Scholarly article on topic 'Strategies to Reconstruct a Functional Urethral Substitute by Self-assembly Method'

Strategies to Reconstruct a Functional Urethral Substitute by Self-assembly Method Academic research paper on "Medical engineering"

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Abstract of research paper on Medical engineering, author of scientific article — Amélie Morissette, Annie Imbeault, Valérie Cattan, Geneviève Bernard, Guillaume Taillon, et al.

Abstract Current urethral reconstruction procedures present many related complications. Moreover, the availability of urologic tissue is limited. Free of exogenous biomaterials, synthetic polymers or acellular matrices, a new tissue engineering technology has been optimized. This technique is known as self-assembly. The fibroblasts produce and assemble their own extracellular matrix to form a tissue very similar to native one. Goals are: to reconstruct a human urethral substitute containing a mature urothelium and a functional vascular network. The same substitute will also be produced using rabbit cells to allow autologous in vivo grafting.

Academic research paper on topic "Strategies to Reconstruct a Functional Urethral Substitute by Self-assembly Method"

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Procedía Engineering 59 (2013) 193 - 200

Procedía Engineering

www.elsevier.com/locate/procedia

3rd International Conference on Tissue Engineering, ICTE2013

Strategies to reconstruct a functional urethral substitute by self-

assembly method

Amélie Morissettea,b, Annie Imbeaultb, Valérie Cattana,b, Geneviève Bernarda, Guillaume Taillona, Stéphane Chabauda, Stéphane Bolduca,b*

a Centre LOEX de l'Université Laval, Génie tissulaire et régénération: Centre LOEX du Centre de recherche FRQS du CHU de Québec, 1401,

18e rue, Québec, QC, Canada, G1J1Z4 b Département de Chirurgie, Faculté de Médecine, Université Laval, 1050, avenue de la médecine, Québec, QC, Canada, G1V 0A6

Abstract

Current urethral reconstruction procedures present many related complications. Moreover, the availability of urologic tissue is limited. Free of exogenous biomaterials, synthetic polymers or acellular matrices, a new tissue engineering technology has been optimized. This technique is known as self-assembly. The fibroblasts produce and assemble their own extracellular matrix to form a tissue very similar to native one. Goals are: to reconstruct a human urethral substitute containing a mature urothelium and a functional vascular network. The same substitute will also be produced using rabbit cells to allow autologous in vivo grafting.

© 2013 The Authors. Published by Elsevier Ltd.

Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande.

* Corresponding author. Tel.: +1-418-990-8255; fax: +1-418-990-8248. E-mail address: stephane.bolduc@fmed.ulaval.ca

1877-7058 © 2013 The Authors. Published by Elsevier Ltd.

Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria,

Centro Empresarial da Marinha Grande

doi:10.1016/j.proeng.2013.05.110

1. Introduction

Urinary apparatus could be separated in two main groups: the first part is formed by the kidneys and ureters which filter the blood to eliminate the waste resulting from metabolism, condensate the urine and transport it to the vesical compartment. The second is formed by the the bladder and urethra which store and evacuate the urine outside the organism. Except for the kidneys, the main characteristics of these tissues is their barrier function in order to avoid toxic waste evacuated from the body, this function is mainly due to the epithelial layer, the urothelium. This feature can only be properly achieved by a complex differentiation of the superficial cells, the umbrella cells. The tightness of junctions between cells and the presence of mature uroplakin plaques are markers of this status. Tissues used to date do not present these characteristics and then did not allow an optimal reconstruction. Even if ureters, bladder and urethra substitutes are now reconstructed using tissue engineering techniques, only the last one is discussed here.

1.1. Urethral anatomy and pathologies and current surgical techniques

After bladder filling, the urine has to be evacuated outside of the body. Smooth muscle contraction and sphincter relaxation allow the urine to pass through the urethra. Multiple layers of tissues constitute the urethra[1]. From the outside to the lumen, this tubular structure is formed by a thick longitudinal muscular inner layer and a thin circular muscular outer layer with intra-fascicular connective tissue, a submucosa or lamina propria with collagen fibres and microvascularization and finally, the urethral epithelium lays on the basal lamina. In males, the urethra can be divided in four sections of different length, position and role. The proximal part, also known as pre-prostatic urethra, is embedded by a sphincter. It carries the urine into the prostatic urethra whereas several channels release secretions such as the ones contained in semen. Following this segment, the membranous urethra begins. It is a structure surrounded by a striated muscled sphincter. Finally the spongy urethra allows the evacuation of the urine outside the organism. The urothelium coating the inner urethra is transitional, then pseudostratified, except in the last two distal centimeters where it becomes pavimenteous and stratified such as the skin. Female urethra is most simple with only two parts: if the first proximal third (1 cm) is roughly similar to the male one, the last 2 cm are more comparable to the epidermis with a pluristratified epithelium.

Pathologies affecting urethra usually are classified in two major groups[2]. Urethral diverticulum, lack of penis, micropenis, hypospadias, epispadias and double urethra are example of congenital urethral pathologies whereas urethral strictures, fistulaes and cancers can be found in the group of acquired pathologies. Epispadias describes abnormal urethral end in an dorsal penile location[3]. Hypospadias, where the urethral meatus is on the ventral side of the penis, also needs complex reconstructive surgeries. For now, patients are treated with autologous graft of penile skin or foreskin[4]. Some complications are observed like contraction, fistulae, stenosis, ectopic hairs, etc. Oral mucosas are also used with similar troubles[5].

1.2. Tissue engineering models

The lack of an ideal tissue to reconstruct urethra led to the development of tissue engineering strategies. Several approaches have been tested until now, for example, synthetic polymers[6-11], acellular matrices[12-19] and self assembly technique[20-22].

The main advantage of synthetic biomaterials such as PLLA or PLGA, is their ability to form biocompatible 3D-organs and to give rapid and reproducible results at a low cost. As the stroma is synthetic, these techniques reduce the need of tissue with poor availability and the risk of the presence of biologic contaminants. Mechanical properties and degradation rates can also be controlled. This last point raises the problem of the behaviour of hydrolytic products of the materials in the body. Another and more important challenge for this synthetic scaffold is to provide an environment for epithelial cell differentiation into a well organized tissue which is a capital point. If these techniques eliminate the risk of stone encrustation or hair in the urethral lumen that can be found with the hair baring skin, several problems appeared after grafting such as microfistulas or a lack of achieved urothelial

differentiation. For now, despite their potential advantages, no convincing models have been established using biomaterials in a long term experiment.

Acellular matrices are prepared from native tissue which are decellularised and sterilised with physical, enzymatic or chemical protocols. It is expected that the process keeps mechanical properties and biochemical environment similar to the living tissue. This feature helps organ development, repair and regeneration. But if inadequate protocols are used, often in order to remove all cell parts, decellularisation and sterilisation techniques could be too aggressive and the extracellular matrix properties are lost. The challenge is to discard all immunogenic or potentially toxic factors but without the lost of beneficial factors. Urethral reconstruction used Small Intestine Submucosa (SIS) and Bladder Acellular Matrix Graft (BAMG), but several problems similar to the one encountered with synthetic polymers were encountered. Nevertheless, this approach is constantly improving and remains very promising.

2. Self-assembly method for urological regenerative medicine

Almost all research groups using tissue engineering for urologic reconstruction work with either synthetic or acelluar biologic matrices. In front of several problems, such as inappropriate differentiation of epithelium, other techniques have to be investigated to achieve in-vitro reconstructed urinary tissues. The self-assembly technique is a tissue engineering method developed by Dr François A. Auger at LOEX, a Canadian research center, mostly for heavily burn patients[23]. With time, it proves to be useful for tissue reconstructions ranging from skin to blood vessels.

2.1. The skin model

The principle of self-assembly technique is very simple. As major problems of inflammation and graft rejection is due to heterologous materials or cells, a solution to avoid these effects is to only use cells from the patients. Supplemented in ascorbic acid, dermal fibroblast cultures produce and assemble an extracellular matrix similar to the stroma of the skin. Neither exogenic nor acellular biologic material are required. When a stroma with sufficient mechanical properties to be manipulated is obtained, several sheets of matrix are superrimposed and keratinocytes, obtained from the same biopsy, were seeded on the top of the construction. Culture at the air-liquid interface allows adequate differentiation of the epidermis. The autologous skin equivalent obtained could be grafted to patients. This technology is used with impressive results for burned patients since 1986. Organization and composition of stroma play an essential role in tissue engineering not only due to its structuring function, but also because extracellular matrix serves as a reservoir of key signalling molecules. In this technique, cells receive appropriate signalling for their differentiation. As demonstrated by several LOEX teams, physiological[22,24-34] or pathological[35-37] conditions can be recapitulated. Several improvements have been obtained with the self assembly approach. Notably, the vascularization of the graft proved its beneficial effects[38-39]. Microvascular endothelial cells, also obtained from the initial skin biopsy, could greatly enhance the survival of the graft in the first week following the surgery, by increasing the nutrients and oxygen availability.

2.2. The blood vessel model

After a decade of successful graft for heavy burned patients with the reconstructed human skin produced in LOEX, Dr François A. Auger investigated the feasibility of blood vessel using the self-assembly approach[30]. Whereas matrix sheets were superimposed to increase the thickness in the case of the skin equivalent, the extracellular matrix layer was rolled around a mandrel to form a tubular structure. Endothelial cells were then seeded in the lumen to obtain a tissue engineered blood vessel. In a second step, different layers of cells were produced and rolled to generate the muscular and the stromal compartments of the vessel. These structures demonstrate not only a good mechanical resistance to burst pressure, but also contractile properties in response to pharmacological agents.

2.3. The first urethral model

Tissue engineering reconstruction of small tubes able to replace a urethra would facilitate largely the work of the urologic surgeons, because they often lack of the necessary tissue for the penile reconstruction surgeries. The Labotaroire d'Organogenèse des Tissus urologiques, LOETU- a department of LOEX, dedicated to urological tissue reconstruction under the supervision of Dr Stéphane Bolduc, a pediatric urologist, was then created and take advantages of the Dr Auger skin models to produce bladder substitute and from blood vessel model to create reconstructed urethral tissue. Magnan et al. [22] had demonstrated the feasibility of a human autologous tube. Histologic analysis of the reconstructed tube showed an homogenous extracellular matrix and the presence of urothelial cells. Mechanical characteristics of this model are roughly similar or even better than the native tissue (Table 1).

Table 1 Increasing strength of tubular structure after maturation

Maturation period Burst pressure (mmHg)

2 weeks 803

3 weeks 1133

4 weeks 1801

Porcine urethra 418

Despite these impressive results, the problem of the adhesion and right differentiation of the urothelium, which is essential to have a functional urethral epithelium, remains. This step is necessary in order to protect the graft from the urine toxicity and its consequences.

2.4. Dynamic culture to improve stromal and urothelial characteristics

The next step is the work of Cattan et al.[20] Tubes were placed in constant flow after epithelial cell seeding. Cell culture medium circulated inside the tube and stimulated the differentiation of the urothelial cells. Histologic analyses demonstrated the presence of a pseudostratified urothelium under dynamic culture conditions. Uroplakin 2 (table 2) and tight junction protein ZO-1 were expressed and formed near native structures when tubes were stimulated by flow. In these conditions, CK20 was present as a marker of a right differentiation of the urothelial cells. Moreover, tubes were less permeable when maturated using a circulating flow inside the lumen (table 2) and these measures correlated with organization of the uroplakin plaques at the surface of cells as assessed by electron microscopy.

Table 2 Mechanical stimuli increase watertightness of tubular structures

Conditions Cumulative amount Level of UPK2 of 14C-urea permeated3 expressionb

Static 7 days +++++ -

Static 14 days ++++ -

Dynamic 7 days +++ +

Dynamic 14 days + +++

Porcine urethra +(+) n.d.

a amount was determined using Franz cells at 8h, when tubes cultivated in static conditions for 7 days reached a plateau.

b transcript levels were normalized to b2-microglobuline, n.d. not determined

2.5. Vascularization

As it was done for the reconstructed skin, vascularization of the tubular structure was tried. As demonstrated by Imbeault et al.[21], a vascular network developed in the tissue and was functional allowing an early inosculation of the host and graft network and blood cell circulation inside the reconstructed tissue (Table 3).

Table 3 Vasularization of urethral substitute after in-vivo subcutaneous implantation in mice

Days post graft Presence of blood cells

Without HMVEC With HMVEC

Day 0 0 0

Day 7 + ++

Day 14 ++ ++++

Day 28 +++ ++++++

HMVEC: human microvascular endothelial cells

2.6. Translation of human to rabbit model

To avoid unnecessary complications for patients, the use of animal models is required before human implantation. In the case of urethral reconstruction, due to their roughly similar urethral organisation, rabbits were chosen. A similar choice had been done by the Dr. Frey's team which reconstructs rabbit urethra [40] using collagen gels [41], but without epithelial layers. To produce a tube showing a good rigidity and to compare it with the human model already established by Cattan in 2010 remain the goal of this objective.

2.7. Extracellular matrix production in rabbit cell cultures

The conditions of cell cultures have to be modified from the original protocols. For example, an increase in the temperatures during culture similar to the internal rabbit temperature is required to mimic internal rabbit organism. Further investigations to obtain a sufficient amount of extracellular matrix and render the stroma handleable should be presented soon by Morissette et al. Several inducers of collagen synthesis, secretion and deposition have been tested such as insulin or adenosine. Cell seeding technique was also modified with a greater benefit. This extracellular matrix, once produced by the cells themselves, allows strengthening of the constructions. Because the texture of reconstructed tissues from rabbit cells is different from the human cells, some tissue engineering protocols used previously had to be adapted.

2.8. Urothelial cell culture

As urologic tissues from rabbits are thinner than the human tissues, techniques of extraction and isolation of urothelial cells had to be revised. The goal of this step is to obtain a reliable source of urothelial cells. Not only a pure population is needed, but technique should give reproductively a sufficient amount of cells to produce equivalents.

Fig. 1. Urothelial cells cultured with a feeder layer of irradiate rabbit fibroblasts, 3 days post-seeding. 40X

3. Conclusion

The production of a urethral equivalent, reconstructed by tissue engineering in a rabbit model, leads research towards the preclinical step and forward to the human transplantation of the self assembly substitute. Tissue engineering is a constantly evolving domain of research: Applications, not only to tissue reconstruction, but also to help elucidation of complex biological phenomenon, render possible to believe that many problems will find accurate solutions. Regenerative medicine must respond to the increasing number of pathologies expected in the next decades. For now, self assembly method, because it is free of exogenous material, seems to be the closest to physiological conditions among those available, raising the possibility to reconstruct more appropriate tissue and more accurate model to study normal and pathologic conditions.

Acknowledgements

Special thanks for Jean-Michel Bourget, Dr. Karine Vallières and Alexandre Rousseau for helpful discussions. Authors thank the Réseau ThéCell of FRQS for facilities. These researches were supported by FRQS, CIHR, CUA Scolarship Fund, Kidney foundation of Canada and Astellas Research Competition.

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