Acellular matrix in urethral reconstruction

ADR-12715; No of Pages 9 Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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Acellular matrix in urethral reconstruction☆ Leopoldo Alves Ribeiro-Filho a,⁎, Karl-Dietrich Sievert b a b

Division of Urology, University of São Paulo, São Paulo, Brazil Department of Urology, University of Lübeck, Lübeck, Germany

a r t i c l e

i n f o

a b s t r a c t The treatment of severe urethral stenosis has always been a challenge even for skilled urologists. Classic urethroplasty, skin flaps and buccal mucosa grafting may not be used for long and complex strictures. In the quest for an ideal urethral substitute, acellular scaffolds have demonstrated the ability to induce tissue regeneration layer by layer. After several experimental studies, the use of acellular matrices for urethral reconstruction has become a clinical reality over the last decade. In this review we analyze advantages and limitations of both biological and polymeric scaffolds that have been reported in experimental and human studies. Important aspects such as graft extension, surgical technique and cell-seeding versus cell-free grafts will be discussed. © 2014 Elsevier B.V. All rights reserved.

Available online xxxx Keywords: Tissue engineering Scaffold Graft Regeneration Urethroplasty

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . Acellular matrix: principles and rationale Types of matrices . . . . . . . . . . Cell-free versus cell-seeded matrices . . Animal models . . . . . . . . . . . 5.1. Cell-free . . . . . . . . . . . 5.2. Cell-seeded . . . . . . . . . . 6. Human studies . . . . . . . . . . . 6.1. Cell-free . . . . . . . . . . . 6.2. Cell-seeded . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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1. Introduction Although the male urethra is a single tubular structure, it is composed by various heterogeneous segments: prostatic, membranous and spongy urethra. Each part has very distinct characteristics. The prostatic urethra is lined with transitional cell epithelium (urothelium)

Abbreviations:LS, Lichensclerosis; PGA, polyglycolicacid; SIS, small intestinal submucosa; GAG, glycosaminoglycan; UAMG, urethral acellular matrix grafts; ECM, extracellular matrix. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Regenerative Medicine Strategies in Urology”. ⁎ Corresponding author at: Av. Dr. Enéas de Carvalho Aguiar, 255, 7° andar, sala 710 F Cerqueira César, São Paulo, SP 05403-000, Brazil. E-mail address: leopoldofi[email protected] (L.A. Ribeiro-Filho).

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0 0 0 0 0 0 0 0 0 0 0 0

and is surrounded by smooth muscle layers and prostatic tissue. The prostatic urethra is usually affected by benign prostatic hyperplasia, but rarely becomes a target for reconstructive urology. On the other hand, the membranous urethra, which spans from the apex of the prostate to the bulbar region, is firmly fixed to the ischio and pubis, rendering this portion of the urethra susceptible to disruption with pelvic fractures. The spongy urethra is located concentrically within the corpus spongiosum and is divided into bulbar and pendulous segments. Blunt straddle injury to the perineum, for instance, is often a common cause of bulbar strictures. Both membranous and spongy urethras are lined with stratified columnar and pseudostratified epithelium. Stratified squamous epithelium is seen distally close to the meatus [1,2]. Besides trauma, infections, inflammatory conditions, congenital malformations, cancer and also iatrogenic lesions may induce scar tissue deposition and, consequently, promote narrowing or even complete

http://dx.doi.org/10.1016/j.addr.2014.11.019 0169-409X/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019

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L.A. Ribeiro-Filho, K.-D. Sievert / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

blockage of the urethral canal. Severe urethral stenosis may have devastating consequences in terms of quality of life and may lead to renal failure and infertility, since both urine and semen flow may be interrupted. Urethral reconstruction has always been a challenge even for skilled urologists. The type of urethroplasty depends on stricture cause, stricture length determined by retrograde urethrogram, location and number of previous urethral surgeries. In terms of urethral reconstruction, success is usually defined as a patent urethra with no need for further invasive or surgical interventions such as dilatation or optical urethrotomy [3]. This strict concept was used to establish the urethroplasty success rate in this review. All other outcomes, ranging from partial to complete restenosis, were considered failures. Success rates vary dramatically (70–90%) and a considerable number of patients need to undergo multiple procedures [4]. After recurrent surgical failures, in some dramatic cases, when classical operative techniques have been exhausted, abdominal urinary diversion may be indicated [5]. Classical treatment options for severe urethral strictures include vascularized skin flaps, full-thickness free skin grafts, or buccal mucosa grafts, but there is no single technique that is appropriate for all stricture cases [6]. Patients with Lichen sclerosis (LS), a chronic inflammatory condition of unknown etiology that induces progressive preputial, glanular and urethral fibrosis, for instance, should not undergo local skin flap procedures due to the high risk of restenosis secondary to eventual LS progression onto the flap [7]. Although buccal mucosa has become the most commonly used substitute material in the treatment of urethral strictures and its success is documented in numerous series of patients who have undergone urethral reconstruction [8], donor site availability represents its principal limitation. Some authors indicate buccal mucosa to treat long urethral strictures; however, harvesting multiple grafts simultaneously may increase morbidity, especially in those who are heavy smokers [9]. Literature on hypospadia, a congenital defect in which the incomplete development of the urethra results in an ectopic meatus located on the ventral part of the penile shaft/perineum proximal to the tip of the glans penis, has hundreds of different repair techniques described, but salvage of a multi-operated and skindeficient ‘cripple’ case is often hard to achieve [10]. For decades, urologists have tested many other different autologous tissues for urethral reconstruction: artery [11], vein [12], ureter [13], appendix [14], tunica vaginalis [15], bladder mucosa [16], and even lyophilized human dura mater [17]. Unfortunately, results of these numerous creative attempts were disappointing. Furthermore, the large number of reports published on different biological tissues indicates that finding the ideal urethral substitute has always been a main goal and a difficult task for reconstructive urology. Over the last two decades, with the advances in the field of tissue engineering, multiple strategies have been proposed for urethral regeneration. In this review, we will focus on the use of acellular matrices for urethral reconstruction. 2. Acellular matrix: principles and rationale The ideal material for urethral replacement must act as a frame for the progressive in-growth of all host bladder wall components and finally become an integrated part of the urethral wall with the same mechanical and functional properties as the host. Furthermore, this biomaterial must not elicit immune response, fibrosis or tissue contraction. In other words, the ideal urethral substitute should provide the creation of a suitable microenvironment for tissue regeneration. Tissue regeneration is well documented in certain reptiles and amphibians. Salamanders, for example, have the ability to regenerate amputated limbs by formation of a mound of progenitor cells called the limb blastema [18]. Apparently, on the other hand, until recently, mammal regenerative capabilities had been considered lost during the evolutionary process. Nevertheless, in 1982, Yannas demonstrated that a graft based on a collagen scaffold was able to induce dermic regeneration in full-thickness wounds in adult guinea pigs [19]. This

represented solid evidence that tissue regeneration in mammals was limited but still present. Several independent studies using decellularized matrices reported promising results with a large variety of organs. Badylak, in 1989, successfully tested the use of autogenous small intestinal submucosa matrix (SIS) as a vascular graft in the infrarenal aorta of 12 dogs [20]. Significant regenerative results were obtained in large abdominal wall defects [21], larynx [22], bladder [23] and urethra [24]. In the decellularization process, cells and antigenic epitopes are removed from native tissue, resulting in a 3-D structure that is basically constituted by collagen fibers and, thus, immunologically welltolerated [25]. Since the mechanisms by which biological scaffold materials promote appropriate tissue regeneration are still not well understood, there is a justifiable controversy concerning the relevant importance of the composition vs. structure of these materials. In terms of composition, besides different types of collagen, biological acellular matrices used for urethral reconstruction also contain elastin and a variety of glycosaminoglycans (GAGs), including heparin, heparin sulfate, chondroitin sulfate and hyaluronic acid [26–28]. Elastin is a crucial component of the extracellular matrix that not only preserves the native structure of soft tissues, based on the property of elastic recoil following deformation, but also regulates the cellular response via biomechanical transduction to maintain tissue homeostasis. Since most adult cells present a poor ability to synthesize elastin and to remodel elastic matrix structures, a biological scaffold that preserves elastin content after its preparation may improve adequate tissue regeneration when compared, for instance, to synthetic scaffolds [29]. Although, the absence of cell-synthesized elastin potentially can be resolved with techniques that incorporate recombinant elastin and synthetic elastin-like peptides into artificial constructs [30], tissue homeostasis may be impacted adversely at the same time, specifically due to the lack of several microfibrillar components of native elastic fibers, which, together with elastin, enable cell–fiber interactions and modulate cell behavior [31]. The decellularization method may determine the amount of GAGs remaining in a treated tissue. For example, ionic detergents often used in the decellularization process can remove GAGs from the matrix [32]. Other components found in an acellular matrix are: adhesion molecules such as fibronectin and laminin, proteoglycans, glycoproteins, and various growth factors such as transforming growth factorb, basic fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF) [33]. Several of these growth factors have been shown to retain their bioactivity even after terminal sterilization and long-term storage [34]. On the other hand, the ultrastructure and 3-D architecture of collagen fibers of the matrix play important roles in modulating the cells' ability to migrate into the scaffold [35] or by influencing tissue specific cell phenotype [36]. For instance, acellular matrices can dramatically affect the differentiation pathway of human embryonic stem cells and selected progenitor cell populations [36]. In summary, biological acellular matrix scaffolds have a complex composition with a variety of active molecules and also a peculiar ultrastructure that together seem to play an important role in tissue regeneration. 3. Types of matrices The term acellular matrix may be applied to different types of scaffolds generally used in regenerative medicine. Biological matrices may be animal- or human-derived, with all cells removed from the original organ during preparation. Different decellularization methods (physical, chemical, and enzymatic) may be used. Obviously, different protocols have distinct effects on the extent of cell removal and extracellular matrix (ECM) composition and structure [37]. In other words, the same type of tissue treated by different decellularization protocols may present variations in GAGs and growth factor composition, resulting in distinct regenerative profiles. Feil et al. examined adherence and viability of human urothelial cells seeded on commercially available small intestine submucosa (SIS) [38]. In comparison with the good

Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019

L.A. Ribeiro-Filho, K.-D. Sievert / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

published results of SIS processed in the laboratory [39], there is an evident difference in commercially available, end-processed SIS maybe as a result of biosafety concerns. Clinical grade SIS production includes extra steps such as freeze-drying, ethylene oxide gas sterilization and pH reduction in order to inactivate porcine and murine viruses. Besides that, the high amount of porcine DNA residues detected in industrymade SIS may also be responsible for some of the cytotoxic effects observed on urothelial cells. On the other hand, acellular matrices consist of the structural and functional molecules secreted by the resident cells of each tissue and organ from which they are derived. Therefore, the specific composition and distribution of the ECM constituents will vary depending on the tissue source [40]. This is the rationale for studies based on organ-specific allografts defended by Tanagho and Dahiya [23, 41]. The use of biological matrices raise legal and ethical issues regarding human organ donation and this may be a major drawback in some countries [42]. Some authors also comment on the possibility of disease transmission, although it has never been well-documented. Actually, decellularization protocols routinely use hyperosmolar solutions and enzymes that kill the vast majority of infectious agents. At the University of São Paulo, Brazil, we had performed more than 200 urethral reconstructions with acellular matrices in humans over the last 10 years. These matrices were produced at our lab by enzymatic conversion of human cadaveric urethras and bladders with a protocol based on the use of DNAses. All pre-operative matrix cultures were negative and there was no evidence of graft–host transmission of any kind of infectious disease to the present time [41]. Synthetic matrices are totally manufactured polymer-based scaffolds. Some authors have tested non-degradable materials, such as silicone [43] and polytetrafluoroethylene (PTFE) [44] for urethral reconstruction with poor results. They reported erosion, calcification, and fistula formation. There was no adequate tissue regeneration. On the contrary, scaffolds made of biodegradable polymers such as polyglycolic acid (PGA), polylactic acid (PLA), and polylactic coglycolic acid (PLGA) have produced some good results when used for urethral reconstruction specially on a cell-seeded human model, since just one patient out of five needed transurethral incision [45]. These biomaterials are degraded by hydrolysis and particles removed through metabolic pathways [46]. The degradation products of synthetic polymeric materials, such as PLGA, are usually acidic, a fact that may interfere with the adequate growth of the surrounding cells [47]. Synthetic polymers have peculiar characteristics that enable researchers to reproduce the 3D shape of organs, control the porosity and mechanical properties of the matrix, in a quantitative and reproducible way, and at relatively low cost [48]. Biomaterial technology has greatly evolved in recent years to produce scaffolds that can be used to direct cell phenotype and behavior by providing a specific cellular microenvironment that mimics the characteristics of native ECM [49]. Emerging nanoparticle delivery systems [50], for instance, may contribute to influence several scaffold parameters such as cell adhesion regulation [51], spatio-temporal availability of typical ECM-bound cell signaling growth factors [52], and scaffold biodegradation rate [53]. However, the promising potential of these technologies has not yet been fully achieved probably due to the extreme biocomplexity of the ECM composition, structure and cell-interactions. Finally, one major advantage of polymeric scaffolds is the lack of concerns about legal aspects of human tissue donation.

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cells for tissue regeneration appears to be 0.5 cm”. This conclusion may be partially true for tubularized bladder submucosa acellular grafts, but may not be extrapolated to onlay organ-specific reconstructions (Fig. 2). Most researches in regenerative urology use rabbits for urethral reconstruction protocols. We had previously worked with rabbits for matrix experimental studies, but recently we prefer murine or porcine models. Rabbits often produce milky or cloudy urine due to high levels of calcium present in many commercial feeding pellets [55]. Acellular matrices have a strong adsorption capacity. Salts present in urine and even the cotton filaments of gauze and sponges used in surgery can be easily impregnated into the collagen network. Furthermore, hypercalciuria and hyperuricosuria were associated with a higher failure rate in our human patients who had undergone urethral reconstruction with acellular matrices [56]. In summary, several aspects may influence the regeneration range in acellular scaffolds such as matrix preparation protocol, host urine salt composition, matrix origin (organ-specific vs. non-specific), infection and surgical technique. Some authors suggest that the use of cell-free matrix should only be indicated in short strictures when a healthy and well vascularized part of urethral wall exists [48]. But this situation is rarely seen in multi-operated patients who represent the main target of regenerative urology. Actually, at our institution, we have used human cadaveric urethral acellular matrices grafting as a salvage procedure to treat very long and complex strictures (3 to 25 cm) with a success rate of 75% [56]. An essential point to mention is that, in all these cases, urethral reconstruction was performed as an onlay ventral grafting procedure (Fig. 2). Since urethral graft width is around 1.5 cm (regardless the length of the stricture), the most central part of the graft will be within less than 1.0 cm from any of the edges of the native urethra. Several authors consider 1.0–1.5 cm as an average range for the adequate regeneration onto the acellular matrix in mammals (see Table 1). Our experience in organ-specific matrices for bladder augmentation in humans revealed that a regeneration range of 2.0 cm from the borders of the graft is usually reached in those patients [57]. The cell-free matrices have some very attractive advantages such as ease of production, storage, and transportation and may be considered an “off the shelf” material. Since additional surgical procedures for graft harvesting may not be needed, operative time and morbidity may be reduced. Homologous (cadaveric) or heterologous tissue maybe used for matrix preparation, since all cells will be removed. Cells, membranes and DNA induce a strong immune response. Since acellular matrix is cell-free, the treated scaffold does not elicit immune reaction. On the other hand, heterologous or even homologous cells, if seeded, will induce tissue rejection. In humans, only autologous cells have been used for urethral tissue engineering.

4. Cell-free versus cell-seeded matrices One important aspect of the scaffold-based urethroplasties that often generates some debate among researchers is the use of cell-free versus cell-seeded matrices. Dorin et al. [54] published results on tubularized urethroplasties (Fig. 1) performed in 12 male rabbits using acellular matrices of bladder submucosa at varying lengths (0.5, 1, 2, and 3 cm). They concluded that “the maximum defect distance suitable for normal tissue formation using acellular grafts that rely on the native

Fig. 1. Tubular grafting procedure. A. The stenotic urethral segment is completely excised. B. A tubular graft is sutured, replacing the excised segment. Final aspect of a tubular grafting urethroplasty.

Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019

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L.A. Ribeiro-Filho, K.-D. Sievert / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

Fig. 2. Ventral onlay grafting procedure. A. A longitudinal urethral incision is made through the stenotic segment. B. The graft is sutured covering the created defect. There is no excision of native urethral tissue. C. Final aspect of an onlay grafting urethroplasty.

Cell-seeded matrices represent a very attractive strategy especially for tubular implants [45]. Biological scaffolds have been seeded with several different types of cells such as mesothelial (collected from omentum) [58], autologous bladder epithelial and smooth muscle cells [59], and bone marrow mesenchymal stem cell [60]. Coculture protocols may have some advantages. Oral keratinocytes are easily harvested and could be converted to the uroepithelium in a urological environment. Fibroblasts, for instance, could improve the mechanical properties of the grafts with efficiency in keratinocyte expansion, possibly by the release of growth factors, cytokines and different types of collagen [61]. However, cell-seeded scaffolds, although promising in initial clinical trials, consist in extremely complex procedures in terms of logistics. If urothelial cells are selected to be used (preferred by most of the authors) [45], for instance, for each patient treated, a cystoscopy is required for harvesting autologous urothelial tissue from the bladder. Clean cell culture facilities are mandatory, especially for human treatment. Once the construct has been adequately cellularized, prompt surgical implantation must be scheduled, since delays may lead to graft failure. The total cost of a cellseeded graft can be up to six times greater than that of cell-free matrix [62]. Thus, the future clinical use of cell-seeded matrices may be restricted to a selected group of patients.

5. Animal models 5.1. Cell-free Porcine small-intestinal submucosa (SIS) was used by Kropp et al., in 1998, for urethral repair in a rabbit model to determine whether this material might promote tissue regeneration. The SIS onlay grafts were shown to induce the regeneration of normal rabbit epithelium supported by a vascularized collagen and smooth-muscle backing [39]. In 1999, Chen et al. reported good results on porcine acellular bladder submucosa matrix grafting for urethral reconstruction in 10 rabbits. A ventral segment measuring 1 × 0.7 cm (approximately one half of the urethral circumference) was excised in all rabbits. The acellular collagen matrix was trimmed and placed over the urethral defect in an onlay fashion. Urethral stents were not used. All animals demonstrated a patent and functioning urethra, as evidenced by radiographic and histologic analyses. The urethral segments reconstructed with the acellular matrices showed a normal cellular organization, indistinguishable from the native urethral tissue. Graft contracture or strictures did not occur in any of the animals [63]. After converting rabbit thoracic aorta into a homologous acellular matrix, Parnigotto et al. [64], in 2000, seeded the scaffolds with

Table 1 Experimental studies with acellular matrices. Author

Year

Type of matrix

Source of matrix

Animal model

Cell-seeded

Graft extension

Technique

Success ratea

Kropp [39] Chen [63] Parnigotto [64] Sievert [23] Sievert [24] Shokeir [65] Shokeir [66] Fu [69] Dorin [54] Nuininga [70] Feng [67]

1988 1999 2000 2000 2001 2003 2004 2007 2008 2010 2011

Biological Biological Biological Biological Biological Biological Biological Biological Biological Synthetic Biological

SIS Porcine bladder submucosa Homologous aorta Homologous urethra Canine urethra Homologous urethra Homologous urethra Homologous bladder submucosa Homologous bladder submucosa Collagen type I Porcine urethra

Rabbit Rabbit Rabbit Rabbit Rabbit Canine Canine Rabbit Rabbit Rabbit Rabbit

1.0 cm 1.0 cm 1.0 cm 1.0–1.5 cm 1.0–1.5 cm 3.0 cm 3.0 cm 1.5 cm 0.5–3.0 cm 1.0 cm 1.5 cm

Onlay Onlay Tubular Tubular Tubular On lay Tubular Tubular Tubular Tubular Onlay

8/8 (100%) 10/10 (100%) 14/14 (100%) 30/30 (100%) 14/14 (100%) 13/13 (100%) 0/14 (0%) 9/9 (100%) 3/12 (25%) 18/18 (100%) 6/6 (100%)

Gu [58] De Filippo [59] Orabi [71] Xie [72]

2012 2012 2013 2013

Biological Biological Biological Synthetic

Homologous bladder Homologous bladder Heterologous bladder Silk fibroin

Rabbit Rabbit Canine Canine

No No Yes (urothelial) No No No No Yes (epidermal) No No (but with growth factors) Yes (corporal smooth muscle cells and lingual keratinocytes) Yes (mesothelial) Yes (urothelial + detrusor) Yes (urothelial + detrusor) Yes (keratinocytes and fibroblasts)

1.5 cm 1.0 cm 6.0 cm 5.0 cm

Tubular Tubular Tubular Onlay

9/9 (100%) 9/9 (100%) 15/15 (100%) 5/5 (100%)

a

Success rates based on urethral patency rates provided by authors.

Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019

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urothelial cells. Fourteen rabbits had 1.0 cm of their pendulous urethras excised and substituted by the tubular cell-seeded matrices in end-toend anastomosis. Regeneration was observed in all implanted matrices, with no rejection and minimal fibrosis. Although they had cell-seeded the scaffolds, there was a very emblematic statement on their paper: “coverage of the matrix with urothelial cells, however, does not seem to be mandatory before the implant, because at 10 days after surgery the implanted area already appeared completely covered by urothelium, formed by epithelial cell migration from the edges of the graft”. Also in 2000, Sievert et al. reported the first experimental study on organ-specific acellular matrix for urethral reconstruction [23]. Thirty rabbits received a homologous tubular urethral scaffold (1.0–1.5 cm). All tissue components were seen in the grafted matrix after 3 months, with further improvement over time; however, the smooth muscle in the matrix was less than in normal rabbit urethra and was not well oriented. The following year, the same author compared homologous and heterologous matrices to determine if this fact would imply differences in tissue regeneration [24]. At 6 months more than a third of the homologous grafts had smooth muscle bundles but the heterologous grafts had only poorly disseminated smooth muscle. These findings suggest that homologous organ-specific matrices may induce better tissue regeneration. In an interesting experimental study, Shokeir et al. [65] excised half of the circumference of a 3 cm segment in 21 male dogs. In 13 dogs the defect was covered by an organ-specific acellular matrix on an onlay fashion and in 8 animals the urethral defect was not covered by any type of tissue. Defect regeneration was complete in the study group by 20 weeks. Surprisingly, the control group also showed partial tissue regeneration with minimal fibrosis, showing that the urethra may have outstanding regeneration capabilities. On the other hand, when they tried to use a tubular organ-specific graft to reconstruct a 3-cm long segment that was completely excised, failure rate was 100% [66]. These findings confirmed that the presence of a urethral bed is essential for lengthy onlay acellular grafts. Furthermore, long tubular grafts should be avoided. 5.2. Cell-seeded Feng et al. compared the mechanical properties and biocompatibility of biomaterials, including bladder submucosa, small intestinal submucosa (SIS), acellular corpus spongiosum matrix, and PGA, to identify the optimal scaffold for urethral tissue engineering. Cytotoxicity assays, tensile mechanical properties, and pore size were determined. Also, smooth muscle cells were seeded on biomaterials to evaluate differences in cell infiltration. They concluded that acellular corpus spongiosum matrix had better overall performance, indicating that an organ specific matrix may be the more adequate type of scaffold for urethral reconstruction [67]. The same group obtained good results when testing porcine urethral acellular matrices seeded with both autologous corporal smooth muscle cells and lingual keratinocytes (coculture) to regenerate urethral defects in onlay reconstructions in rabbits [68]. In order to evaluate the importance of cell-seeding in urethral reconstruction with tubular biological matrices, Fu et al. [69] compared 2 groups of rabbits. In all animals, a 1.5-cm penile urethral mucosal defect was induced in the anterior urethra. The study group received tubular grafts seeded with autologous foreskin epidermal cells labeled with 5bromo2′-deoxy-uridine (BrdU). This cell proliferation marker was used to identify whether the graft cells are incubated epidermal cells or extensions from surrounding transitional cells. The control group had the urethras reconstructed with cell-free tubular matrices. Histology showed a better regeneration pattern in the cell-seeded group. In the control group, a single layer of transitional epithelium with disorganized muscle fiber bundles in the submucosa was observed. In the study group, immunofluorescence for BrdU confirmed the presence of

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implanted epidermal cells at 2 months after grafting. At 6 months, there were several layers of epidermal cells with no signs of BrdU staining. Some points of this experiment may be highlighted. First, it seems that the limit for tissue regeneration in cell-free tubular grafts is around 1.5 cm. Second, the labeled epidermal cells implanted onto the matrix survived and the graft assumed the structure typical of epidermal rather than transitional cells 6 months after grafting, evidencing that regeneration was induced by the seeded cellular components. Type I collagen biomatrices with and without growth factors (GFs) were constructed with fibrils obtained from pulverized bovine Achilles tendon. Nuininga et al. [70] evaluated in a rabbit model for tubular urethral reconstruction of a 1-cm long defect. The GF-containing biomatrices (vascular endothelial GF, fibroblast GF-2, and heparinbinding epidermal GF) showed an increase in extracellular matrix deposition, neovascularization, urothelium, glands, granulocytes, and fibroblasts, compared with biomatrices without GF. These findings suggest that GF may be used to improve tissue regeneration. Orabi et al. compared the outcomes of cell-seeded versus cell-free tubular constructs for bulbar urethral reconstruction in a canine model. Autologous bladder epithelial and smooth muscle cells from 15 male dogs were grown and seeded onto tubular biological matrices (decellularized porcine bladder). Twenty-one dogs had a 6-cm long segment of bulbar urethra resected and reconstructed with cellseeded (n = 15) and cell-free (n = 6) constructs. While all urethroplasties with cell-seeded constructs were successful, the six control animals that received the cell-free tubular matrix presented with urethral stenosis. Fluorescence cell-labeling was also performed and determined that both cell types were shown to contribute significantly to the multilayered tissue structure formed 3 months after implantation. The authors hypothesized that seeded cells may accelerate the development of an urothelial barrier along the luminal surface of the urethra, preventing urine leakage into the suburothelial tissue and associated fibrosis [71]. Xie et al. [72] performed urethral reconstruction in a canine model using tissue-engineered buccal mucosa based on electrospun silk fibroin matrices with autologous keratinocytes and fibroblasts. Previous studies have shown that silk fibroin has excellent biocompatibility and low inflammatory potential [73]. Onlay grafts were implanted to reconstruct a 5-cm urethral defect created between the bladder and the pubic symphysis. The cocultured silk constructs induced tissue regeneration while the synthetic scaffolds alone resulted in fibrosis and urethral strictures. Table 1 summarizes the scaffold characteristics and results of the key experimental articles mentioned in this review. 6. Human studies 6.1. Cell-free In 2002, Mantovani et al. [74] reported the first use of porcine small intestine submucosa (SIS) in urethral reconstruction in human patients. Four men with long strictures (N10 cm) and a woman with a 3.0 cmlong stricture had undergone SIS grafting procedures on a dorsal onlay fashion. All patients preserved urethral patency at six months of follow-up. The same group published good long-term results in 2010 with a series of 40 patients [75]. Various authors also reported on SIS urethroplasties in humans usually with favorable results. Palminteri et al. had 85% success rate with short-term follow-up (mean 21 months). A dorsal inlay graft (Fig. 3) was performed in 14 cases, ventral onlay graft in 1, and dorsal inlay plus ventral onlay in 5. The three failures were penile repairs with long strictures (over 5 cm) [76]. This group updated their series in 2012, publishing long-term results (71 months) with an overall patency rate of 76% [77]. All patients with strictures longer than 4 cm had failed. For Fiala et al. [78] porcine SIS matrix urethral repair midterm results (31 months) are comparable to skin flaps and mucosal grafts.

Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019

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L.A. Ribeiro-Filho, K.-D. Sievert / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

Fig. 3. Dorsal inlay grafting procedure. A. A longitudinal urethral incision is made through the stenotic segment. B. Dorsal incision is made to release the stenotic rings. The margins of the incised dorsal urethra are dissected from the tunica. C. Graft is sutured covering the dorsal elliptical raw area. D. Final aspect of an inlay grafting urethroplasty.

Forty out of 50 patients (80%) presented very good results. No complications such as rejection, fistula, wound or urinary infections were observed. Failures occurred in the first six months of follow-up. Donkov et al. [79] performed dorsal onlay augmentation urethroplasties with SIS in nine men with strictures 4–6 cm. Only one had re-stricture at 6 months due to urethral infection. Hauser et al. [80] reported poor results with long strictures (3.5 to 9 cm) using SIS. Four out of 5 (80%) patients had a recurrent stricture after a mean follow-up of one year. A randomized comparative study between buccal mucosal and acellular bladder matrix grafts was conducted by el-Kassaby et al. [81] in thirty patients with anterior urethral strictures. The length of the strictures ranged from 2 to 18 cm (average 6.9 cm). All grafts were implanted as ventral onlay patches. The patients were followed for a mean period of 25 months. When the patients had one or no previous interventions, the success rate between both groups was similar (100% for buccal mucosa vs. 89% for acellular matrix). However, if a history of 2 or more previous operations was present, patients treated with a buccal mucosal graft showed a more successful outcome during follow-up (100% for buccal mucosa vs. 33% for acellular matrix). Based on this study, onlay acellular matrix grafts would be well-indicated for early strictures with an apparently healthy urethral bed and minimal spongiofibrosis due to the material being an off-the shelf nature. For this author, multi-operated cases should undergo buccal mucosa grafting. Ribeiro-Filho et al. [41,56,82] have been performing urethral reconstructions with human cadaveric acellular matrix grafts over the last 10 years. Both urethral and bladder matrices have been used in more than 200 procedures in human patients. In 2014, our group reported longterm outcomes of the 44 initial patients treated with ventral onlay urethral acellular matrix grafts (UAMG) [56]. These patients presented long and complex strictures (3 to 18 cm) and history of multiple previous urethral procedures (3 to 30 procedures/patient). The urethral bed was fibrotic in the vast majority of patients and had moderate to gross calcifications in seven individuals. Median follow-up was 42 months. Cystoscopy performed in fourteen patients showed a vascularized neourethra covered by urothelium with a wide-open urethral lumen (the limits between the graft and the native urethra could not be determined). Six patients (14%) needed an endoscopic urethrotomy to treat partial restenosis (0.5 to 1.0 cm long) 2 to 8 months after the matrix procedure. Complete restenosis of the graft occurred in 5 patients (11%), who were submitted to a new UAMG procedure. Two out of these five patients who underwent a UAMG salvage procedure were able to urinate, but they need urethral dilations. These results indicate that onlay acellular organ-specific matrices may be used even for long strictures with unhealthy urethral beds.

6.2. Cell-seeded Fossum et al. [83] surgically treated six boys aged 14–44 months with severe hypospadias with autologous urothelial cell constructs. A two-staged procedure starting with repair of the chordee was performed in all cases. Urothelial cells were harvested via bladder lavage during the first operation and seeded on acellular dermis. The seeded scaffold was implanted in a second surgery in an onlay fashion. These children were followed up for 3.5 to 5 years. One patient developed a partial stricture treated conservatively and another one developed an obstruction in the proximal anastomosis that was managed successfully with internal urethrotomy. Two other children developed fistulas requiring surgical correction. Bhargava et al. [84] repaired urethral stenosis secondary to LS in 5 human patients with de-epidermized dermis seeded with cultured keratinocytes and fibroblasts. Buccal mucosa biopsies were obtained from each patient for cell harvesting. These grafts were used for urethroplasty in a one-stage (n = 2) or a two-stage procedure (n = 3). Stenosis extension ranged from 4 to 11 cm. Fibrosis and contraction occurred in two patients, resulting in complete or partial graft excision. The other three patients required some form of instrumentation after 33 months of follow-up. Five boys with post-traumatic posterior urethral strictures were included in a report by Raya-Rivera et al. in 2011 [45]. Autologous muscle and epithelial cells were expanded and seeded onto tubularized PLGA scaffolds. Three months after implantation, urethral biopsies showed that the engineered grafts had developed a normal appearing architecture. Although very time consuming, this approach seems promising as just one patient out of five needed transurethral incision. One important advantage of this tissue engineering technique is the lack of major legal, ethical and disease transmission concerns since autologous cells are seeded onto an artificial scaffold. Human studies mentioned in this article are listed in Table 2.

7. Conclusions Almost two decades ago, initial urethral regenerative studies in both humans and animal models were almost exclusively based on biological acellular matrices, a fact that is perfectly comprehensible. At that time, ECM understanding and biomaterial technologies probably could not produce a highly efficient polymeric scaffold. Converting a harvested tissue into acellular matrix is a simple and cheap process that takes only one to two weeks to complete, resulting in a scaffold that contains not only collagen, but also elastin, GAGs, and residual growth factors. In

Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019

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Table 2 Human studies with acellular matrices. Author

Year

Type of matrix

Source of matrix

Cell-seeded

Stricture extension

Technique

Follow-up

Success ratea

Mantovani [74] Ribeiro-Filho [82] Donkov [79] Hauser [80] Palminteri [76] Fiala [78] Fossum [83] el-Kassaby [81] Bhargava [84] Mantovani [75] Raya-Rivera [45] Palminteri [77] Ribeiro-Filho [56]

2002 2006 2006 2006 2007 2007 2007 2008 2008 2011 2011 2012 2014

Biological Biological Biological Biological Biological Biological Biological Biological Biological Biological Synthetic Biological Biological

SIS Human urethra SIS SIS SIS SIS Dermis Bladder Dermis SIS PLGA SIS Human urethra

No No No No No No Yes No Yes No Yes No No

3–10 cm 3–18 cm 4–6 cm 3.5–10 cm 2–8 cm 4–14 cm 4–6 cm 2–18 cm 5–11 cm 3–10 cm 4–6 cm 2–8 cm 3–18 cm

Dorsal onlay Ventral onlay Dorsal onlay Ventral onlay Dorsal/ventral inlay Onlay Onlay Ventral onlay Onlay Ventral onlay Tubular Dorsal/ventral inlay Ventral onlay

6 months 25 months 18 months 12 months 21 months 31 months 60 months 25 months 33 months 120 months 71 months 71 months 42 months

5/5 (100%) 7/7 (100%) 8/9 (89%) 1/5 (20%) 17/20 (85%) 40/40 (80%) 3/6 (50%) 8/9 (89%) 3/5 (60%) 40/40 (100%) 4/5 (80%) 19/25 (76%) 33/44 (75%)

a

OBS: success rate was calculated by the number of patients who did not need an extra surgical procedure.

summary, acellular matrices, especially those obtained from human cadaveric urethras (homologous, organ-specific), provide a favorable microenvironment for the ingrowth of urethral wall cell components. In order to evaluate the success of a urethral grafting procedure, the most important parameter is the patency of the urethral canal. Long term follow-up should be performed since post-operative restenosis rates increase over the years. In humans, this can be assessed by voiding symptoms, residual urine volume, uroflowmetry tests and imaging studies as well as by the need for urethral dilations or surgery. Urethral biopsies should not be performed in humans, since they can induce scar tissue deposition and restenosis. In animal models, the urethra is harvested for immunocytochemical and histological analyses. Clinically, cell-free acellular matrices are safe, easy to work with, “offthe-shelf”, and have demonstrated long term success rates between 70 and 80% in most series [56,75,76,78]. Unseeded biological acellular matrices represent the most common type of scaffold used in humans to the present time. Hundreds of patients have undergone this type of procedure in several countries [56,75,76,63]. SIS is commercially available and has already been used even in private medical practice. One criticism about commercially available SIS is that some specimens still contain porcine DNA residues which may have cytotoxic effects on urothelial cells [38]. Cadaveric organ-specific acellular matrix is also very effective for urethral reconstruction and has become the preferred treatment option for complex and recurrent stricture cases at the University of Sao Paulo, Brazil [56,82]. In terms of surgical technique, cell-free matrices should always be implanted as onlay/inlay grafts, since tubular reconstructions present a high rate of failure. The reason for that is the limited range for adequate tissue regeneration onto the matrix from the borders of the native urethral wall. This limit is approximately 1.5 cm according to various reports [23,24,65,66] — surgeons must have to take this on account when using these unseeded matrices (Fig. 4). In our opinion, cell-free

biological matrices represent a very good alternative when classical urethroplasty approaches fail even in long and complex strictures. On the other hand, successful cell-seeded matrices for urethral reconstruction have been reported more recently, probably reflecting the progress in cell biology and innovations in the development and characterization of natural and synthetic biomaterials for use as scaffold components [67,70,72]. Although biomaterial technology is still not able to fully recreate the complex ECM microenvironment, urethral cellseeded animal studies have successfully incorporated synthetic scaffolds [55,57], growth-factor delivery [70] and cell coculture [67, 72]. In terms of cell-seeded scaffolds used for human urethroplasties, there are only three reports with limited number of patients. Acellular dermis seeded with keratinocytes and fibroblasts was used to treat 5 individuals with LS, but urethral strictures recurred in all patients [84]. One possible explanation for this complete failure could be the LS itself, since it is a progressive and incurable disease. Of course, other aspects may have contributed to this dramatic outcome such as matrix preparation, type of cell chosen to be seeded, and surgical technique. Hypospadia repair with acellular dermis seeded with urothelial cells was performed in six children [83]. In this study, one child needed a surgical intervention due to urethral restenosis and other two patients had undergone urethro-cutaneous fistula correction. Human studies with seeded biological acellular matrices failed to reproduce the excellent results obtained in animal models. On the other hand, the emblematic protocol conducted by Raya-Rivera et al. [45] demonstrates that complete organ replacement is feasible for urethral reconstructive surgery. Five boys received tubular constructs totally produced in vitro. PLGA scaffolds seeded with autologous muscle and epithelial cells were able to maintain urethral patency without further surgery in 4 out of 5 patients. This is a very promising preliminary result, although larger series with more patients are needed to consolidate the technique.

Fig. 4. Biological acellular matrix urethral grafting (cross sections). The rationale for onlay/inlay procedures. A. The distance between the edge of the native urethra and the center of the graft must be less than 1.5 cm (distance X–Y), but usually is less than 1.0 cm. The arrows indicate the direction of the cellular ingrowth. B. If this rule is observed, then the matrix will induce proper regeneration of the urethral wall.

Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019

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L.A. Ribeiro-Filho, K.-D. Sievert / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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Please cite this article as: L.A. Ribeiro-Filho, K.-D. Sievert, Acellular matrix in urethral reconstruction, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.11.019