James J. Yoo

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.

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.

Urethral Replacement Using Cell Seeded Tubularized Collagen Matrices

PURPOSE Acellular collagen matrices derived from bladder submucosa have been used successfully as an off-the-shelf biomaterial for urethral replacement, experimentally and clinically in an onlay fashion. We investigated whether collagen matrices, either alone or with autologous cells, could be used for tubularized urethral replacement. MATERIALS AND METHODS Acellular collagen matrices were processed and tubularized. Ten rabbits underwent an open bladder biopsy with subsequent cell expansion. Autologous bladder cells were grown and seeded onto the pre-configured tubular matrices. A 1 cm. long urethral segment was excised in 24 male rabbits. Urethroplasty was performed with the tubularized collagen matrices seeded with cells in 12 animals and without cells in 12. Serial urethrography was performed preoperatively and at 1, 2, 3 and 6 months postoperatively. Retrieved urethras were analyzed grossly, histologically, immunocytochemically and with Western blots. Contractility and the presence of neurotransmitter receptors were confirmed with organ bath studies. RESULTS Serial urethrography confirmed the maintenance of a wide urethral caliber without any signs of strictures in animals implanted with the cell seeded matrices. The urethral segments replaced with the collagen scaffolds without cells demonstrated strictures and graft collapse at all time points. The implanted cell seeded matrices had a normal urethral architecture by 1 month, consisting of a transitional cell layer surrounded by muscle cell fiber bundles with increasing cellular organization with time. Epithelial and smooth muscle phenotypes were confirmed immunocytochemically and with Western blot analyses using pancytokeratins AE1/AE3 and smooth muscle specific alpha-actin antibodies. Formation of a transitional cell layer was confirmed in the matrices implanted without cells but only scant unorganized muscle fiber bundles were present, mostly at the anastomotic sites. Organ bath studies demonstrated the capacity for contractility along with cholinergic and adrenergic specific receptors in the tissue engineered scaffolds compared to controls. CONCLUSIONS These results show that collagen matrices seeded with cells form normal urethral tissue can be used for tubularized replacement, whereas tubularized collagen matrices alone without cells lead to poor tissue formation and strictures. The collagen matrices seeded with cells may offer a useful alternative in the future for patients requiring a tubularized urethral segment replacement.

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.

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.

Autologous Penile Corpora Cavernosa Replacement using Tissue Engineering Techniques

PURPOSE The availability of engineered tissues would be beneficial to patients undergoing penile reconstruction. We explored the possibility of replacing an entire cross-sectional segment of both corporal bodies with autologous engineered tissues in rabbits, and investigated the structural and functional integrity of the neo-corpora. MATERIALS AND METHODS Acellular corporal collagen matrices were obtained from donor rabbit penis. Autologous corpus cavernosal smooth muscle and endothelial cells were harvested, expanded and seeded on the matrices. An entire cross-sectional segment of protruding rabbit phallus was excised, leaving the urethra intact. A total of 26 matrices, including 18 seeded with cells and 8 without cells, were interposed into the excised corporal space. An additional 4 rabbits that did not undergo surgical intervention served as normal controls. Functional and structural parameters (cavernosography, cavernosometry, mating behavior and sperm ejaculation) were followed for 6 months. Gross examination, and histochemical, immunocytochemical and Western blot analyses were performed at 3 and 6 months after implantation. RESULTS The experimental corporal bodies demonstrated intact structural integrity on cavernosography and decreased maximal intracavernosal pressures on cavernosometry compared to normal controls. Mating activity in animals with engineered corpora normalized by 3 months postoperatively. The presence of sperm was confirmed during mating and was present in all rabbits with engineered corpora but in only 2 with the matrix alone. Histologically sinusoidal spaces and walls lined with endothelial and smooth muscle cells were observed in the engineered grafts. Each cell type was identified immunocytochemically. Grafts without cells contained fibrotic tissue and calcifications with sparse corporal elements. Western blot analysis of engineered grafts showed nitric oxide synthase activity similar to normal controls. CONCLUSIONS Autologous corpus cavernosal smooth muscle and endothelial cells seeded on collagen matrices can form corpora cavernosa tissue structures in a rabbit model. Engineered corpora cavernosa achieved adequate structural and functional parameters. This technology may be applicable to patients who require additional tissue for phallic reconstruction.

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.

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.

Experimental and clinical experience using tissue regeneration for urethral reconstruction

Abstract 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.

Renal therapy using tissue-engineered constructs and gene delivery.

Abstract 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.