Chapter 98 TISSUE ENGINEERING FOR RECONSTRUCTION OF THE URINARY TRACT AND TREATMENT OF STRESS URINARY INCONTINENCE

Urinary incontinence is associated with impairment of quality of life, social isolation, and depressive symptoms.¹ A conservative estimate is that urinary incontinence affects approximately 20% of women. However, the prevalence may be as high as 50% among older women, with a peak at 47 years old.²^–⁴ In the United States the estimated annual direct cost of caring for patients with urinary incontinence is more than $16 billion.⁵

In general, treatment plans are usually directed toward the specific type of urinary incontinence, and the treatment option with the lowest risk for adverse complications is usually offered. Lifestyle intervention, pelvic floor muscle training, vaginal devices (i.e., pessaries), and pharmacologic treatments are usually considered before surgical intervention.¹

Surgical options include endoscopic injection of bulking agents, colposuspension (e.g., Burch procedure), sling operations, traditional transvaginal needle suspension, and tension-free vaginal tape. However, many of the current surgical treatment options only offer short-term relief of urinary incontinence, and the overall success of these therapies is limited by the complications, including infection, graft erosion, bladder and bowel perforation, prolonged catheterization, and vascular injury.² The shortcomings of current therapies have led to the application of regenerative medicine and tissue engineering to the field of urinary incontinence.

Regenerative medicine encompasses various areas of technology, such as tissue engineering, stem cells, and cloning. Tissue engineering, one of the major components of regenerative medicine, follows the principles of cell transplantation, materials science, and engineering toward the development of biologic substitutes that can restore and maintain normal function. Tissue engineering strategies generally fall into two categories: the use of acellular matrices, which depend on the body’s natural ability to regenerate for proper orientation and direction of new tissue growth, and the use of matrices with cells. Acellular tissue matrices are usually prepared by manufacturing artificial scaffolds or by removing cellular components from tissues by mechanical and chemical manipulation to produce collagen-rich matrices.⁶^–⁹ These matrices tend to slowly degrade on implantation and are generally replaced by the extracellular matrix (ECM) proteins that are secreted by the ingrowing cells. Cells can also be used for therapy by injection alone or with carriers such as hydrogels.

When cells are used for tissue engineering, a small piece of donor tissue is dissociated into individual cells. These cells are implanted directly into the host or are expanded in culture, attached to a support matrix, and then reimplanted into the host after expansion. The source of donor tissue can be heterologous, allogeneic, or autologous (Table 98-1). Ideally, structural and functional tissue replacement will occur with minimal complications. The preferred cells to use are autologous cells. For this method, a biopsy of tissue is obtained from the host, the cells are dissociated and expanded in culture, and the expanded cells are implanted into the same host.⁹^–¹² The use of autologous cells, although it may cause an inflammatory response, avoids rejection, and the deleterious side effects of immunosuppressive medications can be avoided.

Table 98-1 Origin of Tissue

Source of Donor Tissue	Definition
Autologous	Same individual
Homologous (allogeneic)	Same species, different individual
Heterologous	Different species (bovine)

Most strategies for tissue engineering depend on a sample of autologous cells from the diseased organ of the host. However, for many patients with extensive end-stage organ failure, a tissue biopsy may not yield enough normal cells for expansion and transplantation. In other instances, primary autologous human cells cannot be expanded from a particular organ, such as the pancreas. In these situations, stem cells are envisioned as being an alternative source of cells from which the desired tissue can be derived. Stem cells can be derived from discarded human embryos (i.e., human embryonic stem cells), from fetal tissue, or from adult sources (e.g., bone marrow, fat, skin). Therapeutic cloning has also played a role in the development of the field of regenerative medicine. Therapeutic cloning, which has also been called nuclear transplantation and nuclear transfer, involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate an embryo with a genetic makeup identical to that of the donor. Stem cells can be derived from this source, which may have the potential to be used therapeutically.

Major advances have been achieved within the past decade. Regenerative medicine may extend the treatment options for urinary incontinence. However, like every new field, regenerative medicine and tissue engineering are expensive. Several of the clinical trials involving bioengineered products have been placed on hold because of the costs involved with the specific technology. With a bioengineered product, costs are usually high because of the biologic nature of the therapies involved. As with any therapy, the costs allowed by the medical health care system for a specific technology must be limited. The costs of bioengineered products must be lowered for them to have an impact clinically. This issue is being addressed for many tissue-engineered technologies. As the technologies advance over time and the volume of the application is considered, costs will naturally decrease.

NATIVE CELLS

One of the limitations of applying cell-based regenerative medicine techniques to organ replacement has been the inherent difficulty of growing specific cell types in large quantities. By studying the privileged sites for committed precursor cells in specific organs and by exploring the conditions that promote differentiation, researchers may be able to overcome the obstacles that limit cell expansion in vitro. For example, urothelial cells have been grown in an institutional setting in the past, but only with limited expansion. Several protocols developed over the past 2 decades identified the undifferentiated cells and kept them undifferentiated during their growth phase.¹³^–¹⁶ Using these methods of cell culture, it is possible to expand a urothelial strain from a single specimen that initially covered a surface area of 1 cm² to one covering a surface area of 4202 m² (the equivalent of one football field) within 8 weeks.¹³

These studies indicated that it should be possible to collect autologous bladder cells from human patients, expand them in culture, and return them to the donor in sufficient quantities for reconstructive purposes.^13,¹⁵^–²⁰ Major advances have been achieved within the past decade on the possible expansion of a variety of primary human cells, with specific techniques that make the use of autologous cells possible for clinical application.

ANGIOGENIC FACTORS

The engineering of large organs requires a vascular network of arteries, veins, and capillaries to deliver nutrients to each cell. One possible method of vascularization is the use of gene delivery of angiogenic agents such as vascular endothelial growth factor (VEGF) with the implantation of vascular endothelial cells to enhance neovascularization of engineered tissues. Skeletal myoblasts from adult mice were cultured and transfected with an adenovirus encoding VEGF, and these cells were combined with human vascular endothelial cells.²¹ The mixtures of cells were injected subcutaneously in nude mice, and the engineered tissues were retrieved up to 8 weeks after implantation. The transfected cells formed muscle with neovascularization, as determined by histology and immunohistochemical probing, with maintenance of their muscle volume, whereas engineered muscle of nontransfected cells had a significantly smaller mass of cells, with loss of muscle volume over time, less neovascularization, and no surviving endothelial cells. These results indicate that a combination of VEGF and endothelial cells may be useful for inducing neovascularization and volume preservation in engineered tissue. The use of angiogenic factors may support cell-based therapy in patients with postirradiation incontinence or patients with large fibrotic degeneration of the urinary sphincter.

BIOMATERIALS

For cell-based tissue engineering, the expanded cells are seeded onto a scaffold synthesized with the appropriate biomaterial. In tissue engineering, biomaterials replicate the biologic and mechanical function of the native ECM found in tissues in the body by serving as an artificial ECM. Biomaterials provide a three-dimensional space for the cells to form into new tissues with appropriate structure and function, and they allow the delivery of cells and appropriate bioactive factors (e.g., celladhesion peptides, growth factors) to desired sites in the body.²² Because most mammalian cell types are anchorage dependent and will die if no cell-adhesion substrate is available, biomaterials provide a cell-adhesion substrate that can deliver cells to specific sites in the body with high loading efficiency. Biomaterials can provide mechanical support against in vivo forces such that the predefined three-dimensional structure is maintained during tissue development. Bioactive signaling agents, such as celladhesion peptides and growth factors, can be loaded along with cells to help regulate cellular function.

The ideal biomaterial should be biodegradable and bioresorbable to support the replacement of normal tissue without inflammation. Incompatible materials are destined for an inflammatory or foreign body response that eventually leads to rejection and necrosis. Degradation products, if produced, should be removed from the body by means of metabolic pathways at a rate that keeps the concentration of these degradation products in the tissues at a tolerable level.²³ The biomaterial should also provide an environment in which appropriate regulation of cell behavior (i.e., adhesion, proliferation, migration, and differentiation) can occur such that functional tissue can form. Cell behavior in the newly formed tissue is regulated by many interactions of the cells with their microenvironment, including interactions with cell-adhesion ligands²⁴ and with soluble growth factors.²⁵

Biomaterials provide temporary mechanical support while the cells undergo spatial tissue reorganization. The properly chosen biomaterial should allow the engineered tissue to maintain sufficient mechanical integrity to support itself in early development, and in late development, it should have begun degradation so that it does not hinder further tissue growth.²² Three classes of biomaterials have been used for engineering tissues (Table 98-2): naturally derived materials (e.g., collagen and alginate), acellular tissue matrices (e.g., bladder submucosa, small intestinal submucosa), and synthetic polymers such as polyglycolic acid (PGA), polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA). These classes of biomaterials have been tested in terms of their biocompatibility.^26,²⁷ Naturally derived materials and acellular tissue matrices have the potential advantage of biologic recognition. However, synthetic polymers can be produced on a large scale with controlled properties of strength, degradation rate, and microstructure.

Table 98-2 Classes of Biomaterials

Class	Example
Naturally Derived Materials	Collagen and Alginate
Acellular tissue matrices	Bladder submucosa or small intestinal submucosa
Synthetic polymers	Polyglycolic acid (PGA), polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA)

Naturally Derived Materials

Collagen is the most abundant and ubiquitous structural protein in the body, and it can be readily purified from animal and human tissues with an enzyme treatment and salt or acid extraction.²⁸ Collagen implants degrade through a sequential attack by lysosomal enzymes. The in vivo resorption rate can be regulated by controlling the density of the implant and the extent of intermolecular cross-linking. The lower the density, the greater the interstitial space and the larger the pores for cell infiltration, leading to a higher rate of implant degradation. Collagen contains cell-adhesion domain sequences (e.g., RGD) that may assist to retain the phenotype and activity of many types of cells, including fibroblasts²⁹ and chondrocytes.³⁰

Alginate, a polysaccharide isolated from seaweed, has been used as an injectable cell-delivery vehicle ³¹ and a cell-immobilization matrix³² because of its gentle gelling properties in the presence of divalent ions such as calcium. Alginate is relatively biocompatible, and it is approved by the U.S. Food and Drug Administration (FDA) for human use as material to dress wounds. Alginate is a family of copolymers of D-mannuronate and L-guluronate. The physical and mechanical properties of alginate gel are strongly correlated with the proportion and length of polyguluronate block in the alginate chains.³¹

Acellular Tissue Matrices

Acellular tissue matrices are collagen-rich matrices prepared by removing cellular components from tissues. The matrices are often prepared by mechanical and chemical manipulation of a segment of tissue.⁶^–⁹ The matrices slowly degrade on implantation, and they are replaced and remodeled by ECM proteins synthesized and secreted by transplanted or ingrowing cells.

Synthetic Polymers

Polyesters of naturally occurring α-hydroxy acids, including PGA, PLA, and PLGA, are widely used in tissue engineering. These polymers have gained FDA approval for human use in a variety of applications, including sutures.³³ The ester bonds in these polymers are hydrolytically labile, and the polymers degrade by nonenzymatic hydrolysis. The degradation products of PGA, PLA, and PLGA are nontoxic natural metabolites and are eventually eliminated from the body in the form of carbon dioxide and water.³³ The degradation rate of these polymers can be tailored from several weeks to several years by altering crystalinity, initial molecular weight, and the copolymer ratio of lactic to glycolic acid. Because these polymers are thermoplastics, they can be easily formed into a three-dimensional scaffold with a desired microstructure, gross shape, and dimension by various methods, including molding, extrusion,³⁴ solvent casting,³⁵ phaseseparation techniques, and gas-foaming techniques.³⁶ Many applications in tissue engineering require a scaffold with high porosity and ratio of surface area to volume. Other biodegrad-able synthetic polymers, including poly-anhydrides and polyortho-esters, can be used to fabricate scaffolds for tissue engineering with controlled properties.³⁷

TISSUE ENGINEERING OF SPECIFIC STRUCTURES

Investigators around the world, including those at our institution, have been working toward the development of several cell types and tissues and organs for clinical application. In the following sections, we describe the engineering of tissues that may have an impact on the reconstructive strategies for urinary incontinence.

Urethra

The urethra consists of layers of longitudinal and circular smooth muscle. Weakening of the urethral muscle wall by compression damage during delivery or trauma can result in incontinence.² Various biomaterials without cells, such as PGA and acellular collagen-based matrices from small intestine and bladder, have been used in animal models for the regeneration of urethral tissue.^6,³⁸^–⁴² Some of these biomaterials, such as acellular collagen matrices derived from bladder submucosa, have also been seeded with autologous cells for urethral reconstruction. Our institution has been able to replace tubularized urethral segments with cell-seeded collagen matrices.

Acellular collagen matrices derived from bladder submucosa by our institution have been used experimentally and clinically. In animal studies, segments of the urethra were resected and replaced with acellular matrix grafts in an onlay fashion. Histologic examination showed complete epithelialization and progressive vessel and muscle infiltration, and the animals were able to void through the neo-urethras.⁶ These results were confirmed in a clinical study of patients with hypospadias and urethral stricture disease.⁴³ Decellularized cadaveric bladder submucosa was used as an onlay matrix for urethral repair in patients with stricture disease and hypospadias. Patent, functional neourethras were confirmed in these patients, with up to 7 years of follow-up. The use of an off-the-shelf matrix appears to be beneficial for patients with abnormal urethral conditions, and it obviates the need for obtaining autologous grafts, decreasing operative time and eliminating donor site morbidity.

These techniques are not applicable for tubularized urethral repairs. The collagen matrices are able to replace urethral segments only when used in an onlay fashion. However, if a tubularized repair is needed, the collagen matrices should be seeded with autologous cells to avoid the risk of stricture formation and poor tissue development.^44,⁴⁵ Tubularized collagen matrices seeded with autologous cells can be used successfully for urethra replacement.

Bladder

Gastrointestinal or colon segments are commonly used as tissues for bladder replacement or repair. However, both tissues are designed to absorb specific solutes, whereas bladder tissue functions as a barrier to prevent resorption of waste products. Because of the problems encountered with the use of gastrointestinal segments, numerous investigators have attempted using alternative materials and tissues for bladder replacement or repair.

The success of cell transplantation strategies for bladder reconstruction depends on the ability to use donor tissue efficiently and to provide the right conditions for long-term survival, differentiation, and growth. Urothelial and muscle cells can be expanded in vitro, seeded onto polymer scaffolds, and allowed to attach and form sheets of cells.⁴⁶ These principles were applied in the creation of tissue-engineered bladders in an animal model that required a subtotal cystectomy with subsequent replacement with a tissue-engineered organ in beagle dogs.⁴⁷ Urothelial and muscle cells were separately expanded from an autologous bladder biopsy and seeded onto a bladder-shaped biodegradable polymer scaffold. The results from this study showed that it is possible to tissue engineer bladders that are anatomically and functionally normal. Clinical trials for the application of this technology are being conducted.

Vagina

Several pathologic conditions, including congenital malformations and malignancy, can adversely affect normal vaginal development or anatomy. According to the hammock hypothesis, the vagina provides a firm backdrop for the urethra to rest against when the intra-abdominal pressure increases, preventing urinary leakage.

Vaginal reconstruction has been challenging because of the paucity of available native tissue. The feasibility of engineering vaginal tissue in vivo has been investigated.⁴⁸ Vaginal epithelial and smooth muscle cells of female rabbits were harvested, grown, and expanded in culture. These cells were seeded onto biodegradable polymer scaffolds, and the cell-seeded constructs were then implanted into nude mice for up to 6 weeks. Immunocytochemical, histologic, and Western blot analyses confirmed the presence of vaginal tissue phenotypes. Electrical field stimulation studies in the tissue-engineered constructs showed similar functional properties to those of normal vaginal tissue. When these constructs were used for autologous total vaginal replacement, patent vaginal structures were confirmed in the tissue-engineered specimens, whereas the non–cell-seeded structures were stenotic.⁴⁹