saline antibiotic solution was injected daily into the palpable injection port 4 weeks after surgery, dilating the neo-reservoir through the silicone catheter. Baseline and weekly cystograms were performed. Urodynamic studies of the neo-reservoirs were done immediately before sacrifice. Animals were sacrificed 3 months after the initial intervention and the bladder was examined grossly and microscopically. RESULTS: Within 30 days after progressive dilation, the neo-reservoir volume was expanded at least 10-fold according to radiography and cystometrograms. Urodynamic studies of the dilated neo-reservoirs showed normal compliance in all animals. Microscopic examination of the expanded neo-reservoir tissue revealed normal histology. A series of immunocytochemical studies demonstrated that the dilated bladder tissue maintained normal phenotypic characteristics. CONCLUSIONS: The system of progressive dilation is effective in expanding bladder tissue which is able to retain normal phenotypic and functional characteristics.
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Bladder augmentation using allogenic bladder submucosa seeded with cells.
OBJECTIVES: The search for a suitable material to reconstruct the genitourinary tract has been a challenging task. Bowel has been widely used for urinary tract reconstruction, despite its subsequent complications. We investigated the possibility of using allogenic bladder submucosa, a tissue consisting of nonimmunogenic acellular collagen, either with or without cells, as a material for bladder augmentation. METHODS: Partial cystectomies were performed in 10 beagle dogs. Both urothelial and smooth muscle cells were harvested and expanded separately in 5 animals. The allogenic bladder submucosa obtained from sacrificed dogs was seeded with muscle cells on one side and urothelial cells on the opposite side. All beagles underwent cruciate cystotomies on the bladder dome. Augmentation cystoplasty was performed with the allogenic bladder submucosa seeded with cells in 5 animals and with the allogenic bladder submucosa without cells in 5. The augmented bladders were retrieved 2 and 3 months after augmentation. RESULTS: Bladders augmented with the allogenic bladder submucosa seeded with cells showed a 99% increase in capacity compared with bladders augmented with the cell-free allogenic bladder submucosa, which showed only a 30% increase in capacity. All dogs showed a normal bladder compliance, as evidenced by urodynamic studies. Histologically, all retrieved bladders contained a normal cellular organization consisting of a urothelial lined lumen surrounded by submucosal tissue and smooth muscle. Immunocytochemical analyses confirmed the urothelial and muscle cell phenotype and showed the presence of nerve fibers. CONCLUSIONS: These results show that allogenic bladder submucosa seeded with cells appears to be an excellent option as a biomaterial for bladder augmentation.
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Cartilage rods as a potential material for penile reconstruction.
PURPOSE: Sex assignment is made in patients with ambiguous genitalia, genital trauma or iatrogenic injury after a thorough diagnostic evaluation and careful consultation with the family. In numerous instances a decision is made to rear the child as the female gender due to inadequate genitalia regardless of karyotype. Although a silicone penile prosthesis is accepted treatment in adults who require penile reconstruction, it has not been generally used in the pediatric population, mainly due to associated long-term problems. We determine the feasibility of creating natural penile prostheses of cartilage which, if biocompatible and elastic, may be used in patients who require genital reconstruction. MATERIALS AND METHODS: Cartilage was harvested from the articular surface of calf shoulders. Chondrocytes were isolated, grown and expanded in vitro. Cells were seeded onto preformed cylindrical polyglycolic acid polymer rods 1 cm. in diameter and 3 cm. long at a concentration of 50 x 10(6) chondrocytes per cm.3. A total of 40 polymer scaffolds were implanted in the subcutaneous space of 20 athymic mice. In each mouse 2 implantation sites consisted of a polymer scaffold seeded with chondrocytes and a control (polymer alone). Mice were sacrificed 1, 2, 4 and 6 months after implantation, respectively. Stress relaxation studies to measure biomechanical properties, including compression, tension and bending, were performed on the retrieved structures. Histological analyses were done with hematoxylin and eosin, aldehyde fuchsin-alcian blue and toluidine blue staining. RESULTS: Gross examination revealed well formed, milk-white rod-shaped solid cartilaginous structures the same size as the initial implant. Compression, tension and bending studies demonstrated that the cartilaginous structures were readily elastic and withstood high degrees of pressure. Histochemical analyses showed mature, well formed chondrocytes in all implants. There was no evidence of cartilage formation in the controls. CONCLUSIONS: Chondrocytes seeded on preformed biodegradable polymer structures form cartilage rods. The use of an entirely autologous system composed of biodegradable polymers and chondrocytes precludes an immunological reaction. This technology appears to be useful for the creation of a biocompatible malleable penile prosthesis, which may be useful in children with ambiguous genitalia and patients undergoing penile reconstruction.
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Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder.
The epidermal growth factor receptor (HER1) has been implicated in regenerative growth and proliferative diseases of the human bladder epithelium (urothelium), however a cognate HER1 ligand that can act as a growth factor for normal human urothelial cells (HUC) has not been identified. Here we show that heparin-binding EGF-like growth factor (HB-EGF), an activating HER1 ligand, is an autocrine regulator of HUC growth. This conclusion is based on demonstration of HB-EGF synthesis and secretion by primary culture HUC, identification of HER1 as an activatable HB-EGF receptor on HUC surfaces, stimulation of HUC clonal growth by HB-EGF, inhibition of HB-EGF-stimulated growth by heparin and of log-phase growth by CRM 197, a specific inhibitor of HB-EGF/HER1 interaction, and identification of human urothelium as a site of HB-EGF precursor (proHB-EGF) synthesis in vivo. ProHB-EGF expression was also detected in the vascular and detrusor smooth muscle of the human bladder. These data suggest a physiologic role for HB-EGF in the regulation of urothelial proliferation and regeneration subsequent to mucosal injury. Expression of proHB-EGF is also a feature of differentiated vascular and detrusor smooth muscle in the bladder. Because proHB-EGF is known to be the high affinity diphtheria toxin (DT) receptor in human cells, synthesis of the HB-EGF precursor by human urothelium also suggests the possibility of using the DT-binding sites of proHB-EGF as an in vivo target for the intraluminal treatment of urothelial diseases.
Bacterial Proteins↗
Detachable self-sealing membrane system for the endoscopic treatment of incontinence.
PURPOSE: The endoscopic treatment of urinary incontinence is limited by the injectable substances currently available. The ideal injectable material should be able to conserve its volume, be nonmigratory and nonantigenic. Toward this goal we developed a system for the treatment of incontinence in which a catheter with an inflatable and detachable self-sealing silicone membrane fits through a standard cystoscopic needle. We present our experience with this system. MATERIALS AND METHODS: A total of 20 female beagle dogs underwent cystoscopy and in 15 a self-sealing membrane was placed endoscopically in the submucosal region of the proximal urethra at the 3 and 9 o'clock positions. Each membrane was inflated through the delivery catheter with 0.2 cc povidone. Coaptation of the urethra due to the relative bulking effect of the inflated membrane was confirmed endoscopically in each animal. Five control animals received only an injection of saline. Four animals were sacrificed at 1, 3, 6, 12 and 18 months, respectively. At sacrifice gross and histological examinations were performed. RESULTS: At retrieval up to 18 months after implantation the membrane remained inflated and at the same position at which it had been initially placed. There was no evidence of povidone volume loss or extravasation. Membranes were encapsulated by a fibrous capsule. Only a few inflammatory cells surrounded the capsule by month 1 and there were none by month 3. Histological examination of periurethral tissue and distant organs showed no evidence of particle migration. CONCLUSIONS: The detachable self-sealing membrane system is easily implantable, nonmigratory, nonantigenic and able to conserve its volume. These studies indicate that this system may be effective for the endoscopic treatment of incontinence.
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A novel gene delivery system using urothelial tissue engineered neo-organs.
PURPOSE: Presently gene delivery is most effectively achieved by ex vivo gene transfer, which includes removal of the target tissue, in vitro gene delivery to the target cells, possible selection to enhance the proportion of transfected cells and reintroduction of the gene modified cells. Reintroduction of transformed cells in vivo has been a challenging task. Based on the feasibility of tissue engineering techniques in which cells seeded on biodegradable polymer scaffolds form tissue when implanted in vivo, we explored the possibility of developing a neo-organ system for in vivo gene therapy. MATERIALS AND METHODS: Normal human urothelial cells were harvested, expanded in vitro and seeded on biodegradable polymer scaffolds. The cell-polymer complex was then transfected with PGL3-luc, pCMV-luc and pCMV beta-gal promoter reporter gene constructs. The transfected cell-polymer scaffolds were then implanted in athymic mice and the engineered tissue was retrieved 0, 1, 3, 5 and 7 days after implantation. RESULTS: The reporter gene assay demonstrated an expression of luciferase activity at days 1, 3, 5 and 7 with the peak at day 5. X-gal and beta-galactosidase antibody assays stained positive on the deoxyribonucleic acid treated transfection. CONCLUSIONS: Successful gene transfer can be achieved using biodegradable polymer scaffolds as a urothelial cell delivery vehicle. The transfected cell-polymer scaffold forms an organ-like structure with functional expression of the transfected genes. This study demonstrates that urothelial tissue engineered gene transfer is safe and effective.
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