60: Tissue Engineering—Current Status and Future

Este capítulo levará aproximadamente 16 minutos para ler.

Por Darren Ha, MS 1  & Kelly T. Harris, MD 2, 3
  1. University of Colorado School of Medicine, Aurora, CO, USA
  2. Department of Pediatric Urology, Children's Hospital Colorado, Aurora, CO, USA
  3. Department of Surgery, Division of Urology, University of Colorado, School of Medicine, Aurora, CO, USA

Introduction

Tissue engineering is the science of transplanting cells or directing regrowth of healthy cells in order to create tissues or organs that mimic the native form and function to treat a myriad of diseases.1 Tissue engineering in the field of pediatric urology has focused primarily on regenerating bladder and urethral tissue for replacement or augmentation.2,3,4 The current use of intestine for bladder replacement and augmentation pose significant risks and complications, including intestinal obstruction, metabolic derangements, stone formation, mucus production, urinary incontinence, and recurrent infections.3,4 The use of mucosal or skin grafts for urethral replacement and augmentation pose risks such as strictures, fistulas, graft failure, graft contracture, donor site morbidity, and recurrence of chordee.5,6 Tissue engineering holds promise in the effort to provide durable alternatives for children.

Work in this field has progressed from growth of these tissues ex vivo to animal studies to more recent trials in a limited number of pediatric patients. While there have been significant advancements in scaffolds and cell cultures along with promising results from animal studies, there are barriers that remain which limit the application of this technology to large-scale patient use. This review will aim to summarize and present clinical results from work in tissue engineering within pediatric urology and discuss barriers and future directions in this field.

Biomaterials and Cells

Tissue engineering principles dictate the need for a scaffold and seeded cells to populate the scaffold.7 Numerous scaffolds exist and have been used in urological tissue engineering, including acellular small intestinal submucosa (SIS), collagen, and composite collagen-polyglycolic acid (PGA) mesh.2,8 Work in generating a decellularized bladder extracellular matrix (BEM) using porcine bladders has also shown success with satisfactory removal of cells and genetic material without affecting the underlying collagen or elastin structure, with preservation of angiogenic properties.9

Sources of seeded cells can include autologous urothelial cells and stem cells. While autologous cells can be obtained from a bladder biopsy and expanded in vitro, underlying bladder pathology is a potential limitation as cells from diseased bladders had decreased survival on cell culture compared to healthy adult bladder controls. Additionally, urothelium from bladders with pathology were shown to form weaker and less efficacious epithelial borders.10 Numerous stem cells have also been investigated, including bone marrow-derived, mesenchymal, urine-derived, embryonic, and induced pluripotent stem cells.11,12,13 The process is outlined in Figure 1.

Figure 1
Figure 1 Schematic diagram of the process to harvest host cells (a), expand them in vitro and implant on a suitable tissue scaffold (b), and then implant in the body (c).

Vascularization of Engineered Organs

Similar to native tissue, survival of seeded cells on scaffolds is reliant on adequate vascularization. Innovations in biomaterials have allowed for creation of larger constructs which invariably exceed the diffusion limit for gases and nutrients.14 Once implanted, establishing a vascular network may take weeks, depending on the size of the graft, during which time transplanted cells may experience hypoxia and/or nutrient deficiencies. Furthermore, the process of angiogenesis can result in formation of oxygen and/or nutrient gradients that preferentially support growth of superficial cells and may result in non-uniform growth of matrices and potential structural weakness.15 Strategies to overcome the size constraints of tissue engineered organs include: pre-vascularization of scaffolds prior to cell seeding, seeding of scaffolds with both transplant and endothelial cells, and addition of angiogenic factors prior to graft implantation.16 Another option that has shown potential is the use of an omental wrap over implanted bladder tissue to serve as a vascular pedicle; the functional capacity of these grafts can arguably be seen as a proxy for cell survival and function.17 However, this latter option is not feasible for implanted matrices outside the reach of omentum. Additional work is necessary to address this matter of vascular viability of engineered tissue, especially with goals for whole-organ regeneration.

Animal and Human Studies in Bladder Tissue Engineering

Previous animal studies in bladder regeneration have shown encouraging results with use of unseeded scaffolds alone. Caione et al studied the use of an SIS scaffold for bladder regrowth in a porcine model of partial cystectomy. 40-60% of the bladder wall of six piglets were removed and replaced with an SIS scaffold. Blood vessels were noted to be migrating from native bladders into the graft five weeks after surgery. Histologic examination of the graft after three months demonstrated transitional epithelium, smooth muscle, and nerves. Grafts had a collagen:smooth muscle ratio of 72:28 compared to 56:44 in native bladders, consistent with an absence of increased bladder capacity and compliance in all animals.18 In a later study by Roelofs et al, they compared the use of collagen scaffolds for bladder augmentation in a sheep model of bladder exstrophy versus healthy sheep bladder. At the one-month postoperative timepoint, bladders of lambs with exstrophy had lower capacity and compliance compared to augmented healthy bladders. Additionally, bladder grafts from healthy controls had more neovascularization and smooth muscle present compared to grafts from lambs with bladder exstrophy. At the six-month postoperative mark, bladder volumes were similar between the two but compliance remained lower in the exstrophy model; meanwhile regenerated bladders had similar urothelium with nerve regrowth in both groups.19 The early postoperative results corroborate in vitro studies of impaired regeneration in bladders with underlying pathology, however the six-month postoperative findings may be a reassuring sign of longer term potential, although there are limited studies to substantiate this.10

Emboldened by initial successes in animal studies, pilot studies in a limited number of children have been performed to evaluate use of both cell-seeded and unseeded scaffolds for bladder regeneration. Caione et al evaluated the use of unseeded SIS scaffolds in five pediatric patients who did not desire bladder augmentation with bowel segments after repair of exstrophy-epispadias complex. Histologically, there was migration of all three tissue types into the scaffold, including nerve growth. The grafted portions of bladders had a greater proportion of collagen to muscle compared to native bladders. There was no significant change in bladder leak point pressures after placement of the graft, but three of the five patients had improvements in their bladder capacity at the six-month postoperative timepoint. There was a general increase in the length of time between incontinent episodes and a progressive increase in bladder volumes in all patients two to three years after surgery. However, no patient achieved full urinary continence.20

A study in 2006 by Atala and colleagues used autologous seeding of scaffolds for cystoplasty in seven pediatric patients with a history of myelomeningocele who had high-pressure, low compliance bladders. Patients underwent cystoscopic evaluation with bladder biopsy to retrieve samples for in vitro cellular expansion. Cells were seeded on either collagen or collagen-PGA mesh scaffolds and anastomosed with or without omental wrap. There were no postoperative complications and patients were followed for a mean of 46 months. During the first 3-12 months postoperatively, bladder leak point pressures decreased in all patients while capacity and compliance exhibited variable responses in comparison to preoperative urodynamic findings. For the subset of patients who received augmentation with collagen-PGA scaffolds with omental wrap, there was a 1.58-fold increase in postoperative bladder capacity, which was not seen in the collagen scaffolds with or without omental wrap. Functionally, all patients benefitted from an increase in the average daytime dry interval. On histologic examination of the bladder grafts, the border between native and implanted bladders were indistinguishable and all three tissue layers were evident by 31-months after surgery. Notably, there was no evidence of re-innervation since all patients had to perform clean intermittent catheterization.17

Despite these promising results, a subsequent prospective phase II study using a similar tissue engineering protocol, including omental wrap, was unable to replicate these outcomes. The authors assessed the use of autologous cell-seeded scaffolds for augmentation cystoplasty in children with NGB secondary to spina bifida at four tertiary children’s hospitals. Four out of 10 patients experienced decreased bladder capacity at one year after surgery without return to preoperative baseline even after 36 months. There was no convincing or consistent increase in bladder capacity or compliance 36 months after surgery and urodynamic changes were neither statistically nor clinically significant. Notably, four of the 10 patients had significant postoperative adverse events (i.e., bowel obstruction and/or bladder rupture). Ultimately, five of the 10 patients underwent traditional augmentation enterocystoplasty. The authors concluded that their intervention did not yield statistical nor clinical improvement overall and was associated with high rates of adverse events.21 The divergent outcomes in these two studies is likely multifactorial. The patients in Atala’s cohort were a very select subset of patients, which limits the applicability of this technique.22 Others have posited that absence of presurgical normal bladder cycling may predispose patients to implant failure, thereby excluding a large proportion of patients with bladder pathology for whom this therapy is intended for.23

Results from animal studies demonstrated potential efficacy in bladder engineering, with regrowth of epithelium, smooth muscle, vasculature, and nervous tissue. However, regenerated tissue did not histologically resemble native bladders due to excessive fibrosis. The early progress in animals has not yet translated to overwhelming success in limited patient trials. Evidence of satisfactory bladder histology was equivocal and clinical outcomes were not better than traditional augmentation cystoplasty.24,25

Animal and Human Studies in Urethral Tissue Engineering

Given that the primary function of the urethra is to serve as a conduit for passage of urine from the bladder to the external environment, the benchmark of success in urethral tissue engineering has been measured mainly based on integrity and patency of grafts rather than histologic regrowth of tissue types, as is the case for bladder engineering.3 Various techniques for urethral reconstruction exist in pediatric urology, including primary anastomosis and skin and mucosal grafts.5,26 Tissue engineering is being explored as an alternative to these methods, particularly for repair of longer defects.

Dorin et al investigated the maximal length of native urethral tissue regeneration that could be achieved with urethral onlay of unseeded tubular bladder submucosa grafts in rabbits. They found normal urothelial growth and luminal patency in the 0.5 cm graft at one, two, and four-weeks postoperatively. The 1, 2, and 3 cm grafts all developed strictures by four weeks after surgery. Only the 0.5 cm length grafts had adequate ingrowth of epithelium and smooth muscle; all other lengths experienced fibrosis. It was concluded that unseeded tubular grafts could repair a maximal urethral defect length of 0.5 cm.27

A later study by Orabi and colleagues compared efficacy of cell-seeded versus unseeded matrices in the repair of 6 cm urethral defects in dogs. Cells used for seeding were obtained through bladder biopsy for autologous cell expansion. Cell-seeded matrices remained patent 12 months after surgery while unseeded matrices developed strictures by three months. The seeded matrices had full layers of urothelium and smooth muscle, which the unseeded matrices lacked. It was hypothesized that the success of seeded matrices was due in part to an initial layer of protective urothelial cells which limited urine extravasation into the subepithelial space and causing subsequent fibrosis.28

In an early human trial of urethral tissue engineering, Fossum et al investigated the use of autologous cell-seeded collagen scaffolds for the treatment of severe hypospadias in six boys ages 14-44 months with 3-5.5 years of follow up. Five out of six patients had appropriate uroflow curves after surgery. Four patients developed complications, which consisted of two strictures and two fistulas, which were all corrected in an uneventful manner. Subsequent cystoscopy demonstrated patent neourethras in all patients. There was urothelium lining the neourethras of three patients, while two patients had epithelium-lined neourethras. At last follow-up, all patients voided without straining and had low post-void residuals.29 The investigators continued follow-up with these six boys until prepubertal ages and reported cosmetically appropriate outcomes. All patients continued to void with an adequate stream without straining. Five boys had bell-shaped urine flow curves with maximal flow rates ranging from 8.5-28.3 mL/s. One patient had a flat, extended urine flow curve but voided without issue. Cystoscopy demonstrated patent neourethras in all patients and artificial erections were without curvature. Given the complication rates associated with severe hypospadias, the authors were optimistic about their results as a possible alternative treatment modality. Follow-up of these patients is scheduled to continue during puberty and after full genital maturation.30

In a subsequent study, autologous cell-seeded PGA scaffolds were used in complex posterior urethral reconstruction in five boys with a median follow-up of 71 months. Grafts varied in length from 4 to 6 cm and normal histological tissue architecture was observed by three months after surgery. All neourethras remained patent 12 months after surgery and at the last follow-up. The mean end maximum flow rate was 25.1 mL/s as assessed by uroflow and there were no reported adverse events (e.g., infection, diverticula, dysuria, straining). The authors concluded that engineered neourethras can be successfully used in urethral repairs and that their results in posterior urethral reconstruction further bolster the success as the posterior urethra in boys is more delicate due to the lack of a mature prostate, necessitating more complex repairs than adult males.31

Barriers and Future Directions

Limited human studies have shown mixed results within the field of tissue engineering in pediatric urology with implications for future improvement. Currently, there lacks consistent, reproducible results in both bladder and urethral regeneration that yield compelling clinical and functional improvement without significant risks or complications. In order for tissue engineering to become a feasible treatment option for patients in the future, several barriers must be addressed to ensure optimal tissue growth and function. There needs to be a reliable source of cells that can be expanded in vitro for seeding of scaffolds; grafted tissue must be able to survive the requisite urinary environment of the genitourinary tract; and ideally dependable methods for nerve growth should be identified, particularly for bladder mucosal regeneration, to ensure the best chance for functional outcomes in children.

Animal and human trials using cell-seeded grafts tended to produce more durable results.17,27,31 Thus, it would not be unreasonable to presume that cell seeding may continue to play a role in future tissue engineering endeavors. While autologous cells, obtained through bladder biopsy for instance, may be convenient since it minimizes the risk of graft incompatibility, cells obtained from bladders with disease had limited survivability in vitro compared to healthy bladder controls. Healthy urothelium is mitotically quiescent with low cell turnover as demonstrated by a low Ki-67 index and presence of UPK3a, a molecular marker of terminal urothelium differentiation. However, cells from bladders with pathology have been shown to exhibit high proliferation rates (high Ki-67 indices) and low expression of UPK3a. This pattern was present in biopsies from patients with neurogenic bladder, posterior urethral valves, bladder dysfunction, and epispadias, suggesting that various pathologies of the genitourinary tract are capable of negatively impacting the ability of cells to expand and survive for use in autologous seeding of matrices.10,11 These results, coupled with the breadth of conditions that warrant surgical repair and reconstruction in pediatric urology, may lead to suboptimal results from tissue engineering using autologous cells.

Additional sources for cellular seeding may therefore benefit a large proportion of children. CD34+ hematopoietic stem cells induce angiogenesis and are implicated in peripheral nerve growth, while mesenchymal stem cells are a potential source for bladder smooth muscle. Together, they have been used to recapitulate bladder tissue in vivo and may be potential options.13 Another emerging avenue being explored is the use of urine-derived stem cells due to the lower cost and noninvasive nature of collection and their relatively high potential for differentiation.11,32 They have been induced into ectodermal, mesodermal, and endodermal cell lineages and hold potential for use in the field of urology, although the full spectrum of their biological characteristics still warrants further investigation.32

Regardless of the source of cells that are used for seeding of matrices, they must be able to withstand the cytotoxic environment of urine. Previous studies have demonstrated that the simple addition of urine to growth medium led to decreased cell proliferation and survivability in various stem cells and even cultured urothelial cells.11,12,33 Additionally, tissues must also withstand radial stress and fluid shear forces associated with storage and emptying of urine, respectively. While cellular growth and seeding onto scaffolds in ideal growth conditions in vitro may establish a sufficient barrier to tolerate stressors of the urinary tract and alleviate the aforementioned issues, this adds additional time to the overall process, which currently already takes four to seven weeks.31 An option that has been proposed is the use of urinary diversion during the early postoperative period to allow implanted tissues adequate time to establish cell-cell connections while minimizing contact with urine.11 However, this predisposes patients to additional procedures that may increase the risk for infections.34 Adamowicz and colleagues proposed seeding cells on the external surface of the scaffold to reduce their contact with urine during the initial postoperative healing phase.12 Regardless of which method is ultimately pursued, Qin et al cautioned against postoperative transurethral catheterization as it was shown to mechanically disrupt grafted matrices, particularly in cases of urethral regeneration.35 Thus, another consideration for future work is reconciling the tradeoff of catheterization to decrease graft contact with urine with the sloughing of seeded cells from scaffolds.

Lastly, one of the goals of tissue engineering is to re-establish native function of the organ; in the case of the bladder, this is reliant on appropriate nerve growth and activity. To date, there have been mixed results in the realm of nerve growth.17,18,19,20 Despite evidence of nerve regeneration even with the use of unseeded scaffolds, there is no current evidence to demonstrate that these neurons have the capacity to function in a similar manner as native nervous tissue. The spinal pathways that mediate micturition involve bladder afferents, spinal cord efferents, spinal interneurons, and cortical projections that modulate these reflex circuits, components which are unlikely to be completely captured with bladder grafts alone.36 Furthermore, functional outcomes point toward deficits in nerve function since patients still experienced urinary incontinence and relied on clean intermittent catheterization.17,20,21 Work outside of pediatric urology in nerve tissue engineering has identified important characteristics of scaffolds that promote peripheral nerve growth: conductivity to enhance neuronal communication via action potentials, hydrophobicity to improve cell attachment, and larger pore size to stimulate cell proliferation and migration.37 As the field continues to advance, a delicate balance of scaffold porosity must be attained to foster nerve growth without causing extravasation of urine into the subepithelial space. Collaborating with experts in nerve engineering and drawing on the advancements in their field may lead to significant clinical outcomes for pediatric patients with bladder dysfunction.

Conclusion

Significant breakthroughs in pediatric urology tissue engineering have been made in the last few decades. Advances in biomaterials and cellular engineering have led to early success in tissue regeneration in animal models of urologic diseases. However, limited early human trials of similar interventions have not yielded conclusive, reproducible improvements in patients’ clinical function while minimizing risks. While tissue engineering has the potential to drastically alter the field of pediatric urology through tailored therapeutics, numerous pre-translational barriers still need to be addressed in order to optimize graft function in vivo. Additional long-term animal and human studies are necessary to better understand outcomes and complications, and multidisciplinary collaboration at all stages will undoubtedly be a key to future success.

Key Points

  • Tissue engineering for urology patients is still under investigation with only preliminary early, translational studies having been completed.
  • Current challenges include revascularization of small vessel networks and incomplete neuralation within a host.
  • Should the field advance, however, it may dramatically improve outcomes from a number of congenital urological conditions.

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