Tissue engineered extracellular matrices (ECMs) in urology: Evolution and future directions

Autologous gastrointestinal tissue has remained the gold-standard reconstructive biomaterial in urology for >100 years. Mucus-secreting epithelium is associated with lifelong metabolic and neuromechanical complications when implanted into the urinary tract. Therefore, the availability of biocompatible tissue-engineered biomaterials such as extracellular matrix (ECM) scaffolds may provide an attractive alternative for urologists. ECMs are decellularised, biodegradable membranes that have shown promise for repairing defective urinary tract segments in vitro and in vivo by inducing a host-derived tissue remodelling response after implantation. In urology, porcine small intestinal submucosa (SIS) and porcine urinary bladder matrix (UBM) are commonly selected as ECMs for tissue regeneration. Both ECMs support ingrowth of native tissue and differentiation of multilayered urothelial and smooth muscle cells layers while providing mechanical support in vivo. In their native acellular state, ECM scaffolds can repair small urinary tract defects. Larger urinary tract segments can be repaired when ECMs are manipulated by seeding them with various cell types prior to in vivo implantation. In the present review, we evaluate and summarise the clinical potential of tissue engineered ECMs in reconstructive urology with emphasis on their long-term outcomes in urological clinical trials. © 2017 Royal College of Surgeons of Edinburgh (Scottish charity number SC005317) and Royal College of Surgeons in Ireland. Published by Elsevier Ltd. All rights reserved.


Introduction
More than 400 million people worldwide suffer from congenital and acquired anomalies of the urinary tract that may necessitate surgical intervention. Surgical repair for end stage upper and lower urinary tract defects often necessitates autologous, vascularised, mucus-secreting gastrointestinal tissue to either replace the diseased organ or to augment inadequate tissue. 1 Postoperatively, the compliance of the Abbreviations: ECM, extracellular matrix; SIS, small intestinal submucosa; UBM, urinary bladder matrix; PTFE, polytetrafluoroethylene; 3D, three-dimensional; ESRD, end-stage renal disease; ADSCs, adipose derived stem cells; SMC, smooth muscle cell; VECM, vascular extracellular matrix; PGA, polyglycolic acid; PGA/PLA, polyglycolide/polylactide; BAMG, bladder acellular matrix graft; ADM, acellular dermal matrix. bowel is often sufficient to restore the basic shape, structure and function of the urinary tract, however lifelong postoperative complications are common. 1,2 Long-term comorbidities that result from interposition of gastrointestinal tissue in the urinary tract are classified as metabolic or neuromechanical and their incidence approaches 100% due to the mucus-secreting nature of the bowel epithelium. 3 Tissue engineering in urology focuses on developing alternative strategies for reconstructing injured, diseased and congenitally absent cells, tissues and organs within the urinary tract. 4 Throughout the twentieth century synthetic scaffold biomaterials were investigated in reconstructive urology as alternatives to gastrointestinal tissue. In the 1950s nonbiodegradable synthetic materials like polytetrafluoroethylene (PTFE), silicone, rubber, polyvinyl, and polypropylene were applied but were found to rapidly encrust with prolonged urinary contact. 5 Additionally, these materials were susceptible to bacterial colonisation and foreign body reactions. 6 The debilitating comorbidities associated with autologous gastrointestinal tissue and synthetic biomaterials may be mitigated by the availability of tissue-engineered readily available, porcine derived extracellular matrix (ECM) scaffolds.

Tissue engineered extracellular matrices (ECMs)
ECMs are decellularised, biocompatible, biodegradable biomaterials usually derived from porcine organs. 7,8 They are prepared by mechanical, chemical and enzymatic treatments to yield tissue that is minimally immunogenic but retains its basic structural elements. 9 Collagen, glycosaminoglycans, fibronectin, laminins and other intrinsic growth factors are retained after the preparation process to allow for host derived cellular attachment, growth and differentiation. 4 Appropriately prepared urological ECMs aim to provide a biologically active tissue/organ substitute that can effectively integrate into host tissue, maintain urinary tract compliance and functionally replace a defective urinary tract segment after a pre-established duration. 10 An integrated tissueengineered ECM should restore or preserve the normal function of the urinary tract structure it is augmenting or replacing. 3 Furthermore, it should be biocompatible to minimise rejection and inflammatory reactions.

Urological ECMs
In urology, porcine small intestinal submucosa (SIS) and porcine urinary bladder matrix (UBM) are selected as the more favourable ECMs for tissue reconstruction. 8,11 Their harvesting sites differ; however both can be utilised as interposition grafts within the urinary tract as they support ingrowth of native tissue, proliferation and differentiation of multilayered urothelial and smooth cells layers while maintaining a robust connective tissue layer to provide mechanical support to the urinary tract. 12 SIS is harvested from porcine small bowel and contains significant amounts of collagen type I with lesser amounts of collagen types III, IV, V and VI. 4 UBM is harvested from the porcine urinary bladder and contains an intact basement membrane layer with large quantities of collagen type VII. 13 Manipulating tissue-engineered ECMs Currently, there are two techniques for manipulating biodegradable ECM scaffolds after their preparation process. These approaches are referred to as unseeded and seeded techniques. The unseeded method involves the use of a bare ECM scaffold in vivo to provide a framework for ingrowth and regeneration of native tissue. In general, unseeded ECMs are reserved for reconstructing smaller (i.e. 1 cm) urinary tract segments 14 (Fig. 1). The seeded method requires the in vitro culture and expansion of various cell types on an ECM scaffold to create a composite tissue for grafting in vivo (Fig. 2). 11,15,16 Seeded cells function in a paracrine manner once implanted and recruit endogenous cells to regenerate the native tissue across the scaffold (Fig. 3). 17,18 Structural, functional and biological properties of cell-seeded ECM scaffolds should be accurately characterised in vitro to confirm that the ECMs mechanical and physiological properties are similar to the intended urinary tract segment that is being replaced. 9,19e21 Urinary tract applications of tissue-engineered ECMs Repairing defective urinary tract structures with ECM scaffolds has been widely reported with many successes and failures described. Unseeded ECM scaffolds are effective for repairing small urinary tract defects through the release of stimulatory growth factors. Cell seeded or composite ECMs are more robust than unseeded ECMs and are therefore capable of repairing larger urinary tract segments and structures. 22,23 At the turn of the millennium most studies on urological ECMs were described in animal models. Recently, phase 1 and phase 2 human clinical trials are becoming more prevalent with some encouraging short-and intermediate-term follow-up results. 22,24 Recent progress in whole organ tissue-engineering strategies in urology is an exciting development that may be clinically translatable soon. 2

Kidney
Renal transplantation is the definitive treatment option for patients with end-stage renal disease (ESRD). 25 However, only a minority of patients with ESRD receive kidney transplants due to a global shortage of deceased donor organs. 26 Researchers are currently developing three-dimensional (3D) cell culture techniques to construct tissue-engineered kidney ECMs in vitro as potential alternatives to renal allografts. 2,27,28 Preliminary studies have demonstrated the feasibility of decellularising and recellularising renal ECMs in rat models. 29 However, development of an intricate and sophisticated vascular network has limited clinical progression. 30,31 Vascular endothelial cell-seeding techniques have recently been pioneered to address this limitation by coating the decellularised vasculature of porcine kidney ECMs with an endothelial lining. Pre-seeding with a precursor to a vascular endothelium enhances vascular patency of the implanted scaffold in vivo. 28,32 Challenges such as difficulties in expanding sufficient quantities of cell lines in vitro and thrombosis in vivo are 2 areas that need to be addressed to ensure the feasibility of these approaches in the near future. 32e35

Ureter
Reconstruction of long ureteral segments may necessitate autologous ileum to restore the mechanism for draining urine from the kidney to the urinary bladder. 36 Early studies on porcine ureters reported encouraging histopathological outcomes when SIS was utilised to replace a 7 cm ureteral segment after two thirds of the periphery of the upper third of the left ureter was excised. 37 Vascularised segments of collagen and smooth muscle were found in the 8 porcine models at autopsy after 7 weeks. There was no macroscopic or microscopic features of anastomotic strictures, ureteral stenosis or inflammation. The control group was the contralateral ureter where a Davis ureterotomy was performed. There was no evidence of epithelial regeneration at 7-weeks in the control group and all porcine models had radiological evidence of ureteral stenosis. Subsequently, porcine SIS was utilised to replace a 2 cm ureteral deficit (encompassing half the circumference) in 9 porcine models. 38 Again, there were no macroscopic or microscopic features of stenosis at autopsy after 9 weeks. Histopathology demonstrated regenerated urothelial and smooth musculature at the site of the implanted SIS. Discouraging findings were described in other studies where complete circumferential ureteral defects were replaced. Shalhav et al. reported a 100% failure rate when acellular SIS replaced the ureter in 6 Yucatan mini pigs after 12-weeks. 39 More recently, Zhao et al. investigated the cell seeded approach by isolating adipose derived stem cells (ADSCs) from rabbits and stimulating the cell line to differentiate into a smooth muscle cell (SMC) phenotype. 40 SMCs were then seeded onto a vascular extracellular matrix (VECM) and implanted into rabbit models to replace a 3 cm full circumferential ureteral defect. Histopathology demonstrated an organised smooth muscle layer with a stratified differentiated urothelium and no evidence of ureteral stricture disease after 16 weeks' follow-up. 40 The success of this recent novel approach is attributable to an abundant cell source and improved implantation techniques. 36,41 Bladder The urinary bladder can be compromised by neoplastic diseases and debilitating medical conditions such as bladder exstrophy, spinal cord injury, myelomeningocele, multiple sclerosis and interstitial cystitis. Bladder augmentation with a tissue engineering approach has been described in 22 patients to date in 3 different clinical trials. 22,24,42 Cell-seeded ECM scaffolds cultured in conjunction with biodegradable synthetic materials did show early promise for augmenting or replacing the urinary bladder as exemplified in a phase 2 clinical trial study by Atala et al. in 2006. 22 Native urothelial and smooth muscle cells were cultured onto ECM scaffolds or a composite scaffold composed of collagen and polyglycolic acid (PGA) in paediatric patients requiring augmentation cystoplasty for myelomeningocele (n ¼ 7). The tissue-engineered scaffolds were implanted with or without an omental wrap and no postoperative complications were noted after 46 months. Furthermore, postoperative cystograms and urodynamic studies demonstrated an increase in bladder capacity and compliance values that were 1.58-folde2.79-fold improved compared to baseline values. Mean bladder leak point pressure at capacity decreased postoperatively by 56% (67e37.5 cm H 2 O). 22 Notably, one other recent phase 2 clinical trial by Joseph et al. was unable to replicate these encouraging results when an autologous cell seeded polyglycolide/polylactide (PGA/PLA) Fig. 2 e Construction of cell-seeded ECM. In the cell-seeded approach, donor cells are harvested and expanded in vitro in large quantities. Exposing a mesenchymal stem cell line to a physiological urinary tract environment allows stem cells to differentiate into urothelial and/or smooth muscle cell lines. Differentiated cells are then cultured onto a decellualrised ECM and expanded again for a period of weeks. The differentiated cell-seed ECM can then be implanted in vivo to repair larger urinary tract defects. Fig. 3 e Cell-seeded ECMs over time. Viability/cytotoxicity fluorescence assays with three-dimensional confocal microscopy illustrating viable and proliferating urothelial (UCs) and smooth muscle (SMCs) cell lines on urinary bladder matrix (UBM) after 6, 10, and 14 days of dynamic culture (A, B, and C, respectively). 12 The cell-seeded scaffolds are stained with calcein and ethidiumhomodimer-1 (Ethd-1; Molecular Probes, Eugene, OR, USA). Calcein is actively converted to calcein-AM in viable cells, which appear green under a fluorescence microscope. Ethd-1 accumulates in dead cells and on the ECM scaffold, which appears red. 12 composite scaffold was utilised for augmentation cystoplasty in patients with spina bifida (n ¼ 10). 42 There was no improvement in bladder capacity on urodynamics after 1-year or 3-years and serious adverse events occurred in 4 patients with 5 patients requiring re-operation in the form of a conventional ileocystoplasty. 42 Such findings demonstrate that further prospective studies are needed to demonstrate the clinical effectiveness of tissue-engineering for reconstructing the urinary bladder.
Most recently, urologists have developed a PGA urinary conduit scaffold as an alternative to a conventional ileal conduit for urine drainage after cystectomy. The 'neo-conduit' was seeded with autologous smooth muscle cells (SMCs), grown from adipose derived mesenchymal stem cells, for patients undergoing radical cystectomy for bladder cancer. 43,44 Eight patients have been enrolled in this phase 2 clinical trial to date and early findings have demonstrated regeneration of urothelium, smooth muscle and neuronal tissue on histopathology. 44 Long-term functional results are currently awaited.

Urethra
The anterior urethra frequently requires reconstruction to treat ischaemic strictures caused by trauma or inflammation and congenital hypospadias. 45 Substitute urethroplasty, with a skin flap or graft, is most often required in instances of strictures/defects greater >2 cm, recurrent strictures or strictures of the penile urethra. Oral mucosa, derived from buccal or lingual tissue, is the gold standard grafting biomaterial in cases where there is insufficient penile skin to utilise as a flap. 46 Limitations with this graft harvesting technique are donor site morbidity, necessity for >1 harvesting site in cases of longer strictures/defects and the absence of sufficient donor tissue in the cases of repeat urethroplasty. 47 Presently, urethral reconstruction using tissue-engineered ECMs is at the forefront of regenerative medicine in urology. 48 Urethral reconstruction utilising tissue engineering techniques, have been used in hundreds of human patients to date ( Table 2). A tissue engineered urethral graft alternative has the potential to eliminate donor site morbidity, decrease operative time and provide graft material in cases of repeat urethroplasty. Table 1 summarises 21 clinical studies that implant a range of tissue engineered scaffolds to treat urethral stricture and hypospadias. Decellularised urethral scaffolds are derived from porcine SIS, cadaveric bladder acellular matrix graft (BAMG), and human acellular dermal matrix (ADM). A naturally autologous collagen based matrix (Mukocell © ) has also been utilised in addition to the synthetic polymer polyglycolic acid (PGA). Five studies have investigated the effect of seeding autologous cells onto tissue engineered urethral scaffolds. These cells are expanded from bladder urothelial cells (BUC), bladder smooth muscle cells (BSMC), oral keratinocytes (OK) and oral fibroblasts (OF). Table 2 also demonstrates that tissue engineered urethral scaffolds demonstrate favourable clinical outcomes under certain conditions. Specifically, the success of the graft is dependent on the length of the stricture and condition of the urethral bed whereby shorter strictures and healthier urethral beds are more conducive to clinical success. Furthermore, outcomes with bulbar grafts are superior to penile Table 1 e Summary and clinical outcomes of studies that describe tissue-engineering methods for reconstructing the urinary bladder in human patients.   urethral grafts due to the reduced urethral bed present in the penile urethra whereby the scaffold is sutured to surrounding skin rather than within the more abundant corpus spongiosum present in the bulbar urethra. This decreases the vascularity of the scaffold and therefore limits tissue regeneration. Furthermore, although cell seeded scaffolds have shown promise in initial clinical studies, the cost of producing such grafts is estimated to be 6 times that of acellular scaffolds therefore reducing the attractiveness of this approach. Future studies should focus on reducing the cost of cellularisied scaffold production or, alternatively, encourage the use of acellular tissue engineered scaffolds under clinical conditions known to be conducive to successful outcomes.

Future perspectives and conclusions
Significant clinical progress has been made with tissue engineered ECMs in urology in recent years. Acellular ECMs are useful for repairing small urethral defects and cell-seeded ECMs can augment or replace defective tissue segments in the ureter and bladder. Currently, composite scaffolds composed of cell seeded ECMs and synthetic degradable biomaterials are being investigated as regenerative scaffolds to replace solid organs like the urinary bladder and kidney. Soon, it is envisaged that three-dimensional (3D) bioprinting technology may ultimately lead to whole organ development in tissue-engineering. 3D bioprinting technology is garnering attention as it has enabled 3D printing of ECMs, cell lines and supportive growth factors into living tissues in a 3D format. Limitations with 3D bioprinting in its present format are decreased mechanical strength and poor tissue integration of constructs when implanted in vivo. Concerns over in vivo mechanical durability and biocompatibility need to be addressed to ensure that the progression of tissue engineered ECMs into urological practise continue. Furthermore, differences in the long-term clinical outcomes associated with implanting tissue-engineered constructs in the human urinary tract indicate that the intricacies of the relationships between cell lines, ECM scaffolds and the host's physiological environment are not yet fully understood among researchers and clinicians. These relationships need to be fully clarified and defined before tissue-engineering in urology progresses from bench to bedside in mainstream reconstructive urology.