Scientific Advances in Reconstructive Urology and Tissue Engineering

Scientific Advances in Reconstructive Urology and Tissue Engineering is a comprehensive overview of reconstructive urology that spans male and female, pediatric, neuro-urology and trauma. Traditionally, this field has been focused solely on surgical principles with little attention to basic and translational research. However, recent advances in acellular matrices, scaffolds, stem cells and tissue engineering have changed this focus. This publication is the perfect bridge and definitive tome of the advances that will move the translational and basic science of reconstructive urology forward and allow readers to implement the findings in clinical practice.

Through the use of both current case studies and future implications, the content bridges the gap from bench to bedside making it the perfect reference for translational benign urology researchers who wish to move the field of reconstructive surgery forward.

• Includes real-world applications of current research to help readers get rapidly up-to-speed on which research results are appropriate for immediate implementation
• Presents the underlying mechanism of urologic disease processes pertinent to reconstructive urology for comprehensive understanding within the field
• Provides independent guidance to manage specific clinical conditions while also presenting approaches that are applicable to all conditions

• Language: English
• Publisher: Academic Press
• Release date: Mar 31, 2022
• ISBN: 9780323915205

Book preview

Preface

Matthias D. Hofer, Urology San Antonio, San Antonio, TX, United States

Research is an important part of all medicine-driving innovation and progress. This also holds true for urology and in particular for reconstructive urology. In the past two decades, reconstructive urology has emerged as an integral subspecialty in urology and as one of its most innovative disciplines.

Research in reconstructive urology is founded on several principles. The first principle is to increase knowledge about pathophysiology of disease, and this principle is mutual to research in other medical specialties including urology as a whole. The second principle is clinical research to improve patient outcomes. Again, optimizing clinical management based on research results is also a mutual cornerstone of all medical research. The third principle, which is unique to reconstructive urology, is the research on interventions that improve wound healing which is essential for the success of any reconstructive surgery. Finally, the fourth principle is tissue engineering, a wide term encompassing the development of the tissue material from a variety of sources including synthetic, biologic, or autologous origin which in application extends from the graft material all the way to organ substitution. This, also, is a unique aspect of research in reconstructive urology, and along with the third principle depends on results that have had their origins in labs rather than patient populations. Given that reconstructive urology not only covers reconstruction of several unique organs such as urethra, bladder, and ureter but also addresses restitution of organ function, for example, the treatment of erectile dysfunction or incontinence, the demand for basic science research is extensive.

However, translating basic research results into patient care is ultimately necessary as a clinical application is integral for promoting treatment success of our patients. This translation can pose its own challenge as lab and clinical environments are different entities. While there often appears to be a dichotomy of researchers focusing on either lab or clinical research, these boundaries are more fluent in reconstructive urology where research teams are composed of both. This textbook is an attestation as its chapters describe the research discoveries made by research teams comprising clinicians and basic science researchers and in many cases clinician-scientists.

The aim of this textbook is to describe the current research in reconstructive urology by those who have undertaken this research. The topics recapitulate the principles described above: research in pathophysiology, in optimizing wound healing, in tissue engineering, and in restoration of organ function thus covering the wide spectrum of research performed in reconstructive urology. This book is intended for researchers and clinicians alike, not only to share knowledge of the current state of the research but also to share knowledge about research methods and translation of lab results into clinical urology. Because while propagation of research results is one objective of this book hoping that urologist can utilize this for treatment of their patients, another and equally important objective is to enthuse interested researchers and clinicians to reproduce reported approaches and methods to further expand treatment options for our patients. And indeed, this book should not be limited to reconstructive urology but applicable to all urologists and other medical specialties alike.

Chapter 1

The cell as a tool to understand and repair urethra

Virginia Sceberras¹, Federica Maria Magrelli¹, Davide Adamo², Eleonora Maurizi¹, Eustachio Attico², Vincenzo Giuseppe Genna¹, Massimo Lazzeri³, Guido Barbagli⁴ and Graziella Pellegrini¹,²,    ¹Holostem Terapie Avanzate S.r.l., Modena, Italy,    ²Stefano Ferrari Regenerative Medicine Center, University of Modena and Reggio Emilia, Modena, Italy,    ³IRCCS Humanitas clinical and Research Hospital, Rozzano, Milano, Italy,    ⁴The Center for Reconstructive Urethral Surgery, Arezzo, Italy

Abstract

The term Regenerative Medicine was introduced at the end of the 20th century to describe an innovative field of medical research, aiming to restore, replace, and regenerate tissues or even whole organs starting from cells and biomaterials. The regeneration of self-renewing tissues, as the urethral epithelium, requires specific adult stem cells and genetic correction in the case of inherited genetic diseases. Generally, the urothelial stem cell identification, fate and molecular phenotype have been studied, but not in relation to tissue regeneration. Tissue engineering proposed in urology for many decades had limited clinical applications, despite technical success in the laboratory. This chapter reviews cell sources, biomaterials, regulatory rules, and the related preclinical and clinical applications as lessons learnt, providing support for improvement and standardization of the cure for urethral disabling diseases.

Keywords

Regenerative medicine; tissue engineering; urethra; cell; biomaterial; stem cells

Introduction

The term Regenerative Medicine (RM) was introduced at the end of the 20th century to describe an innovative field of medical research, aiming to restore, replace, and regenerate tissues or even whole organs starting from cells and biomaterials.¹

An example of RM is tissue engineering (TE), which integrates expertise from cell biology, engineering, biomaterials, and clinics. These different areas cooperate and share competences to address and solve relevant medical needs and reach a successful therapeutic approach. In addition, in disorders requiring genetic correction of the tissue, a TE approach may not be resolutive. Therefore, it might be necessary to use a combined gene therapy–TE approach.²

To understand a TE strategy, it is essential to consider two main components: scaffold selection and cell source. In addition, in the case of complex biological tissue reconstruction (e.g., with different cell subtypes), the use of bioreactors and pharmacological support should be integrated.³

The scaffold is a structural component of the bioengineered construct. It should be biocompatible, nonimmunogenic, nontumorigenic, nontoxic, and capable of supporting cell adhesion, proliferation, migration, and differentiation, mimicking the ultrastructure of the original tissue.¹

The scaffold must retain its precise biomechanical properties. For instance, in cardiovascular applications, bioresorbable scaffolds are applied to provide short-term benefits typical of permanent stents, along with complete long-term reabsorption.⁴ Moreover, the scaffold should promote innervation and vascularization to integrate it into the whole organism.⁵ In other cases, it needs to be tubularized to maintain tissue functionality and structure, as in urethra and airway reconstruction.

There are different scaffold types: natural (derived from either natural polymers or obtained from decellularization of tissues/organs), synthetic (constituted by synthetic polymers), or hybrid (generated from the combination of natural and synthetic materials).⁶

In several TE approaches, the scaffold is colonized in vitro with different cell types to help proper restoration of the damaged or defective tissue in the recipient organism. The cell source employed for each application must be accurately selected and characterized through extensive preclinical studies.

The main categories of cells investigated for TE approaches include pluripotent stem cells [e.g., embryonic stem cells (ESC) and induced pluripotent stem cells (iPSc)], adult somatic stem cells (amniotic fluid stem cells, hematopoietic stem cells, and mesenchymal stem cells), and pluripotent tissue-committed stem/progenitor cells (epithelial stem cells, chondrocytes, muscle, and endothelial cells).⁷

It is essential to perform an extensive analysis of both the cell–scaffold interaction and the different cell types that can coexist in the bioengineered graft before any clinical application. Moreover, the evaluation of stem cell maintenance and their quantification within the construct is crucial to guarantee long-term clinical success.⁸

To reconstruct complex three-dimensional (3D) structures composed of different cell types, bioreactors can simulate the physiological environment through rotation and perfusion. Dynamic seeding conditions can be preferred over static techniques to promote a more homogeneous distribution of nutrients and oxygen.³

A deeper understanding of the molecular mechanisms of cells provides a chance to properly administrate pharmacological support to the bioengineered graft.⁵ The scaffold surface could be functionalized with bioactive molecules, or the molecule could be locally or systemically administered to the patient after reconstructive surgery. An example of how scaffold functionalization may help promote vascularization while decreasing fibrosis comes from Jia et al., who showed how vascular endothelial growth factor (VEGF)-bound scaffolds have better performances than those without it.⁹

To obtain a successful TE approach, all of the components mentioned above must be optimized and integrated to guarantee the patient’s well-being. The clinical outcomes of TE approaches highlight the necessity of an extensive characterization of the biology of the system.¹⁰,¹¹

TE solutions have been proposed in the field of urology for many decades; however, their clinical applications have been modest despite technical success in the laboratory.¹² Moreover, there is only one example of urethral TE in humans that has been approved by the German regulatory body/board, MukoCell.¹³

In conclusion, scientific and technical expertise, together with preclinical and clinical trial design, create safe alternative solutions to fulfill unmet medical needs, significantly improving the patient quality of life.

Preclinical studies

The existence of more than 300 surgical techniques for urethral repair in patients affected by urethral strictures or hypospadias¹⁴ reflects the difficulties related to the correction of these pathological conditions. Moreover, surgical reconstruction becomes more challenging for long defects of the urethra that cannot be treated through the end-to-end anastomosis. To solve complications derived from conventional urethroplasty treatments, TE has emerged as an important and promising approach for correcting urethral defects. To date, a large number of cell types and scaffolds have been proposed in preclinical studies to identify better solutions for urethral reconstruction. Scaffolds in urethral TE can be classified as (1) scaffolds made of natural polymers such as collagen and silk, (2) natural scaffolds derived from decellularized tissue, for example, amniotic membrane (AM), bladder acellular matrix (BAM), small intestinal submucosa (SIS) and decellularized urethra, and (3) synthetic polymeric scaffold.¹⁵ All these scaffolds have been used alone or in combination with cells, producing advantages and drawbacks for urethral defects treatments.

Some examples are bi-layer acellular scaffolds made by silk fibroin (BLSF), used in 2014¹⁶ to repair a urethral injury induced in male rabbits and compared with a SIS graft. Three months after treatment, a similar extent of regenerative process was observed in both groups of animals without reduction of urethral caliber. However, the BLSF scaffold showed a minimal inflammatory response suggesting the possibility to use it as an alternative biomaterial to SIS.

A few years later, another study used a similar rabbit model of onlay urethroplasty. In this experiment, animals’ urethra was previously damaged by electrocoagulation, in order to induce a local fibrosis to simulate the pathological condition of patients affected by urethral strictures.¹⁷ Three months after the operation, BLSF scaffold appeared to be able to reduce stenosis severity. However, it didn’t enable a complete regeneration of smooth muscle tissue suggesting that an injured environment can hamper the healing process.

Concerning natural scaffolds, some attempts to improve regeneration were represented by binding growth factors (VEGF or bFGF, basic Fibroblast Growth Factor) to collagen scaffolds. Neovascularization and regeneration processes were observed, although strictures and fistulas were found in all treated animal models, suggesting that further studies must identify the optimal combination of scaffolds and growth factors.⁷

The second group of biological scaffolds for urethral TE is obtained from decellularized tissue such as BAM and SIS, both widely applied for urethral reconstruction in preclinical models. A single-layer graft of SIS was used in a rabbit model to correct both onlay and tubular defect of urethra. The following histological evaluation revealed the presence of strictures and fistulas in animals treated with tubularized SIS and a limited regeneration in animals with onlay SIS compared to spontaneous repair. This suggests that the SIS scaffold itself was unable to guarantee a proper urethral regeneration.¹⁸ A different study, performed by Dorin et al. showed that tubularized BAM scaffolds were unable to repair urethral defects longer than 0.5 cm. The authors observed a complete regeneration only at the anastomotic edges and a dense fibrosis at the center of the scaffold, requiring a scaffold colonized by cells to correct a urethral defect longer than 0.5 cm.¹⁹ Indeed, acellular natural scaffold obtained by tissue decellularization have the advantage to maintain the native structure of the original tissue including extracellular matrix proteins and growth factors. However, they cannot be used to repair long defects and present high variability between each other.

Regarding synthetic scaffolds, they have lower batch variability and some of their important properties, such as porosity and tensile resistance, can be easily modulated. However, by-products released by scaffold degradation can induce an inflammatory reaction responsible for fibrosis and scaffold shrinkage.⁶ Synthetic scaffolds commonly used for urethral reconstruction are made of biodegradable polymers such as polyglycolic acid (PGA), polylactic acid (PLA), polylactic co-glycolic acid (PLGA), and poly-L-lactide-co-ε-caprolactone (PLCL). For example, the PLCL scaffold was used in a comparative study with human AM to evaluate its ability to sustain human urothelial cell (UC) viability and proliferation. After 14 days of culture, PLCL showed better results with an increased number of viable cells expressing CK7, CK8 and CK19 markers, confirming scaffold biocompatibility.²⁰

In fact, an additional important pillar of TE is the cell source used for scaffold colonization. The use of cell-seeded scaffolds is associated with an improvement of urothelial barrier development and a reduced inflammatory response with the consequence of a decreased risk to develop fibrosis and strictures.²¹ A large meta-analysis performed by Versteegden et al. confirmed that the use of cell-seeded scaffolds was associated with a reduced probability of side effects for both full and inlay urethral repair and that the most common cell types used are smooth muscle cells and UCs.²²

UCs have been successfully applied to treat long urethral defects in animal models using BAM, resulting in the absence of strictures and fibrosis compared to control groups.²¹,²³ However, an invasive procedure is necessary to isolate UCs from a bladder biopsy as well as for bone marrow stem cells (BMS), a cell source potentially usable for urethral TE due to the capability to differentiate into urothelium-like and smooth muscle-like cells.²⁴ To reduce the use of invasive retrieval procedures, alternative cell types involved in urethral reconstruction include foreskin epithelial cells, adipose-derived stem cells (ADSCs), urine-derived stem cells (UDSCs) and oral mucosa epithelial cells.

Fu et al. seeded foreskin epithelial cells into a BAM scaffold for urethral defect repair in a rabbit model. In contrast to the control group, 6 months later, treated rabbits did not develop urethral strictures and showed a regenerated urethral mucosa with the presence of a foreskin epithelium derived from the implanted cells.²⁵ However, some limitations associated with the use of foreskin include site-specific morbidity due to biopsy retrieval and the impossibility of using this procedure in circumcised men or in patients affected by genital lichen sclerosus.⁶

On the other hand, ADSCs can be easily harvested, possess antiinflammatory and angiogenic properties, and differentiate into several lineages.¹⁵ In a rabbit model of urethral reconstruction,²⁶ these cells were differentiated into epithelial cells in vitro and then seeded on a BAM scaffold that was subsequently implanted to treat a 2 cm long defect of the ventral urethra. After 6 months of follow-up, the study group showed a wide urethra with a multilayered epithelium and no recurrence of strictures or other complications compared to the control groups treated with an acellular BAM or a BAM seeded with undifferentiated ADSCs. Although after induction, rabbit ADSCs showed expression of CK13 and CK19, the expression of the epithelial terminal differentiation marker involucrin was almost absent, suggesting the necessity to optimize the in vitro culture conditions to obtain a well-differentiated epithelium.

UDSCs are another cell source used for urethral regeneration. These cells are easy to collect, and recent studies have described them as capable of bipotent differentiation due to their ability to express urothelial or smooth muscle cell-specific markers when exposed to specific differentiating culture media.²⁷ When these cells were seeded in an SIS scaffold and implanted in a rabbit model for urethral defect repair, faster regeneration was observed in the experimental group, while the control animals showed the development of fibrosis and strictures.²⁸ However, further studies are required to better characterize this recently identified small population of putative stem-like cells.

Proof of principle should be investigated before human application, but in vitro or in vivo models cannot reproduce the whole human system complexity, and they can only give us a broad view of the predicted human homeostasis. The in vitro model allows us to study cell characterization and to explore the regenerative cell ability for a long-term therapy solution. However, in vitro studies cannot replicate the complexity of an organ system; thus in vivo studies can be helpful to compensate those limits.²⁹ In the field of urethral reconstruction, results from in vivo preclinical studies are very promising and the main animal models used are rabbits, whose urethra is structurally similar to that of humans and characterized by a corpus spongiosum surrounding urothelium. However, most of these studies show that the TE products are implanted in a healthy urethra where a portion of tissue is surgically removed and replaced with the seeded/unseeded scaffold. This condition is not representative of what is observed in human patients whose treatment is made more challenging by the presence of a compromised urethra affected by preexisting fibrotic tissue.³⁰ This overview introduces the concept that the successes derived from preclinical studies may not consistently be reproduced in clinical application.

Need for regulatory guidelines

To improve translational TE approaches in clinics, a regulatory system has been proposed. Currently, TE falls within medicinal products and requires deep scientific, technological, and regulatory knowledge and expertise.

Medicinal products have distinctive properties that differentiate them from standard therapies. These products offer extraordinary promise for long-term management in cases of high unmet medical needs (Fig. 1.1). These advanced medicinal therapies drive a new paradigm in both healthcare and science (Advanced Therapies Manufacturing Action Plan³¹ https://www.abpi.org.uk/publications/advanced-therapies-manufacturing-action-plan/. They hold promise as treatments for preceding untreatable and high-burden diseases, which means they can positively impact health in a long-lasting manner, providing durable, life-changing solutions for both patient and society. These treatments rely on traditional surgical practice and could not prescind from it but differ from current surgical practices regarding how they are made/administered and by the type of advantages they may provide.³²

Figure 1.1 Differences between surgical and advanced therapies medicinal products (ATMPs) treatment.

To date, fewer than 200 RM products (RMPs) or advanced therapy medicinal products have been approved worldwide. These products are named advanced therapies medicinal products (ATMPs) in the EU, RMPs in Japan, and competitive generic therapies (CGTs) in the United States.³¹,³³,³⁴,³⁵

In the EU, ATMPs are broadly classified into (1) gene therapy medicinal products (GTMPs), (2) somatic-cell therapy medicinal products (sCTMPs), and (3) tissue-engineered products (TEPs). In addition, some ATMPs can be (4) combined ATMPs, containing one or more medical devices as an integral part of the medicine European Medicines Agency (https://www.ema.europa.eu/en/human-regulatory/overview/advanced-therapy-medicinal-products-overview).

These products are managed by the ATMP Regulation no. 1394/2007 on advanced therapy medicinal products. Among other considerations, the Regulation stipulates that marketing authorization must be achieved prior to the marketing of ATMPs. In addition, the ATMP Regulation authorizes member states to allow the use of advanced therapies not authorized by the Commission, under certain conditions, the so-called hospital exemption European Medicines Agency https://www.ema.europa.eu/en/documents/other/european-commission-dg-health-food-safety-european-medicines-agency-action-plan-advanced-therapy_en-0.pdf; European Medicines Agency https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=COM:2014:0188:FIN:EN:PDF). In this last case, patients can receive an ATMP under controlled circumstances where no other authorized medicinal product is available. Indeed, the hospital exemption must require the application of national requirements on traceability, quality, and pharmacovigilance analogous to those requested for authorized medicinal products European Medicines Agency (https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=COM:2014:0188:FIN:EN:PDF). A specialized committee of the European Medicines Agency (EMA)³⁶–³⁹—the Committee for Advanced Therapies (CAT)—made the evaluations for all the products.

Regulation is essential to protect patients from unsafe treatment European Medicines Agency (https://www.ema.europa.eu/en/documents/other/european-commission-dg-health-food-safety-european-medicines-agency-action-plan-advanced-therapy_en-0.pdf). Medicinal products must have several manufacturing steps that are strictly controlled from production to packaging and from clinical trials to pharmacovigilance. Safety and efficacy follow-up systems are under the control of the pharmaceutical quality system (PQS), aiming to ensure that medicinal products have the standard of quality required for the intended use. Eudralex Vol 4 https://ec.europa.eu/health/documents/eudralex/vol-4_en.

These ATMPs are commonly administered just once or a few times within a short period. However, they are usually paid as a one-time treatment, and they have elaborate manufacturing processes, justifying a high up-front cost.³² They are considered a personalized medicine where product development, clinical trial design and control, patient selection, and stratification are considered (Food and Drug Administration—FDA-2013—Paving the Way for Personalized Medicine: FDA’s role in a new era of medical product development). https://www.fdanews.com/ext/resources/files/10/10-28-13-Personalized-Medicine.pdf.

Another significant impediment is the reimbursement of medicinal products because of their valuable manufacturing, which leads to long-term investment revenues.⁴⁰

In summary, the expected worldwide increase in medicinal product approvals in the upcoming decades will produce a significant financial challenge for patients, public health care, and insurance companies, drastically reducing the up-front cost and providing personalized medicine to each patient.⁴¹

Clinical application

Among the several preclinical approaches mentioned above, only 22 studies achieved the clinical phase (Table 1.1). However, not all of them followed the ATMP directives since the relative EU approval was introduced in 2007 (Directive no. 1394/2007).

Table 1.1

The study of Romagnoli et al. in 1990 represents the first example of a clinical approach by TE in urethral reconstruction. A deeper knowledge of cell biology and epithelial cell culture allowed the production of a stratified urethral epithelium starting from a small biopsy of the urethral meatus to treat hypospadias cases. Autologous adult stem cells located in the basal layer support long-term epithelium regeneration and avoid immunotherapy. The resulting graft showed the same morphology and biochemical and functional characteristics as the starting sample.⁴² Over the years, the graft application was further improved; initially, the epithelium was mounted on a petrolatum gauze. Thereafter, a tubular PFTE (Gore-Tex) support was used.⁴³ Despite the tailored experimental procedure, this challenging reconstructive approach requires invasive biopsy procurement and, in some cases, showed postsurgical complication as small fistula and strictures, necessitating surgical reintervention.

Subsequently, Fossum et al. adopted a similar approach and started a clinical trial that enrolled six patients with severe hypospadias. The treatment consisted of an allogeneic acellular dermal matrix populated by autologous UCs derived from bladder washing. In this study, four of the six patients developed complications, such as obstruction, fistula, and urinary tract infection, which were subsequently surgically treated. The use, firstly, of commercially available human dermal tissue and, later, of normal de-epithelialized human skin represents a study variability and a limitation along with the small group of patients treated. A partial and noncontrolled de-epithelialization procedure could lead to cell persistence. The analysis of biopsies procured after treatment showed a transitional squamous epithelium not identified as urothelium, except in one patient. In two cases, hair growth was observed on the urethral wall.⁴⁶,⁶⁵

The first application of BAM was proposed by Atala et al. in 1999 for the clinical application in four patients suffering from hypospadia.⁴⁴

This technique was further applied to stricture treatment. In a randomized comparative study, 30 patients received oral mucosal graft, considered the gold standard treatment, or BAM (from cadaveric donors). The use of BAM would represent an alternative to oral mucosa treatment because it avoids the invasive procedure of graft withdrawal.⁴⁵ The treatment had shown partial success, especially in those patients with multiple previous interventions. The results emphasize (1) the relevance of surgical bed regarding complications as fibrosis and inflammation and (2) the capability of surrounding cells to colonize the scaffold after implantation vs the use of a pre-seeded graft.

The use of commercially available off-the-shelf material bypasses the problems related to autologous tissue shortage and donor site morbidity in cases of oral mucosa harvesting. An example is represented by SIS, a widely tested scaffold in different clinical studies. Mantovani performed its first application in 2002, in which five patients with strictures were treated; among them, there was a patient aged 72 years with complete urethral stricture.⁴⁸ An open urethroplasty technique was performed, and except for some urethral dilatation and endoscopic urethrotomy due to early recurrences, no other complications were observed. A lack of cell infiltration probably caused the failures into the graft and poor vascularization of the recipient site. During the follow-up visits, a homogeneous transformation of the graft into the native tissue was described by the group as a consequence of the surrounding area