Bladder Acellular Matrix and Its Application in Bladder Augmentation

Abstract

Over the last few decades, both synthetic and natural materials have been utilized to develop bladder substitutes. Most attempts have not been successful because of mechanical, structural, functional, or biocompatibility problems. Bladder acellular matrix (BAM) is obtained by removing cellular components from donor bladders, leaving a tissue matrix consisting of collagen, elastin, fibronectin, glycosaminoglycans, proteoglycans, and growth factors. Multiple BAM-based studies now suggest that tissue engineering techniques may provide efficacious alternatives to current methods of bladder augmentation. Efforts to optimize BAM-based scaffolds are ongoing and would be greatly assisted by feasible means of improving scaffold properties and interaction with cells and tissues. Future applications of BAM will likely include cell-seeded grafts with the eventual hope of producing “off the shelf” replacement materials for bladder augmentation.

Introduction

Current surgical options for patients who have decreased bladder capacity or compliance are augmentation with gastrointestinal segments and catheterized stomas (Mitrofanoff or Monti technique). However, these surgical options often involve complications including infection, urolithiasis, metabolic disfunction, and malignancy.¹ Alternative treatments for bladder dysfunction involving tissue engineering techniques have been tried in an effort to overcome the limitations associated with the conventional methods of augmentation.

Since the introduction of the concept of tissue-engineered bladders in the early 1990s, significant progress has been made toward the goal of clinical translation.² Tissue engineering approaches need scaffolds to provide biochemical signals during bladder tissue regeneration. The ideal scaffold should be biocompatible, promote cell–matrix interaction and tissue development, and have the proper mechanical and physical characteristics. Two main classes of biomaterials have been often used for bladder tissue engineering: synthetic polymers and naturally derived acellular tissue matrices. Synthetic polymers, such as poly glycolic acid, poly lactic-co-glycolic acid, and poly-L-lactide,^3,4 can be produced on a large scale with controlled properties of microstructure, degradation rate, and strength. Naturally derived acellular tissue matrices, including bladder acellular matrix (BAM), small intestine submucosa,^5,6 and so on, may retain their biological activity even after decellularization. The decellularized matrix provides a complex of functional and structural proteins, that is, collagen, elastin, fibronectin, glycosaminoglycans (GAGs), proteoglycans, and growth factors.⁷ In addition, the composition and structure of the extracellular matrix (ECM) strongly influence the processes of cell attachment, growth, and migration, which is unique to individual tissues.^8,9 A three-dimensional (3D) bladder-derived scaffold could be effective in supporting bladder tissue regeneration. To date, acellular matrix derived from bladder can be prepared in large quantities for use. Acellular scaffolds may be used to promote remodeling, or, alternatively, scaffolds seeded with autologous cells may be used for bladder and urethra repair.^2,7,10–15 The terminology for acellular matrices derived from bladder has varied in the literature, and terms such as BAM,^{12,14,16–25} urinary bladder matrix,^26–29 and bladder submucosa matrix have been used.^30–33 In this review, we will use the term “BAM.” The source for the donor tissue used to generate BAM for experimental and clinical studies includes human,^2,23 pig,^19,24,34 dog,^23,35,36 rabbit,^17,20 and rat.^{12,22,25,37–40} The present article provides an overview of the published literature describing pre-clinical and clinical applications of BAM in bladder augmentation.

Preparation of BAM

Preparation of BAM was first described in 1975.⁴¹ It commonly involves a combination of physical methods to delaminate the layers, followed by chemical and enzymatic methods to remove cells. It is well known that the intrinsic properties of any biomaterial can be significantly altered by methods to decellularize and sterilize the material. Currently, common variations to produce BAM include mechanical delamination of the tissue, the types of detergent used for cell removal, and terminal sterilization methods.⁴²

In the last few decades, a number of methods have been described for the decellularization of BAM.^9,26,43 An optimal decellularization protocol would completely remove xenogenic proteins that induce immune-mediated rejection while retaining the biological activity of desirable BAM components. The technique used for the decellularization of BAM can greatly influence the properties of the scaffold. The direction of tissue removal, the region of the bladder harvested, the thickness of the starting biomaterial (full vs. split thickness), and whether the bladder is distended could also affect the scaffold properties.⁴³

Most studies prefer mechanical delamination of the bladder tissues with removal of the muscular and serosal layers before chemical and enzymatic procedures.^{25,26,28,35–38,44} The benefits of mechanical delamination are thought to make the processing that follows more straightforward. A few studies have applied decellularization methods to the full-thickness bladder.^8,17,45 The decellularization techniques often involve chemical and enzymatic agents such as sodium azide,^{23,25,35,37,38,46} hypotonic liquid,¹⁸ sodium desoxycholate,⁴⁶ sodium dodecyl sulfate (SDS),^45,47 Triton X-100+ ammonium hydroxide,^{17,30,32,33,43} and RNAse/DNAse.^{18,25,46,48–50} However, many decellularization methodologies that aim at limiting the matrix immunogenicity are not always entirely successful. Matrices that maintain a certain amount of cellularity may present immunogenic components to the recipient.⁸

Several studies have optimized protocols to completely remove the cellular components while preserving the extracellular bioactive factors. Rosario et al.²⁶ have investigated the effects of the key tissue processing steps on the properties of bladder tissue. A combined mechanical delamination, physical rinsing in hypotonic buffer, 0.1% SDS solution, and 0.1% peracetic acid (PAA) proved to be a highly efficient decellularization agent with the authors demonstrating that this method resulted in minimal effects on stiffness. This procedure produced a BAM with mechanical properties to facilitate growth of urothelial and bladder stromal cells. However, several studies^43,51,52 suggest that ionic detergents (such as SDS) might lead to an alteration of the conformational structure of the sulfated GAGs, resulting in a lower binding affinity between sulfated GAGs and bioactive factors. The use of nonionic detergents, such as Triton X-100, may prevent this important molecule loss. Due to the probable cytotoxic effects of sodium azide, Kajbafzadeh et al.¹⁸ used hypotonic liquid (distilled water) for cell lysis instead of azide solution for the decellularization of BAM, and observed a time-dependent regeneration of various cellular components in the BAM in vivo. Liu et al.³⁰ showed that 3–5% PAA more efficiently reduced xenogenous cellular components, and additionally increased overall porosity of the scaffold. This system provided a 3D cell culture setting that enhanced efficiency, yields, and homogeneity of bladder cell differentiation and promoted cell–matrix infiltration. Yang et al.⁴³ used several preservative techniques, including decreased wash times, supplementation with proteinase inhibitors, and control of the pH value and temperature of the wash buffer. This method maximally retained the extracellular collagen, sulfated GAGs, and bioactive factors such as platelet-derived growth factor BB (PDGF-BB) and vascular endothelial growth factor (VEGF). The preserved bioactive factors may be important in promoting cell proliferation and migration.

Distention of the intact full-thickness bladder generates thin tissue sections. Bolland et al.⁸ were able to ensure complete decellularization by allowing the solutions to permeate throughout the entire tissue. Histological analysis revealed that the underlying histoarchitecture was retained even with the removal of the urothelium and smooth muscle components. With the maintenance of the lamina propria, no statistical differences were observed in the burst pressure of intact BAMs compared with fresh bladders. The investigators also found that the tissues demonstrated a degree of anisotropy, with mechanical results influenced by tissue orientation. In contrast, Rosario et al.²⁶ found significant anisotropy with whole bladder; however, there were no significant differences after delamination. These findings were supported by Freytes et al.,⁵³ indicating that orientation of the smooth muscle bundles is related to the degree of observed anisotropy seen rather than BAM ECM orientation.

Intrinsic Properties of BAMs

Although the BAM scaffold is thinner after decellularization, the basic architecture remains, including collagen, fibronectin, elastin, GAGs, and growth factors. In addition, BAM retains an intact basement membrane and this advantageous feature can contribute to protection against urine extravasation while supporting growth and differentiation of multilayered urothelial cells in vivo.

Collagen fibers form the principal proportion of the BAM compared with normal bladder tissue. The content and ratio of collagen is a critical decisive factor of the BAM mechanical properties. It was determined to be composed primarily of type-I and type-III collagen. Since the ratio of type-I to type-III collagen does not alter during the process of cell removal, the grafted BAM presumably possesses similar mechanical properties to the normal bladder.¹⁸ However, comparing the luminal to outer side, differences were observed in the structure, thickness, and density of the collagen. The urothelial and muscularis mucosa area consisted of small and tightly compact collagen fibers, while the detrusor muscle zone consisted of thicker and loosely oriented collagen fibers.³⁵

In addition, many growth factors have been identified in the BAMs, including PDGF-BB, VEGF, basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), transforming growth factor-β1 (TGF-β1), insulin-like growth factor, and epidermal growth factor. Among them, VEGF, KGF, and PDGF-BB are expressed at detectable levels.^8,32,43 The presence of the bioactive factors is thought to be maintained by the GAGs, and the amount of PDGF-BB and VEGF is positively related with the sulfated GAGs content in the BAM.⁴³ Dahms et al.²¹ suggest that higher expression of TGF-β1 in BAM is essential for the regeneration of bladder-wall components. The soluble BAM extracts showed an effect on cell proliferation in vitro, suggesting that growth factors and ECM in the BAM maintain biological activity after decellularization.³² Bolland et al.⁸ demonstrated a relative increase in the proportion of GAGs after bladder decellularization. This is important, as GAGs associated with the scaffold may act as substrates for cell growth and promote water retention.

BAM appears to possess antibacterial property in being against both Gram-positive and Gram-negative bacteria, which may be related to some low-molecular-weight peptides within BAM.^54,55 Liu et al.²⁹ observed that BAM surfaces have lower adhesion rates of Staphylococcus aureus and Escherichia coli, suggesting that BAM may contain natural antibacterial activity.

Angiogenesis of BAM

Tissues thicker than 0.8 mm require vascularization in order to supply all cellular components with oxygen and nutrients.⁵⁴ Early and rapid generation of a vascular network is imperative to maintain tissue viability and long-term survival.

One of the strategies to address this issue is the use of angiogenic growth factors incorporated in BAM. For a successful application of exogenous angiogenic growth factors, an effective delivery system is required, otherwise the biological activity of these proteins does not persist in vivo.⁵⁵ Kanematsu et al.³⁹ demonstrated that BAM itself functions as a platform for sustained release of exogenously applied bFGF, a growth factor which promotes angiogenesis and proliferation of mesenchymal cells. bFGF protein was effectively incorporated into BAM by rehydration and locally released according to the matrix biodegradation with intact biological activity. The release pattern of bFGF correlated with biodegradation of the BAM. Incorporation of bFGF into BAM may prevent the proteolytic degradation of bFGF. Based on the study just described, Kanematsu et al.⁵⁶ compared the effects of several different angiogenic growth factors (hepatocyte growth factor, PDGF-BB, and bFGF) for BAM incorporation. In vivo release tests revealed that bFGF release was the most sustained according to the matrix biodegradation, followed by hepatocyte growth factor and PDGF-BB. The angiogenic growth factors released from the BAM resulted in angiogenic activity in a dose-dependent manner in vivo.

In order to create a more proper environment for bladder regeneration, Cartwright et al.⁵⁷ incorporated hyaluronic acid (HA) into BAM, and a hybrid construct was created that was significantly less porous than the untreated BAM, thus preventing urine leakage and reducing inflammation. Enzymatic degradation of HA by hyaluronidase can release smaller polymers that can stimulate a strong angiogenic response in vivo.⁵⁸ Over time, incorporated HA would be replaced by cellular ingrowth in vivo and may potentially function as a carrier for other bioactive molecules and growth factors.⁵⁹ In another study,⁶⁰ this group demonstrated improved neovascularization of bladder constructs with VEGF fortification of BAM-HA. In contrast to purified angiogenic factors, incorporation of HA may be a superior carrier to promote angiogenesis in transplanted BAM. Besides the effect of neovascularization, regulation of the host immune response was also observed after the treatment of BAM with HA and VEGF.³⁴

Another strategy used to improve angiogenesis of BAM was reported by Geng et al.⁶¹ This group fabricated a thermal responsive BAM system containing hydrogel-entrapped VEGF-nanoparticles. Results indicate that this novel system provides a tissue-compatible environment and an effective VEGF sustained release approach without acute immune reaction, inflammation, and toxic manifestation.

BAM-Based Bladder Augmentation

Two main approaches to using BAM have been reported in the literature. In the first approach, BAM is implanted as a scaffold to facilitate host cell migration, proliferation, and differentiation. This approach relies on the intrinsic structure's ability to interact with the host. The other approach is that the BAMs serve as a scaffold for seeding with host cells followed by cell growth in vitro. In this approach, autologous cells are grown to confluence into BAM that are then transplanted to enlarge the bladder in vivo.

Cell-free BAMs for bladder augmentation

Animal models with normal bladder

Sutherland et al.⁶² first reported the use of BAM allograft for bladder augmentation in a rat model with partial cystectomy (Table 1). The authors observed complete epithelialization of the mucosa surface of the graft within 4 days, regeneration of vascular and muscle components within 2 weeks, and neural elements formed as early as 4 weeks after grafting. Piechota et al.^22,37,63 also described that these regenerated bladders exhibited 80% of native bladder contractile activity to electrical stimulation and drug administration. In their histological studies, BAM grafts can require approximately 6 months for nerve regeneration, while smooth muscle regeneration is usually complete within 3 months. In a dog model, Probst et al.³⁵ confirmed the potential of the homologous BAM as a bladder augmentation graft with minimal antigenicity. The mean bladder capacity in the augmented canines was significantly higher than in the controls (264 vs. 172 mL, p<0.05). Using host cell labeling technique (KI67), Wefer et al.⁴⁶ observed that the time-dependent increase of muscle cell in a BAM is consistent with the restoration of bladder function. Reddy, Merguerian et al., and Brown et al.^45,47,49 present evidence that large BAM (>40 cm²) allograft implantation is technically feasible in a pig model out to longer time frames (22 weeks). However, toward the central portion, all grafts were lacking organized smooth muscle bundles and a mature urothelium. Significant differences were observed in the elastic modulus and rupture strain of the BAM compared with native bladder tissue. BAM shrinkage averaged 31% over 12 weeks. Recently, Davis et al.²⁸ compared differences between BAM with the autologous ileum for bladder augmentation in an ovine model, and found that BAM resulted in a significant increase in bladder capacity and compliance. Furthermore, these increases were comparable with ileum when a twofold increase in BAM surface area (50 cm²) relative to the resected ileal segment (25 cm²) was applied. To address the ongoing problem of limited autologous tissue reserves, Sievert et al.²³ investigated the effectiveness of heterologous bladder augmentation in a dog model. Seven months postoperatively, although the functional outcome was satisfactory, heterologous BAM (human and monkey) was less effective as when autologous bladder tissue is used.

Table 1.

Cell-Free BAMs for Bladder Augmentation

Author	Animal model	Surgery	Modification of BAM	Follow-up (month)	Important findings	BAMs related complications
1996/Sutherland et al.⁶²	Rat	PC: 25% (0.8×0.8 cm)	No	6.5	Regeneration of urothelium, smooth muscle, blood vessels, and nerves in histology	Bladder stones were noted in 17/27 rats
1999/Piechota et al.²²	Rat	PC: >50%	No	4	Morphologic and functional bladder regeneration, but with a high incidence of bladder stone formation	10/31 rats died from urinary leakage and infection, bladder stones occurred in 16/20 rats
2000/Probst et al.³⁵	Dog	PC: 20–50%	No	7	Morphological regeneration and bladder capacity recovery	1/7 developed an intraabdominal urinoma
2000/Reddy, Merguerian and Brown et al.^45,47,49	Pig	PC: 40.5 cm²	No	3	Completely regeneration in histology	None described
2001/Wefer et al.⁴⁶	Rat	PC: 50%	No	3	Time-dependent increase of muscle cell in a BAM is concordant with the progressive restoration of bladder function	5/41 died from urinary leakage into the peritoneal cavity. The stone formation rate was 78%.
2002/Cayan et al.³⁸	Rat (CC)	PC: ≥50%	No	3	Bladder capacity and compliance are improved. Urothelial and smooth muscle regeneration at 1 months and nerve regeneration at 3 months	6/37 rats died of urinary leakage at the anastomotic site
2003/Kanematsu et al.³⁹	Rat	PC: 66%	bFGF	3	Angiogenesis was promoted, shrinkage was inhibited at 4 weeks and did not after 12 weeks	None described
2005/Youssif et al.²⁵	Rat	PC: 50%	VEGF	3	Enhanced angiogenesis and smooth muscle content compared with VEGF-free group	6/40 rats died of urinary leakage, bladder stones were observed in 7/34 rats
2006/Sievert et al.²³	Dog	PC: >50%	Homologous/Heterologous	7	Histologic outcome of heterologous BAM is not as good as homologous BAM	None described
2006/Obara et al.⁶⁴	Rat (NB)	PC: 50%	No	3	Regenerated urothelium, smooth muscles, and nerve fibers, proper storage function with distensibility	None described
2007/Kajbafzadeh et al.¹⁸	Rat	PC: ≤50%	No	6	A considerable role for the CD34⁺ EPCs in the neo-vasculogenesis of the BAMs in vivo	None described
2007/Urakami et al.⁶⁵	Rat (NB)	PC: 50%	No	2	Improved voiding function was accompanied by histological regeneration	5/31 rats died due to urine leakage at the site of anastomosis; 9/26 rats had bladder stones
2009/Chen et al.¹⁹	Pig	NA	No	3	Morphological regeneration and bladder function recovery	None described
2009/Kikuno et al.¹²	Rat (NB)	PC: 50%	VEGF+NGF	2	Bladder capacity and compliance were much higher in the VEGF+NGF combined group than in the control, or NGF and VEGF-alone groups	None described
2010/Evren et al.³⁴	Pig	4×4 cm	HA-VEGF	2.5	Stimulate recellularization and vascularization regulate immune responses by reducing TLR4, IL-4 and increasing TGF-β1	None described
2010/Loai et al.⁵⁸	Pig	4×4 cm	HA-VEGF	2.5	Increased vascularization, coupled with increased urothelium and smooth muscle cell regeneration,	None described
2011/Davis et al.²⁸	Ovine	NA	Four-ply BAMs	NA	50 cm² BAM resulted in a 40.7% increase in capacity compared with 40.0% with 25 cm² of detubularized ileum	None described
2012/Mitsui et al.⁷⁸	Pig	PC: 50%	Simultaneous implantation of ureters	2	Simultaneous implantation of bilateral ureters into a BAM enhanced epithelialization	Three died from graft rejection, UTI and renal failure, respectively
2013/Zhou et al.⁶⁷	Rabbit	4×5 cm	PDGF-BB, VEGF	6	The vascularization, smooth muscle regeneration, and recovery of bladder function were enhanced by coadministration of PDGF-BB and VEGF	2/12 animals had central graft calcifications

PC, partial cystectomy; CC, chemical cystitis; NB, neurogenic bladder; UTI, urinary tract infection; EPCs, endothelial progenitor cells; BAM, bladder acellular matrix; PDGF-BB, platelet-derived growth factor BB; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; TGF-β1, transforming growth factor-β1; HA, hyaluronic acid; NGF, nerve growth factor; NA, not applicable.

Animal models with diseased bladder

In order to further confirm the effectiveness of bladder augmentation with BAM, some studies were conducted in animal models with diseased bladders to simulate actual situations of clinical treatment. Cayan et al.³⁸ utilized the chemical cystitis rat model to investigate the restoration of the bladder using BAM graft. Bladder capacity and compliance increased in the grafted group compared with the control group. Histologically urothelial and smooth muscle regeneration within the BAM grafts in the chemical cystitis rat bladder was comparable to that in normal bladder, although nerve regeneration was much slower. In a rat model of neurogenic bladder induced by spinal injury, Obara et al.⁶⁴ indicated that host cells are capable of reconstituting bladder tissue in the BAM grafted area. At 2 and 4 weeks postoperatively, regenerated urothelium, smooth muscle, and nerve fibers were identified, and the grafted BAM displayed proper distension to store urine. Similarly, studies performed by Urakami et al.⁶⁵ suggested that the voiding function accompanied by histological regeneration in neurogenic bladder can be improved by augmentation using BAM. It is noteworthy that bladder augmentation decreased bladder capacity in high-capacity rats, increased the bladder capacity in low-capacity rats 8 weeks after augmentation. These studies suggest that BAM augmentation may not only function as a low-pressure reservoir, but also confer functional contractile activity to the bladder tissue.

BAMs revascularization in vivo

Successful bladder regeneration using BAMs appears to be dependent on the size and revascularization rate of the graft. Due to insufficient vascular supply, shrinkage of larger grafts is common. Revascularization plays a critical role in bladder augmentation.⁶⁶ An alternative technique is to cover the tissue-engineered construct with a well-vascularized structure (e.g., omentum) to provide timely and efficient recellularization.² In addition, enhancement of vascularization by incorporating growth factors has been an approach to the problem of angiogenesis in tissue-engineered bladders. Kanematsu et al.³⁹ first demonstrate that BAMs itself functions as a platform for sustained release of exogenously applied bFGF and incorporation of bFGF into the BAM promoted angiogenesis and inhibited graft shrinkage in a dose-dependent manner. Based on the delivery method described earlier, Youssif et al.²⁵ incorporated VEGF into the BAMs by injection and incubation. They demonstrated in a rat model that VEGF leads to accelerated regeneration within the BAM by increasing angiogenesis, neurogenesis, and muscular regeneration at early (4 weeks) but not late periods after grafting. A subsequent study demonstrated that BAM, combined with the administration of nerve growth factor and VEGF, improved bladder capacity and compliance in a rat neurogenic bladder model.¹² This led to aggregated bundles of smooth muscle, regeneration of nerve fibers, and enhancement of bladder function. These changes were stable until 8 weeks. Zhou et al.⁶⁷ showed that smooth muscle regeneration and vascularization could be improved by incorporation of PDGF-BB and VEGF into BAMs, which has a downstream effect on MMP-2 and MMP-9 activity. Loai et al.⁵⁸ assessed the regenerative and angiogenic effects of porcine BAM incorporated with HA and VEGF in mouse and porcine models. Incorporation of VEGF with HA was effective in promoting the recellularization and vascularization of the BAM graft. Increased urothelial and smooth muscle development was noted throughout the center and periphery of the grafts. The graft area showed highest epithelialization in the HA-VEGF BAM group at week 10.

Although BAMs have shown extensive biocompatibility and re-cellularization by host-derived cells, some studies found that naturally implanted grafts remain susceptible to inflammatory reactions, a low muscle-to-collagen ratio, interstitial fibrosis, and urinary infection, which may result in atrophy of grafted BAMs, accompanied with stone formation, decreased bladder capacity, even urinary leakage, and deaths.^{17,22,38,46,49,64,65,68} Evren et al.³⁴ demonstrated that the incorporation of biomimetic material, HA and HA+ VEGF, into BAMs may help modulate host immune responses by specifically reducing IL-4 and TLR4 expression and promoting TGF-β1, leading to reduced graft fibrosis and inflammation.

Bladder augmentation with BAMs seeding cells

The majority of unseeded BAM used for bladder augmentation have been able to induce the formation of a well-developed urothelial layer, especially when used in a small animal model; however, they have been associated with a weak muscular layer and graft shrinkage (Table 2).⁴⁹ Some studies have highlighted the importance of recognizing the cytotoxicity of urine when designing grafts for tissue regeneration. Urothelial cells possess unique and specialized features that enable these cells to have important functional roles in the urinary system. Several important functions include acting as a permeability barrier, protection of the underlying muscle tissues, and expansion to adjust to changes in bladder pressures.⁶⁹ This highlights the importance of cell seeding for successful bladder reconstruction in larger animals or humans.⁷⁰

Table 2.

Cell-Seeding BAMs for Bladder Augmentation

Author	Animal/patient	Model/disease	Seeding/modification	Follow-up (month)	Important findings	BAMs related complications
1998/Yoo et al.³⁶	Dog	PC: 50%	UCs+SMCs	3	BAM with cells: Bladder capacity 99% increase with a normal urothelia and smooth muscle BAM without cells: Bladder capacity 30% increase with graft contraction and shrinkage	None described
2009/Drewa et al.⁶⁸	Rat	0.7 cm²	HFSCs	6	BAM shape was more regular, muscle regeneration was better in the HFSCs-seeded group than in the BAM-alone group	Two died in the BAM alone group due to leakage and stone disease with a coexistent urinary tract infection
2010/Zhu et al.¹⁷	Rabbit	PC: 40–60%	ADSCs	6	Bladder capacity was much higher and smooth muscle was more evident in the ADSCs group than in the BAM-alone group	One rabbit in the BAM-alone group died due to infection secondary to bladder leakage
2011/Chen et al.²⁴	Pig	PC: 40%	EPCs+VEGF gene	6	VEGF-modified EPCs significantly enhanced neovascularization compared with BAM alone	None described
2012/Corcos et al.⁴⁰	Rat	NA	BMSCs	6	Seeding BMSCs onto BAMs is feasible and produces a viable tissue for bladder augmentation	None described
2006/Atala et al.²	Patient	NB	UCs+SMCs	46 (mean)	Morphological regeneration and bladder function recovery	None described

HFSCs, hair follicle stem cells; ADSCs, adipose-derived stem cells; BMSCs, bone marrow mesenchymal stem cells; NB, neurogenic bladder; PC, partial cystectomy; UCs, urothelial cells; SMCs: smooth muscle cells.

Cell-seeded BAM have been used for bladder augmentation in dogs.³⁶ The autologous urothelial cells were seeded on the mucosa surface of the BAMs, and the muscle cells were seeded on the serosa side of bladder. The regenerated bladder tissues displayed a normal cellular organization consisting of urothelium and smooth muscle. The bladders showed a 99% increase in capacity compared with bladders augmented with the cell-free BAM, which showed only a 30% increase in capacity within 3 months after implantation. Brown et al.¹⁶ demonstrated the ability of BAM to support the growth of porcine bladder urothelial and smooth muscle cells. The results of the study suggested that BAM may contain factors that are capable of promoting important bladder cell–cell and cell–matrix interactions.

Cell-seeded BAMs were used for bladder reconstruction in a pilot clinical study in patients with neurogenic bladder caused by myelomeningocele.² All patients were identified as candidates for augmentation cystoplasty. Based on initial preclinical studies, cell-seeded BAM without an omental wrap were used for the first three patients. After realizing that omental wrapping was beneficial to the vascularization of the tissue in vivo from animal studies, the protocol was revised, and one patient underwent implantation with cell-seeded BAM with an omental wrap. The patients with reconstructed bladder tissue showed increased compliance, increased capacities, decreased pressures, and longer dry periods.

Patients with bladder cancer may be unable to benefit from autologous bladder cell seeding because of the neoplastic potential of the cells. Other possible alternative cell types include stem cells, such as bone marrow mesenchymal stem cells (BMSCs), bone marrow-derived endothelial progenitor cells (EPCs), adipose-derived stem cells (ADSCs), or amniotic fluid-derived stem cells.⁷¹ The differentiated phenotype of stem cells relies the substrate on which they are grown. The differentiation of stem cells into specialized cell types can be influenced by ECM constituents and matrix elasticity.⁷² Several studies^30,73,74 have demonstrated that BAM can enhance BMSCs proliferation and differentiation into a smooth-muscle phenotype, especially in the environment created by bladder smooth muscle cells in vitro.

Physiologically, EPCs have been shown to promote neovascularization and cellular regeneration in bladder regeneration with BAM.¹⁸ Chen et al.²⁴ pre-seeded BAM scaffolds with EPCs induced to express VEGF, resulting in improved bladder tissue formation by assisting neovascularization in vivo in a porcine model. Zhu et al.¹⁷ demonstrated that seeding ADSCs onto BAM promotes regeneration of smooth muscle and nerve, and simultaneously increases bladder capacity in a rabbit model. These effects are more evident in seeded BAM than unseeded BAM. Drewa et al.⁶⁸ cultured rat hair follicle stem cells (HFSCs) on a BAM scaffold for grafting into surgically created defects within the anterior bladder wall in a rat model. Bladder reconstruction with HFSC-seeded BAM decreased stone development, and increased the thickness of the muscle layers compared with BAMs alone.

The choice of animal models for bladder augmentation

Evidence of successful new therapeutic approaches in animal models is crucial for the prediction of success in clinical applications. Animal models are chosen to closely mimic the human clinical situation in order to facilitate extrapolation of the preclinical data as much as possible. Usually, small animals are used before proceeding into large animal, and this progression is reflected in many BAM-based bladder augmentation studies. Some groups primarily used the rat models; however, the obvious size differences between these animals and humans presented a challenge. Some authors prefer rabbits as an animal model to perform bladder augmentation. However, the urine of rabbits has a high level of urate and is easy to form stone and encrustation, which might be a relative contraindication to use rabbits in a bladder augmentation model to test biomaterial. The advantage of the rabbit model is the potential to evaluate modulations of the biomaterial to prevent stones and encrustation.⁷⁵

Although both seeded and unseeded BAMs have shown some success in small animal models, these therapies have often failed when tested on larger animals such as dogs and pigs. In larger animals, challenges such as insufficient vascularization, graft shrinkage due to rapid ingrowth of fibroblasts, and limited smooth muscle development became more apparent.^49,70,76 The dog and pig models are well accepted as a surgical model for bladder augmentation, as the size and anatomy is more similar to the human. We and other groups have confirmed that pre-seeded BAMs and rapid establishment of vascular ingrowth are essential for successful bladder reconstruction in the larger animals.^36,58

Future Prospectives of BAM's Application in Bladder Augmentation

Although advances have been made with BAM scaffolds for tissue-engineered bladder regeneration, many challenges remain. First, there are graft size limitations. Grafts longer than 1 cm from any edge do not tend to achieve adequate tissue regeneration. Studies have shown that the addition of growth factors such as VEGF, bFGF, or PDGF can improve specific functional parameters. Pluripotent stem cells and specific bioactive factors combined with the modified BAM may be a valuable way to achieve full tissue regeneration. Future studies directed at BAM functionality may be able to improve the development of the grafts for clinical application. Second, the characteristics of BAM used in the bladder reconstruction are critical for the success of these technologies. BAM prepared from different species or prepared using different methods have been shown to have certain distinctive biological properties. The highly variable methods for BAM preparation and their consequent effects to bladder augmentation are still not very clear. In previous studies, autologous BAM were often used; however, it seems more clinically significant to apply heterologous BAM, which are worthy of investigation, to make the heterologous BAM safe and functional for clinical bladder reconstruction. Third, from the point of ethical consideration, the majority of bladder diseases such as neurogenic bladder, posterior urethral valves, and bladder exstrophy belong to pediatric urological conditions. In the BAM-based bladder augmentation, especially with stem cells seeding or exogenous growth factors loading, something such as tumorgenicity may be a very real concern, as these adverse events may occur in the distant future, arranging for such a long surveillance period may prove to be a challenge. It is imperative to highlight that there are legitimate concerns regarding the long-term fate of BAMs and stem cells before human trials can proceed.⁷⁷

Conclusions

The studies described in this article demonstrate that BAM provides the necessary environment to promote cell migration, growth, and differentiation. With the indepth research in this area, BAM-based bladder augmentation may prove to be an effective surgical alternative to enterocystoplasty in the treatment of bladder dysfunction and become an “off the shelf” bladder substitute for patients.

Disclosure Statement

No competing financial interests exist.

References

Castellan

, Gosalbez

, Bar-Yosef

, and Labbie

Complications after use of gastric segments for lower urinary tract reconstruction. J Urol, 187, 1823, 2012.

Atala

, Bauer

S.B.

, Soker

, Yoo

J.J.

, and Retik

A.B.

Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 367, 1241, 2006.

Salem

S.A.

, Hwei

N.M.

, Saim

A.B.

, Ho

C.C.

, Sagap

, Singh

, et al. Polylactic-co-glycolic acid mesh coated with fibrin or collagen and biological adhesive substance as a prefabricated, degradable, biocompatible, and functional scaffold for regeneration of the urinary bladder wall. J Biomed Mater Res A, 101, 2237, 2013.

Jayo

M.J.

, Jain

, Wagner

B.J.

, and Bertram

T.A.

Early cellular and stromal responses in regeneration versus repair of a mammalian bladder using autologous cell and biodegradable scaffold technologies. J Urol, 180, 392, 2008.

Zhang

, Lin

H.K.

, Frimberger

, Epstein

R.B.

, and Kropp

B.P.

Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder. BJU Int, 96, 1120, 2005.

Zhang

, Kropp

B.P.

, Moore

, Cowan

, Furness

P.D.

3rd , Kolligian

M.E.

, et al. Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology. J Urol, 164, 928, discussion 34, 2000.

Atala

Bioengineered tissues for urogenital repair in children. Pediatr Res, 63, 569, 2008.

Bolland

, Korossis

, Wilshaw

S.P.

, Ingham

, Fisher

, Kearney

J.N.

, et al. Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering. Biomaterials, 28, 1061, 2007.

Gilbert

T.W.

, Sellaro

T.L.

, and Badylak

S.F.

Decellularization of tissues and organs. Biomaterials, 27, 3675, 2006.

10.

Soler

, Fullhase

, and Atala

Regenerative medicine strategies for treatment of neurogenic bladder. Therapy, 6, 177, 2009.

11.

Farhat

W.A.

Bladder regeneration: great potential but challenges remain. Regen Med, 6, 537, 2011.

12.

Kikuno

, Kawamoto

, Hirata

, Vejdani

, Kawakami

, Fandel

, et al. Nerve growth factor combined with vascular endothelial growth factor enhances regeneration of bladder acellular matrix graft in spinal cord injury-induced neurogenic rat bladder. BJU Int, 103, 1424, 2009.

13.

, and Cao

Y.L.

Tissue engineering and stem cell application of urethroplasty: from bench to bedside. Urology, 79, 246, 2012.

14.

, Xu

Y.M.

, Song

L.J.

, Fu

, Cui

, and Yin

Urethral reconstruction using oral keratinocyte seeded bladder acellular matrix grafts. J Urol, 180, 1538, 2008.

15.

, Deng

C.L.

, Liu

, and Cao

Y.L.

Urethral replacement using epidermal cell-seeded tubular acellular bladder collagen matrix. BJU Int, 99, 1162, 2007.

16.

Brown

A.L.

, Brook-Allred

T.T.

, Waddell

J.E.

, White

, Werkmeister

J.A.

, Ramshaw

J.A.

, et al. Bladder acellular matrix as a substrate for studying in vitro bladder smooth muscle-urothelial cell interactions. Biomaterials, 26, 529, 2005.

17.

Zhu

W.D.

, Xu

Y.M.

, Feng

, Fu

, Song

L.J.

, and Cui

Bladder reconstruction with adipose-derived stem cell-seeded bladder acellular matrix grafts improve morphology composition. World J Urol, 28, 493, 2010.

18.

Kajbafzadeh

A.M.

, Payabvash

, Salmasi

A.H.

, Sadeghi

, Elmi

, Vejdani

, et al. Time-dependent neovasculogenesis and regeneration of different bladder wall components in the bladder acellular matrix graft in rats. J Surg Res, 139, 189, 2007.

19.

Chen

B.S.

, Zhang

S.L.

, Geng

, Pan

, and Chen

Ex vivo functional evaluation of isolated strips in BAMG tissue-engineered bladders. Int J Artif Organs, 32, 159, 2009.

20.

Zhu

W.D.

, Xu

Y.M.

, Feng

, Fu

, and Song

L.J.

Different bladder defects reconstructed with bladder acellular matrix grafts in a rabbit model. Urologe A, 50, 1420, 2011.

21.

Dahms

S.E.

, Piechota

H.J.

, Dahiya

, Gleason

C.A.

, Hohenfellner

, and Tanagho

E.A.

Bladder acellular matrix graft in rats: its neurophysiologic properties and mRNA expression of growth factors TGF-alpha and TGF-beta. Neurourol Urodyn, 17, 37, 1998.

22.

Piechota

H.J.

, Gleason

C.A.

, Dahms

S.E.

, Dahiya

, Nunes

L.S.

, Lue

T.F.

, et al. Bladder acellular matrix graft: in vivo functional properties of the regenerated rat bladder. Urol Res, 27, 206, 1999.

23.

Sievert

K.D.

, Fandel

, Wefer

, Gleason

C.A.

, Nunes

, Dahiya

, and Tanagho

E.A.

Collagen I:III ratio in canine heterologous bladder acellular matrix grafts. World J Urol, 24, 101, 2006.

24.

Chen

B.S.

, Xie

, Zhang

S.L.

, Geng

H.Q.

, Zhou

J.M.

, Pan

, et al. Tissue engineering of bladder using vascular endothelial growth factor gene-modified endothelial progenitor cells. Int J Artif Organs, 34, 1137, 2011.

25.

Youssif

, Shiina

, Urakami

, Gleason

, Nunes

, Igawa

, et al. ffect of vascular endothelial growth factor on regeneration of bladder acellular matrix graft: histologic and functional evaluation. Urology, 66, 201, 2005.

26.

Rosario

D.J.

, Reilly

G.C.

, Ali Salah

, Glover

, Bullock

A.J.

, and Macneil

Decellularization and sterilization of porcine urinary bladder matrix for tissue engineering in the lower urinary tract. Regen Med, 3, 145, 2008.

27.

Freytes

D.O.

, Stoner

R.M.

, and Badylak

S.F.

Uniaxial and biaxial properties of terminally sterilized porcine urinary bladder matrix scaffolds. J Biomed Mater Res B Appl Biomater, 84, 408, 2008.

28.

Davis

N.F.

, Mooney

, Callanan

, Flood

H.D.

, and McGloughlin

T.M.

Augmentation cystoplasty and extracellular matrix scaffolds: an ex vivo comparative study with autogenous detubularised ileum. PLoS One, 6, e20323, 2011.

29.

Liu

, Li

, Wang

, Xu

, Ge

, and Liang

Evaluation of the biocompatibility and mechanical properties of xenogeneic (porcine) extracellular matrix (ECM) scaffold for pelvic reconstruction. Int Urogynecol J, 22, 221, 2011.

30.

Liu

, Bharadwaj

, Lee

S.J.

, Atala

, and Zhang

Optimization of a natural collagen scaffold to aid cell-matrix penetration for urologic tissue engineering. Biomaterials, 30, 3865, 2009.

31.

Kim

B.S.

, Atala

, and Yoo

J.J.

A collagen matrix derived from bladder can be used to engineer smooth muscle tissue. World J Urol, 26, 307, 2008.

32.

Chun

S.Y.

, Lim

G.J.

, Kwon

T.G.

, Kwak

E.K.

, Kim

B.W.

, Atala

, et al. Identification and characterization of bioactive factors in bladder submucosa matrix. Biomaterials, 28, 4251, 2007.

33.

Pariente

J.L.

, Kim

B.S.

, and Atala

In vitro biocompatibility evaluation of naturally derived and synthetic biomaterials using normal human bladder smooth muscle cells. J Urol, 167, 1867, 2002.

34.

Evren

, Loai

, Antoon

, Islam

, Yeger

, Moore

, et al. Urinary bladder tissue engineering using natural scaffolds in a porcine model: role of Toll-like receptors and impact of biomimetic molecules. Cells Tissues Organs, 192, 250, 2010.

35.

Probst

, Piechota

H.J.

, Dahiya

, and Tanagho

E.A.

Homologous bladder augmentation in dog with the bladder acellular matrix graft. BJU Int, 85, 362, 2000.

36.

Yoo

J.J.

, Meng

, Oberpenning

, and Atala

Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology, 51, 221, 1998.

37.

Piechota

H.J.

, Dahms

S.E.

, Nunes

L.S.

, Dahiya

, Lue

T.F.

, and Tanagho

E.A.

In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft. J Urol, 159, 1717, 1998.

38.

Cayan

, Chermansky

, Schlote

, Sekido

, Nunes

, Dahiya

, et al. The bladder acellular matrix graft in a rat chemical cystitis model: functional and histologic evaluation. J Urol, 168, 798, 2002.

39.

Kanematsu

, Yamamoto

, Noguchi

, Ozeki

, Tabata

, and Ogawa

Bladder regeneration by bladder acellular matrix combined with sustained release of exogenous growth factor. J Urol, 170, 1633, 2003.

40.

Corcos

, Mahfouz

, Loutochin

, and Galipeau

Bone marrow mesenchymal stromal cell therapy for restoration of bladder wall defects. J Urol, 187, e84, 2012.

41.

Meezan

, Hjelle

J.T.

, Brendel

, and Carlson

E.C.

A simple, versatile, nondisruptive method for the isolation of morphologically and chemically pure basement membranes from several tissues. Life Sci, 17, 1721, 1975.

42.

Crapo

P.M.

, Gilbert

T.W.

, and Badylak

S.F.

An overview of tissue and whole organ decellularization processes. Biomaterials, 32, 3233, 2011.

43.

Yang

, Zhang

, Zhou

, Sun

, Zheng

, Chen

, et al. Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng Part C Methods, 16, 1201, 2010.

44.

Brown

B.N.

, Barnes

C.A.

, Kasick

R.T.

, Michel

, Gilbert

T.W.

, Beer-Stolz

, et al. Surface characterization of extracellular matrix scaffolds. Biomaterials, 31, 428, 2010.

45.

Reddy

P.P.

, Barrieras

D.J.

, Wilson

, Bagli

D.J.

, McLorie

G.A.

, Khoury

A.E.

, et al. Regeneration of functional bladder substitutes using large segment acellular matrix allografts in a porcine model. J Urol, 164, 936, 2000.

46.

Wefer

, Sievert

K.D.

, Schlote

, Wefer

A.E.

, Nunes

, Dahiya

, et al. Time dependent smooth muscle regeneration and maturation in a bladder acellular matrix graft: histological studies and in vivo functional evaluation. J Urol, 165, 1755, 2001.

47.

Merguerian

P.A.

, Reddy

P.P.

, Barrieras

D.J.

, Wilson

G.J.

, Woodhouse

, Bagli

D.J.

, et al. Acellular bladder matrix allografts in the regeneration of functional bladders: evaluation of large-segment (>24 cm) substitution in a porcine model. BJU Int, 85, 894, 2000.

48.

Hodde

Naturally occurring scaffolds for soft tissue repair and regeneration. Tissue Eng, 8, 295, 2002.

49.

Brown

A.L.

, Farhat

, Merguerian

P.A.

, Wilson

G.J.

, Khoury

A.E.

, and Woodhouse

K.A.

22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model. Biomaterials, 23, 2179, 2002.

50.

Farhat

W.A.

, Chen

, Haig

, Antoon

, Litman

, Sherman

, et al. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties. Biomed Mater, 3, 025015, 2008.

51.

Gandhi

N.S.

, and Mancera

R.L.

The structure of glycosaminoglycans and their interactions with proteins. Chem Biol Drug Des, 72, 455, 2008.

52.

Gratzer

P.F.

, Harrison

R.D.

, and Woods

Matrix alteration and not residual sodium dodecyl sulfate cytotoxicity affects the cellular repopulation of a decellularized matrix. Tissue Eng, 12, 2975, 2006.

53.

Freytes

D.O.

, Martin

, Velankar

S.S.

, Lee

A.S.

, and Badylak

S.F.

Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix. Biomaterials, 29, 1630, 2008.

54.

Nomi

, Atala

, Coppi

P.D.

, and Soker

Principals of neovascularization for tissue engineering. Mol Aspects Med, 23, 463, 2002.

55.

Singh

, Berkland

, and Detamore

M.S.

Strategies and applications for incorporating physical and chemical signal gradients in tissue engineering. Tissue Eng Part B Rev, 14, 341, 2008.

56.

Kanematsu

, Yamamoto

, Ozeki

, Noguchi

, Kanatani

, Ogawa

, et al. Collagenous matrices as release carriers of exogenous growth factors. Biomaterials, 25, 4513, 2004.

57.

Cartwright

L.M.

, Shou

, Yeger

, and Farhat

W.A.

Porcine bladder acellular matrix porosity: impact of hyaluronic acid and lyophilization. J Biomed Mater Res A, 77, 180, 2006.

58.

Loai

, Yeger

, Coz

, Antoon

, Islam

S.S.

, Moore

, et al. Bladder tissue engineering: tissue regeneration and neovascularization of HA-VEGF-incorporated bladder acellular constructs in mouse and porcine animal models. J Biomed Mater Res A, 94, 1205, 2010.

59.

Prestwich

G.D.

Hyaluronic acid-based clinical biomaterials derived for cell and molecule delivery in regenerative medicine. J Control Release, 155, 193, 2011.

60.

Cartwright

, Farhat

W.A.

, Sherman

, Chen

, Babyn

, Yeger

, et al. Dynamic contrast-enhanced MRI to quantify VEGF-enhanced tissue-engineered bladder graft neovascularization: pilot study. J Biomed Mater Res A, 77, 390, 2006.

61.

Geng

, Song

, Qi

, and Cui

Sustained release of VEGF from PLGA nanoparticles embedded thermo-sensitive hydrogel in full-thickness porcine bladder acellular matrix. Nanoscale Res Lett, 6, 312, 2011.

62.

Sutherland

R.S.

, Baskin

L.S.

, Hayward

S.W.

, and Cunha

G.R.

Regeneration of bladder urothelium, smooth muscle, blood vessels and nerves into an acellular tissue matrix. J Urol, 156, 571, 1996.

63.

Piechota

H.J.

, Dahms

S.E.

, Probst

, Gleason

C.A.

, Nunes

L.S.

, Dahiya

, Lue

T.F.

, and Tanagho

E.A.

Functional rat bladder regeneration through xenotransplantation of the bladder acellular matrix graft. Br J Urol, 81, 548, 1998.

64.

Obara

, Matsuura

, Narita

, Satoh

, Tsuchiya

, and Habuchi

Bladder acellular matrix grafting regenerates urinary bladder in the spinal cord injury rat. Urology, 68, 892, 2006.

65.

Urakami

, Shiina

, Enokida

, Kawamoto

, Kikuno

, Fandel

, et al. Functional improvement in spinal cord injury-induced neurogenic bladder by bladder augmentation using bladder acellular matrix graft in the rat. World J Urol, 25, 207, 2007.

66.

Kanematsu

, Yamamoto

, and Ogawa

Changing concepts of bladder regeneration. Int J Urol, 14, 673, 2007.

67.

Zhou

, Yang

, Sun

, Qiu

, Sun

, Chen

, Zhang

, and Dai

Coadministration of platelet-derived growth factor-BB and vascular endothelial growth factor with bladder acellular matrix enhances smooth muscle regeneration and vascularization for bladder augmentation in a rabbit model. Tissue Eng Part A, 19, 264, 2013.

68.

Drewa

, Joachimiak

, Kaznica

, Sarafian

, and Pokrywczynska

Hair stem cells for bladder regeneration in rats: preliminary results. Transplant Proc, 41, 4345, 2009.

69.

Zhang

, and Atala

Urothelial cell culture: stratified urothelial sheet and three-dimensional growth of urothelial structure. Methods Mol Biol, 945, 383, 2013.

70.

Horst

, Madduri

, Gobet

, Sulser

, Milleret

, Hall

, et al. Engineering functional bladder tissues. J Tissue Eng Regen Med, 7, 515, 2013.

71.

De Coppi

, Callegari

, Chiavegato

, Gasparotto

, Piccoli

, Taiani

, et al. Amniotic fluid and bone marrow derived mesenchymal stem cells can be converted to smooth muscle cells in the cryo-injured rat bladder and prevent compensatory hypertrophy of surviving smooth muscle cells. J Urol, 177, 369, 2007.

72.

Aitken

K.J.

, and Bagli

D.J.

The bladder extracellular matrix. Part I: architecture, development and disease. Nat Rev Urol, 6, 596, 2009.

73.

Kanematsu

, Yamamoto

, Iwai-Kanai

, Kanatani

, Imamura

, Adam

R.M.

, et al. Induction of smooth muscle cell-like phenotype in marrow-derived cells among regenerating urinary bladder smooth muscle cells. Am J Pathol, 166, 565, 2005.

74.

Tian

, Bharadwaj

, Liu

, Ma

P.X.

, Atala

, et al. Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering. Biomaterials, 31, 870, 2009.

75.

Nuininga

J.E.

, van Moerkerk

, Hanssen

, et al. A rabbit model to tissue engineer the bladder. Biomaterials, 25, 1657, 2004.

76.

Sievert

K.D.

, Amend

, and Stenzl

Tissue engineering for the lower urinary tract: a review of a state of the art approach. Eur Urol, 52, 1580, 2007.

77.

Oerlemans

A.J.

, Feitz

W.F.

, van Leeuwen

, and Dekkers

W.J.

Regenerative urology clinical trials: an ethical assessment of road blocks and solutions. Tissue Eng Part B Rev, 19, 41, 2013.

78.

Mitsui

, Shiina

, Hiraoka

, Arichi

, Yasumoto

, Dahiya

, Tanagho

EA.

, and Igawa

Simultaneous implantation of bilateral ureters into bladder acellular matrix graft after partial cystectomy in a porcine model. BJU Int, 110, E1212, 2012.