Open access peer-reviewed chapter

# Aspects of Urological Tissue Engineering

By Arun K. Sharma and Dorota I. Rozkiewicz

Submitted: November 5th 2010Reviewed: May 4th 2011Published: August 17th 2011

DOI: 10.5772/19863

## 1. Introduction

Factors affecting the homeostasis of the urological system stem from a multitude of different sources that include developmental miscues to aberrant signaling pathways leading to malignant transformations. In order to create functional tissues that can act as surrogate tissues while simultaneously mimicking endogenous anatomical and physiological functions, strategies utilizing regenerative medicine based methodologies must be employed. The urological system is comprised of the kidneys and their corresponding ureters; the bladder and sphincter as well as aspects of the genitourinary system. The complex architectural makeup and intricate physiological processes of the urological system at the gross and microscopic levels make tissue engineering attempts to recapitulate these tissues a very difficult and arduous process. Although great strides have been made in the field of urological tissue engineering over the last two decades, the clinical needs of patients suffering from urological defects have largely gone unfulfilled. However, the advent of novel approaches that span multiple disciplines including the material and clinical sciences as well as the constantly evolving field of stem cell biology have curtailed these issues to some extent. This Chapter will delve into strategies that are currently utilized for urological based tissue engineering while also providing alternative tactics to prevailing approaches.

## 2. Conditions affecting the urological system

### 2.1. The kidney

A simplistic overview of nephrogenesis proceeds as three sequential yet overlapping phases during human embryogenesis. During the third to fourth week of development, primitive cell populations derived from intermediate mesoderm converge upon the cervical region of the embryo to form one of two transitory structures termed the pronephros. (McCrory, 1974) These converging cell groups form rudimentary excretory units or nephrotomes that join with the pronephric duct. Although the pronephros is non-functional in mammals, it serves as a precursor structure for the slightly more defined mesonephros. (McCrory, 1974) The progressive development of the definitive kidney continues as the pronephros regresses and the development of mesenophric ducts again derived from intermediate mesoderm becomes apparent. (Pelliniemi, 1983) The mesonephros is composed of two distinct structures, the mesenophric ducts and the mesonephric tubules. The mesonephric tubules acquire a bed of capillaries and are analogous in physiological function as well as anatomic structure to adult nephrons in the definitive kidney. These elementary nephrons include the glomerulus, renal corpuscles, and Bowman’s capsule. The mesonephric ducts (Wolffian ducts) eventually give rise to the epididymis, the vas deferens, and the seminal vesicles in males and as vestigial organs in females. (McCrory, 1974; Hannema, 2006; Welsh, 2006) An outpocketing from the mesonephric duct arises during the fifth to six week of development to form the ureteric bud which becomes the decisive ureter as well as the collecting system found within the kidney including major and minor calyces, and the renal pelvis within the final stage of kidney formation, the metanephros. (McCrory, 1974; Fischer, 2001) The physiological aspects of kidney function are numerous in nature and non-exclusively include hormone production and regulation, homeostatic balance, pH and blood pressure control, waste removal and nutrient re-absorption. It is therefore imperative that the various physiological aspects of the kidney remain under proper control and maintenance. The introduction of insult in the forms of either trauma or aberrant developmental events can signify a poor outcome for the patient.

Congenital abnormalities of the kidney and urinary tract (CAKUT) occur in 1 out of 500 newborns which constitutes approximately 30% of all prenatal anomalies. (Toka, 2010) These include a plethora of conditions including horse-shoe kidney, ureter malformation/abnormalities, renal hypoplasia, renal agenesis, polycystic kidney disease, and Wilms’ tumor formation and can arise at varying stages of the developmental process. (Toka, 2010; Stahl, 2006; Gimpel, 2010; Acien, 2010) Depending upon the severity of the malformation and organs affected, various strategies can been utilized to treat patients afflicted with CAKUT that include surgical and conservative management. However, even with treatment, kidney and urinary tract functions are typically far from ideal. Certain signaling pathways have been elucidated but only scratch the surface of this complex disorder. (Stahl, 2006; Nakanishi, 2003)

### 2.5. The urethra

Urethral narrowing is the most common affliction affecting the urethra. Physical trauma or insult by bacteria or virus can result in an abundance of scar tissue caused the local invasion of inflammatory cells as the body attempts to resolve the situation at hand resulting in stricture. Clinical presentation includes painful urination, decreased urinary output, dark colored urine, pelvic pain, and incontinence among some of the symptoms. (Ghoniem, 1994; Barbagli, 2007; Mangera, 2010) Depending upon the anatomical location and the severity of the stricture, different techniques have been utilized to remedy stricture exacerbation. Urethroplasty has been successfully utilized as a treatment option to repair the stricture. One method is to excise the stricture from the urethra and then rejoin the opposing ends. A second approach requires tissue grafting from typically autologous sources of buccal mucosa, amniotic membrane, or saphenous vein tissue. (Mangera, 2010; Goel, 2011, Levine, 2007, Shaeer, 2006) Complications associated with this type of surgery include fistula formation, infection, and stricture reformation. (Olajide, 2010) Tissue engineering approaches using synthetic and biologic matrices have also been investigated as alternative approaches to urethral repair. Derivatives of PLGA have been created to mimic buccal mucosa with the added benefit of possessing superior mechanical and elastic properties. (Selim, 2011) Other biomaterials such as SIS, bladder acellular matrix (BAM), and acellular corpus spongiosum matrix (ACSM) have also been utilized in studies with favorable results. (Feng, 2010; Powers, 2010) Since host inflammatory responses appear to be a major culprit behind stricture formation and its persistence, means to address this issue should be explored. One practical solution may be the localized delivery of anti-inflammatory agents into the affected area to prevent further stricture formation. This may be accomplished via a number of delivery vehicles including hydrogel releasing anti-inflammatory agents as well as self-assembling nanomolecules capable of down-regulating the cellular inflammatory response through surface expression of anti-inflammatory epitopes. Current research is focusing upon the creation of peptide amphiphiles that may be capable of such feats. Combined with the anti-inflammatory characteristics of mesenchymal stem cells, this one-two punch to either attenuate inflammation or act in a prophylactic manner may help with stricture development and other biologically relevant other pro-inflammatory situations.

## 3. Stem cells and potential applications in urological regenerative medicine

Stem cells can be described in several different embodiments such as those that are derived from embryonic sources or somatic (adult) in nature. The differential capabilities of these cells can further be sub-categorized as being either totipotent, pluripotent, or multipotent. Totipotent stem cells are a result of a fusion of sperm and egg that have undergone limited divisions during early embryological development in mammalian systems. (Mitalipov, 2009; Seydoux, 2006) At this early stage of development, totipotent stem cells have the potential to become any type of cell in the body which also includes extraembryonic tissue. (Mitalipov, 2009; Seydoux, 2006) As a zygote undergoes further differentiation, totipotent stem cells lose their ability to create whole organisms and become more lineage restricted, hence becoming pluripotent. Pluripotent stem cells can give rise to tissues of endoderm, ectoderm, and mesoderm origin, but not extraembryonic tissue and can be derived from both embryonic and somatic cells. (Pittenger, 1999, Thomson, 1998; Takahashi, 2007; Ko, 2009) Multipotent cells are greatly restricted in their ability to generate specific differentiated cell types and subsequently tissue. These cells retain the ability to self-renew and divide into daughter progeny, but these characteristics are greatly limited. A highly characterized population of cells that fits this definition is mesenchymal stem cells that have been described to differentiate into adipocytes, osteocytes, chondrocytes, and others. (Pittenger, 1999, Phadnis, 2011)

### 3.1. Mesenchymal stem cells

The hematopoietic system and its constituents have been extensively characterized at multiple levels via decades of research and clinical experiences. The hematopoietic system found within the bone marrow of long bones in mammals consists of a hierarchy of cells originating from a primitive hematopoietic stem cell. The hematopoietic stem cell is capable of giving rise to all blood derived cells including those of lymphoid and myeloid origin. (Akashi, 2000) Also residing in the bone marrow are non-hematopoietic multipotent mesenchymal stem cells capable of variable cellular differentiation as previously described. (Takahashi, 2007; Ko, 2009; Phadnis, 2011) The frequency of which bone marrow derived mesenchymal stem cells are found within long bones is approximately 0.001% to 0.1% of nucleated cells which include adipocytes, macrophages, osteoblasts, and reticular connective tissue. (Pittenger, 1999) Therefore, fibroblast cells that reside in the bone marrow are not synonymous with mesenchymal stem cells. The plasticity demonstrated by mesenchymal cells can be utilized in a variety of regenerative medicine settings, especially those targeting urologic defects.

Although the aforementioned studies provided strong evidence for bladder regeneration in hemicystectomized animals, future endeavors will require a great deal of scientific prowess to regenerate a fully functional bladder. Hence, the natural course of action would be the continuance of these studies. One aspect in conjunction with regenerating tissue is to create an environment that can provide a robust blood supply to developing tissue. Genetically modified mesenchymal stem cells carrying pro-angiogenic growth factors under the control of inducible promoters may aid in this function. A second strategy involves the use of nanomolecules capable of delivering much needed growth factors to tissues undergoing regeneration. Rajangam et al demonstrate robust in vivo angiogenesis via the delivery of heparin binding nanostructures. (Rajangam, 2006) The cross-pollination of stem cell biology with nanotechnology derived materials can potentially provide reliable and reproducible results when applied to urologic tissue regeneration which is the basis for future studies.

There are several tissue sources in which mesenchymal stem cells can be identified and isolated. Two of those sources are found within adipose tissue as well as amniotic fluid which bathes the fetus in utero. These cell populations have been shown to function within urological settings. Dyslipidemia has been associated with a variety of poorly compliant physiological conditions including erectile dysfunction. (Huang, 2010) Damage occurs to the cavernous endothelium and associated nerves through mechanisms that are not quite fully understood. In order to facilitate recovery, adipose derived mesenchymal stem cells have been utilized to augment repair in the penis with the understanding that various factors secreted from mesenchymal stem cells promote re-growth of endothelium and improved nerve conduction. Within the context of this study, adipose derived mesenchymal stem cells were injected into the corpus cavernosum of dyslipidemic rats. Following a period of recovery, erectile function was assessed and mean intracavernous pressure/arterial pressures ratios were superior to control samples. Increased levels of nitric oxide synthase and alpha smooth muscle actin were also observed. (Huang, 2010) This study provides preliminary evidence that adipose derived mesenchymal stem cells can be used to ameliorate the urologic complications that arise in urologic settings. In order to obtain continual exposure of these factors in the afflicted areas, seeding these cells upon implantable nanofabricated materials may allow for immediate regeneration accompanied by long term maintenance. Nanofabricated materials can recreate specific cell niches in order to potentially enhance the regenerative effect and this topic will be discussed in greater detail within subsequent sections. (Murtuza, 2009)

Adipose derived mesenchymal stem cells have also been utilized in bladder regenerative settings and provide a means to obtain a multipotent cell source through adipose tissue aspirates. (Jack, 2009) Adipose derived mesenchymal stem cells maintain an approximate 3% of all nucleated cells within a sample of aspirate. Jack et al seeded adipose derived mesenchymal stem cells upon PLGA scaffolds and implanted them in 50% cystectomized athymic nude rats. 12 weeks post-transplantation, physiological measurements were assessed as well histological and immunofluorescent analyses, and organ bath studies. All data derived from this study indicate that the adipose derived mesenchymal stem cells perform similarly or better than control samples in almost every biological aspect. The novelty of this study obviously involves the use of this unique population cells. Future studies involving potentially sub-total cystectomized large animal models will hopefully provide a great deal of physiological information that may be applied to the human condition.

The recently described characterization of amniotic fluid mesenchymal stem cells offers another potential alternative cell source that may be applied to urologic tissue engineering. Initially described by In 't Anker et al, 2ml of second trimester amniotic fluid was obtained through transcervical isolation. (In’t Anker, 2003) Subsequent cell populations were cultured in vitro and coerced to undergo terminal differentiation where they demonstrated differentiation into adipocytes and osteocytes. Transplantation studies also provided evidence that these cells could be used in an allogeneic setting, which suggests that these cells may possess a greater immune-privileged status than their adult counterparts. The application of amniotic fluid derived mesenchymal stem cells in a bladder injury model convincingly demonstrates the wound healing potential of these cells. (De Coppi, 2007) The direct injection of amniotic fluid derived mesenchymal stem cells into the cryo-injured bladder resulted in markedly reduced levels of smooth muscle hypertrophy but also demonstrated a limited level of smooth muscle cell regeneration which may have been attributed to inadequate numbers of injected cells since a dose response curve was not performed. Although both experimental procedures describe this source of cells as potent in function, the means in which they are acquired are highly invasive and pose great risk to fetus and mother. It is estimated that 1 in 300 women experience fetal loss, infection, cramping/vaginal bleeding or other complications during or following amniocentesis. (Simpson, 2007) The isolation and subsequent banking of these cells may allow a convenient means for future therapies in both autologous and allogeneic settings.

### 3.2. Endothelial progenitor cells

The lack of a properly vascularized graft is the bane of many tissue engineering studies regardless of the tissue attempting to be regenerated. Inadequate or improper vascularization leads to poor tissue development or graft failure of implanted cell/scaffold composites. This is evident towards the center of grafted composites as these areas are typically inaccessible to nutrient and gas exchange potentially resulting in a necrotic core. A manner in which to avoid the aforementioned scenarios would be to utilize autologous sources of endothelial progenitor cells. These cells could promote vascular growth even at the most cell dense levels of the graft since the endothelial progenitor cells can simultaneously be seeded along with other bladder cell types thereby encouraging vascular growth. Endothelial progenitors can typically be isolated from either the bone marrow or rather innocuously from the peripheral blood as circulating endothelial progenitor cells.

#### 3.2.1. Bone marrow derived endothelial progenitor cells

It was initially speculated that cells that were destined to become constituents of blood vessels as a result of vascular growth were present throughout embryonic and early stages of fetal development. This was later revealed to be somewhat inaccurate with the advent of specific antibodies utilized to phenotype specific cell populations. The CD34+ hematopoietic stem cell has been greatly characterized over the last several decades and it has been demonstrated that subset populations derived from bone marrow sources have the capacity to either form functional vasculature in or contribute to its remodeling vivo. (Abu El-Asrar, 2009; Ruger, 2008; Baum, 1992; Takahashi, 1999) CD34+/CD133+ cells derived from the bone marrow have provided evidence that this population can give rise to highly purified endothelial cells. (Quirici, 2001) Sharma et al describe the in vivo potential of this cell population as it was seeded within a chicken chorioallantoic membrane model. (Sharma, 2009) Post experimentation analyses of this work clearly demonstrate the angiogenic potential of bone marrow derived CD34+/CD133+ cells as human specific vasculature was formed utilizing this model. Other derivatives of this cell population including CD34+/CD133+/VEGFR2+, CD34+/CD133+/VEGFR3+, CD34+/VEGFR2+, and CD133+/VEGFR2+ cells maintain similar properties. (Salven, 2003; Luppi, 2010) However some may argue that the CD34 epitope is not required to describe an endothelial progenitor/precursor cell, although those groups appear to be in the minority. (Friedrich, 2006) In all cases, the frequency of bone marrow derived endothelial progenitor cells is quite low, hence other alternatives cell sources should be examined for potential regenerative medicine purposes.

#### 3.2.2. Circulating endothelial progenitor cells

The seminal discovery that endothelial progenitor cells were persistent throughout the vascular system gave a tremendous boost to a number of tissue engineering approaches. Approximately 11 years ago, Asahara et al describes a simplistic and non-invasive means to isolate circulating endothelial progenitor cells. (Asahara, 1997) By utilizing CD34+ cells isolated from peripheral blood, the study demonstrates that this population of cells was able to differentiate into endothelial cells in vitro while concurrently incorporating into sites of active angiogenesis in an animal model of ischemia. Data goes onto describe the concept that circulating endothelial progenitor cells can home to areas of pre-existing angiogenesis and contribute to the angiogenic process. Since this revelation, many studies have been performed that confirm the concept of circulating endothelial progenitor cells. (Dubois, 2011; Baker, 2011) One recent example demonstrates the construction of a tri-leaf heart valve seeded with circulating endothelial progenitor cells. (Sales, 2010) Data revealed that endothelial progenitor cells were able to colonize the heart valve and provide both interstitial and endothelial functions to the evolving valve. (Sales, 2010) This again demonstrates the potential utility of endothelial progenitor cells. The use of endothelial progenitor cells is pivotal for tissue regeneration and cannot be understated. The goal of utilizing endothelial progenitor cells for tissue regeneration is definitely not simplistic in the sense by just applying a population of endothelial progenitor cells to a graft and assuming angiogenic events. The shear number of molecules that are involved in the angiogenic pathway need to be carefully balanced in order to prevent aberrant results. This can be demonstrated as an over-abundance of localized vascularization leading to excessive bleeding and subsequent pathological issues. The strengths of using circulating endothelial cells are that they can be isolated in large numbers and more importantly, they can be isolated in an autologous fashion. The latter avoids a battery of immunological events and immunosuppressive therapies that may have to be endured for the lifetime of the patient. Unfortunately, works describing the use endothelial progenitor cells in urological tissue engineering is very limited and should be further investigated as this resource is being squandered.

### 3.3. Neural stem/progenitor cells

The greatest issue involved with urological based tissue engineering strategies (in this author’s opinion) is the recapitulation of functional nervous tissue. Although attempts have been made to provide neural functionality to urologic organs such as the bladder utilizing nerve re-routing techniques, these results have been met with skepticism and concern. (Xiao, 2005; Kurzrock, 2010; Park, 2010) Thus, alternate avenues should be evaluated to create functional neural circuitry for urologic organs such as the use of stem cells. The initial isolation and identification of neural stem cells occurred in 1992 by Reynolds and Weiss. (Reynolds & Weiss, 1992) Neural stem cells were isolated from the striatum of the mouse brain and under specific culture conditions coerced to differentiate into neurons and astrocytes. Further testing demonstrated morphological features that were indicative of functional neural cells. This seminal work provided compelling evidence that argued against the long established belief that brain derived tissue could not undergo neurogenesis after birth or damaging insult. Subsequent in vivo studies exploited the use of neural stem and more differentiated neural progenitor cells in a variety of in vivo settings as cells were also being isolated from peripheral nervous systems. (Gage, 2000) One study in particular describes the transplantation of neural progenitor cells into the cortical region of the rat brain that underwent lesion induction. (Prajerova, 2010) Electrophysiological assessments of brain slices revealed the generation of action potentials created by the neural progenitor cells that were statistically greater than control samples. Evidence was also provided in which transplanted neural progenitor cells exhibited expression of differentiated neural markers by immuno-staining. (Prajerova, 2010)

As of this writing, attempts to utilize bona fide neural stem or progenitor cells have not come to fruition in a urological regenerative medicine setting. One study does report the use of neuronal-glial precursor cells which facilitated the mild improvement in bladder physiological function. These included various urodynamic parameters such as mean voiding pressure, capacity, bladder pressure, and mean contraction amplitude. These values were marginally better than control animals. (Temeltas, 2009) It is quite apparent that a considerable amount of research is required to address the major concerns regarding urologic tissue neuro-regeneration. Cultivating cross-discipline approaches may provide a suitable means to an end.

### 3.4. Embryonic stem cells

The seemingly limitless therapeutic potential of human embryonic stem cells has commanded the attention of the scientific community since its initial discovery. (Thomson, 1998) Derived from the inner cell mass of the developing blastocyst, pluripotent embryonic stem cells can be isolated and possess several unique features. These are typified by high nuclei to cytoplasm ratios, very pronounced nucleoli, the capability of indefinite cellular propagation, and most significantly, the ability to differentiate into cell types composed of all three germ layers. (Thomson, 1998) The isolation and characterization of embryonic stem cells laid the foundation for literally thousands of studies that now spans multiple disciplines and aspects of regenerative medicine. (Birket, 2011; Nistor, 2005; Farzaneh, 2010) The use of embryonic stem cells is slowly making its way into the realm of urological tissue engineering. One study describes an in vitro culture environment in which conditioned media from primitive ureteric bud cells were used to support the differentiation of murine mesoderm cells initially derived embryonic stem cells. (Ren, 2010) The mesoderm cells were primed with activin and retinoic acid and subsequently forcibly differentiated into cells of renal lineage with the aid of the conditioned media. The strong inductive effect of the secreted molecules from the ureteric bud culture provides a means to elucidate potential mechanisms involved with mesoderm-renal cell lineage differentiation. A second, more recent study sheds light upon some of the molecules involved in the differentiation of murine embryonic cells into urothelium. (Mauney, 2010) As previously utilized in the aforementioned study by Ren et al, all trans-retinoic acid had the profound ability to induce pluripotent embryonic stem cells in to urothelial cells through a GATA4 and GATA6 signaling pathway. Stimulation of the embryonic stem cells with all trans-retinoic acid caused a prominent increase in several uroplakin genes including UP1A, UP1B, UP2, and UP3B with a simultaneous down-regulation in the pluripotency factor OCT-4 compared to control cell populations. Concurrently, transcription factors GATA4 and GATA6 were also greatly up-regulated. GATA4/6 null embryonic stem cell lines demonstrated a marked reduction in uroplakin expression when undergoing similar treatments as compared to wild type controls. This data suggests a partial mechanism with regard to the inductive effect of all trans-retinoic acid and its ability to drive embryonic stem cells into urothelial cells. Several other studies have also utilized embryonic stem cells or embryoid body derived cells to generate bladder tissue to varying capacities. (Thomas, 2008; Kinebuchi, 2008; Lakshmanan, 2005) Unfortunately, a lack of follow-up studies has seemed to put a damper on this potentially ground breaking field of research.

The utility of embryonic stem cells is obviously not without debate. Ethical and moral dilemmas have significant impact on political policy and are of grave concern for future studies utilizing embryonic stem cells that use federal funds to support research. 2001 saw restrictions placed upon the use and creation of embryonic stem cell lines, a ban that was later rescinded in 2009. The recent political atmosphere continues to be in a state of flux witnessed most recently by a recent court injunction that stopped the use of federal dollars to be used for embryonic stem cell research, thus effecting thousands of researchers. Although the injunction was temporarily stayed upon an appeal, it could set a damaging precedent for the future. Privatized monies as found within the California Institute of Regenerative Medicine currently finance studies with embryonic stem cells with other states in pursuit. (http://cirm.ca.gov/for-researchers/researchfunding) Nevertheless, several clinical trials set forth by privately held companies such as Geron and Advanced Cell Technology are currently underway. (Strauss, 2010; http://www.advancedcell.com/news-and-media/press-release) The controversies surrounded embryonic stem cell research in part fueled the scientific community to pursue other avenues of research that would lead to the identification of an alternative source of pluripotent stem cells.

### 3.5. Induced pluripotent stem cells

Preliminary observations involving studies with somatic cell nuclear transfer into recipient oocytes resulted in cell populations that displayed features that were highly analogous to embryonic stem cells in morphology and function. (McGrath, 1983; Campbell, 1996) Via successive rounds of a highly selective molecular screening process, four genes were determined to be essential in the de-differentiation of terminally differentiated cells back to a pluripotent, embryonic-like state. Takahashi et al produced the first generation of induced pluripotent stem cells from human adult fibroblasts with the over-expression of four transcription factors including Sox2, Oct3/4, c-Myc, and Klf4. (Takahashi, 2007) These cells possessed embryonic stem cell-like qualities as they could differentiate into cells of endoderm, mesoderm, and ectoderm origin; were able to form teratomas in vivo; and proliferate indefinitely without losing their pluripotency. A subsequent study from Yu et al utilized a similar procedure derived from the Takahashi study but determined that a slightly different cocktail of genes (or proteins) could induce the same effect. (Yu, 2007) OCT4, SOX2, NANOG, and LIN28 were identified as pluripotency factors that had the same effect as the aforementioned gene set. Since a slightly different set of genes was utilized in the second study, it would be easy to speculate that different combinations of genes or yet to be identified factors that are tissue specific may be utilized to create pluripotent cell lines. This groundbreaking work led to a number of other studies focusing on specific cell type and disease states. (Moretti, 2010; Ebert, 2009) However, with each great discovery come certain drawbacks. It has been recently demonstrated that induced pluripotent stem cells outwardly function similarly to embryonic stem cells, but the gene expression signatures possessed by each cell type are quite different. (Chin, 2009) This is most likely a result of differential promoter binding by the reprogramming factors. Other studies also demonstrate the detrimental effects that coerced cellular reprogramming may induce upon target cell populations. (Hussein, 2011; Lister, 2011) The use of induced pluripotent stem cells in urological settings (excluding spinal and nerve injury scenarios) is still in its infancy with a paltry number of studies. (Morizane, 2009) As more data emerges with regards to the molecular machinery involved with the reprogramming process (along with potential consequences), perhaps these data will be applied to future urological regenerative medicine based studies.

## 4. Polymeric materials and their potential applications in urologic regenerative medicine

The choice of scaffold material is of paramount significance in determining the outcome of implanted cell/scaffold composites. There are a wide variety of scaffold materials that vary greatly in composition, texture, mechanical and surface characteristics that could potentially be applied in a urological regenerative medicine setting. A number of these materials have been previously discussed in great detail such as bladder acellular matrix, small intestinal submucosa, varying embodiments of poly(lactic-co-glycolic acid) and polyglycolic acid, and collagen. Similarly, other chemically synthesized nano- and macro- based structures are also pivotal in creating hospitable environments for tissue growth and development. The focus of the forthcoming section will concentrate on relatively newly described elastic based materials that may be utilized for urological tissue engineering along with bioactive peptides and nanomolecules.

### 4.1. Synthetic elastomeric substrates for urological tissue engineering

#### 4.1.2. Elastomeric poly(ester urethane)urea

A second well described elastomeric compound, poly(ester urethane)urea (PEUU), can also be configured into several different forms including those consistent with blood vessels while retaining elastic properties. (Stankus, 2007) By concurrent electrospraying and electrospinning of vascular smooth muscle cells and PEUU, respectively, a tubular conduit containing the cells was formed with uniform cellular distribution in a radial and circumferential manner. Further testing revealed the ability of the material to remain sutured, while maintaining compliancy that was similar to native arteries. Modification of PEUU also demonstrates the ability to control gene expression utilizing a cleverly created gene induction system. (Baraniak, 2011) Further modifications also allow for the release of bioactive molecules of insulin-like growth factor-1 and hepatocyte growth factor. (Nelson, 2011) Although this material has not been utilized in a urological regenerative setting to date, the implications of this material are obvious as this material could be structured for urinary bladder regenerative studies or the creation of synthetic ureters. An overly simplistic study design would include the seeding of porous scaffolds with relevant bladder cell types as previously described by Baraniak et al accompanied by the appropriate functional testing and immunohistochemical analyses. The added benefit of utilizing PEUU is its ability to become modified so that relevant genes such as those found within urologic epithelial stem cells may contribute to regeneration, for example. (Pascal, 2007) It is hopeful that studies involving PEUU and urological tissue engineering will be seen in the not too distant future.

### 4.2. Bioactive peptides and peptide amphiphiles for regeneration and wound healing

Polymeric materials which are enhanced with bioactive peptides show perhaps the greatest degree of promise for application in tissue regeneration scaffolds. Short peptide sequences originating from the cell binding regions of extracellular matrix (ECM) proteins can be incorporated into a graft material to obtain biospecific adhesion of cells. RGD (arginine-glycine-aspartic acid), the most commonly used peptide in cell adhesion studies, originating from fibronectin, collagen, fibrinogen and other ECM proteins, enhances adhesion and spreading of most cell types (fibroblasts, endothelial, and smooth muscle cells) and interacts with several different integrin receptors. (Humphries, 1990; Massia, 1991) It has been shown in the past that migration of smooth muscle cells and fibroblasts is biphasically dependant on the concentration of RGD attached to the migratory surface with intermediate loadings of RGD displaying maximal mobility. (Olbrich, 1996; Mann 2002) Polymers containing RGD sequence have been used in several wound healing in vivo and in vitro applications. For instance, incorporation of RGD peptide into PEG hydrogels partially eliminated inflammatory reaction of a scaffold implanted subcutaneously in immunocompetent in c57bl/6 mice resulting in a reduced presence of macrophages. (Lynn, 2011) Hydrogels containing RGD peptide were able to promote healing of ulcers, partial thickness burn wounds, and encourage ingrowth of glial tissue in the rat. PHSRN (proline-histidine-serine-arginine-glutamate), a cell binding peptide sequence has been found to act synergistically with RGD sequence for cell adhesion. (van der Veen, 2010; Davis, 2001) When applied individually it can stimulate invasion of ECM by keratinocytes and fibroblasts in vitro and it can enhance the re-epithelialization and concentration of dermal wounds in healing-impaired diabetic mice. (Livant, 2000) Other peptides sequences such as laminin-derived YIGSR (tyrosine-isoleucine-glycine-serine-arginine), LRE (leucine-arginine-glutamic acid) or IKVAV (isoleucine-lysine-valine-alanine-valine) have potential application in nerve regeneration. (Fittkau, 2005; Pittier, 2005) YIGSR, unlike RGD and PHSRN, does not interact with the 67 kDa laminin binding protein. (Graf, 1987) In addition, YIGSR promotes the adhesion and spreading of many cell types including endothelial cells, fibroblasts, and smooth muscle cells. (Massia, 2011) It was found that PEG surfaces functionalized with cell binding peptide RGD and YIGSR, significantly increased microvascular endothelial cell migration rates relative to RGD alone. (Fittkau, 2005)

Another group demonstrated that degradation of endothelial ECMs with bacterial collagenase releases proangiogenic peptide fragments which can stimulate epithelial responses to injury and wound healing in vivo. (Demidova-Rice, 2011) It has been shown that sequences such as Col4-1, Col4-2, Fibr2, Fibr3, Ten1, Ten2, and Comb1 significantly increase the rate of sprout formation, which enhances cell-cell interactions and improves endothelial motility. Comb1 and Ten2 peptides contain the GXXPG sequence, which was previously reported to enhance endothelial migration and tube formation on collagen type I in vitro; both induce morphogenesis in a Matrigel-based assay. Comb1, as a larger combinatorial peptide with more complex tertiary structure, and two GXXPG sequences also stimulates cellular proliferation. The aforementioned sequences can also stimulate endothelial response to injury in complex environments where the cells remain viable for a prolonged time (up to one week). The 3D model of injury repair which was applied in that research consisted of two layers of Matrigel collagen mixture and a layer of endothelial cells sandwiched in between. Full thickness injury was created by aspiration, and the defect was filled with matrices containing serum, proangiogenic growth factors, or peptides. It was demonstrated that in addition to identification of promising proangiogenic or antiangiogenic therapeutics, the 3D model of injury repair can be employed to study the stability of wound healing compounds and to evaluate drug delivery systems. As described in this Chapter bioactive peptides play an important role in a wound healing process and tissue regeneration. Fabrication of scaffolds with a high specificity for the adhesion of a certain type of cells might require the use of a combination of bioactive peptides such as RGD, IKVAV, Ten 2, Comb 1, and biomimetic synthetic polymers, depending on the in vivo application.

Peptide amphiphiles (PA) are small molecules that contain a peptide sequence and a hydrophobic segment such as an alkyl tail. (Silva, 2004; Rajangam, 2006) Hydrophobic collapse of these filament-forming molecules under strong screening conditions leads to the display of biological signals on their surfaces in high density (on the order of 1015 signals per cm2). (Cui, 2010) In vivo and in vitro studies have shown that certain PA molecules, bearing bioactive epitopes promote regeneration of spinal cord axons, angiogenesis, bone regeneration, cartilage repair, and selective differentiation of neural progenitor cells into neurons. (Sargeant, 2008; Webber, 2010; Silva, 2004) Previously, PAs were used to coat PGA microfiber scaffold for the improved attachment of smooth muscle cells.

(Harrington,2006; Behanna, 2005) This PGA scaffold was submerged in a suspension of smooth muscle cells in media and modified with PA gel mixed with growth factors as a top layer for the entrapment of cells. The opposite face of the scaffold could them be modified using a second component, which in this case was a mixture of PGA and urothelial cells, since these cells could potentially send necessary signals to smooth muscle cells during regeneration. (Beqaj, 2005; Imamura, 2007) When the human bladder smooth muscle cells and urothelial cells where embedded in a PA-scaffold with a growth factor (bFGF) and incubated in vivo in a subcutaneous nude rat model, it was found that the human bladder smooth muscle cells were retained and composed the majority of the scaffold cellular content. It was also found that the system which uses a combination of PGA, PA, growth factors and cells demonstrated higher levels of phenotypic alpha-smooth muscle actin than control scaffolds made of PGA and cells only. PAs offer a great potential in regenerative medicine due to their nanoscale filamentous assembly resembling the one in ECM, the ability to incorporate any bioactive epitopes into their structure, and propensity to form gels.

### 4.3. Nano- and microstructured surfaces for bladder regeneration

Alternative means to achieve nanostructured membranes for bladder regeneration research utilizes micro or nanomolding. PLGA or PU polymers can be easily cast into molds with pre-fabricated nanometer features made of poly(dimenthylsiloxane) (PDMS). (Thapa, 2003) The procedure relies on pouring a solution of a polymer (for example PLGA or PU) onto a PDMS master mold, curing the polymer until is solid and releasing the polymer from the mold simply by peeling the film off from the mold. The minimum size of the features that could be achieved is defined by the master mold. This process is commonly used in the fabrication of microcontact printing stamps for soft lithography purposes. In this process the mold is usually fabricated in silicon through photolithography or e-beam lithography and the polymer which is used for stamps is usually PDMS. (Xia, 1998) PLGA and PU nanostructured polymers showed advantage in ovine bladder smooth muscle cells adhesion over polymers with larger features.

An interesting method for the bladder surface nanostructuring is the application of nanoparticles to introduce nanoscale roughness or improve certain properties such as permeability of the scaffold. One example was recently introduced where hyaluronic acid (HA) modified PLGA nanoparticles (HA PLGA NPs) were applied for the modification of SIS as a scaffold for bladder regeneration to alter its permeability and simultaneously deliver pro-regenerative compounds. (Roth, 2010) When SIS scaffolds were modified with PLGA NPs of the size in the range of 200-500 nm the scaffold showed a decrease in permeability to urea when compared with unmodified scaffolds. In addition, PLGA modified scaffolds demonstrated significantly higher adhesion of endothelial cells in comparison to unmodified scaffolds. Introduction of pro-regenerative compounds into the SIS scaffold via modification of PLGA NPs with HA (which has been recognized for its ability to promote scarless wound healing), improved the SIS scaffold in vivo performance. HA was attached through electrostatic interaction to the positively charged PLGA NPs which were previously modified with polyethylene imine to produce cationic NPs. The attachment of particles to the SIS scaffold was conducted by incubation and absorption of particles from solution. The modified SIS membrane was investigated for angiogenesis and permeability studies. It was found that HA-NP SIS demonstrated a statistically higher number of blood vessels when compared with unmodified. In addition, HA-NP SIS significantly decreased or even eliminated the propensity to form calcifications, decreased urea permeability and improved full-thickness bladder regeneration.

Electrospinning is one of the most commonly used techniques for the (nano)fiber formation. In this process, nanofibers can be formed through an electrically charged jet of polymer solution or polymer melt. The simplest setup for electrospinning is composed of a high voltage power supply, a polymer solution reservoir (e.g. a syringe with a small diameter needle), and two electrodes (one connected to the needle and the other to the surface of the collecting mat or other substrate). The polymer supply is connected with a power supply through an electrode clipped to the metal needle for electrospinning. Flow control pump can be used to precisely control the flow rate of the polymer solution which is spun onto a substrate. The fiber-collecting surface must be conductive in order to attract the electrospun polymer and can be either stationary or rotating plate for collecting nonwoven or aligned fibers, respectively.

Electrospinning permits fabrication of biodegradable elastomers into matrices that can resemble the scale and mechanical behavior of the native extracellular matrix. There is a great selection of polymers available for the electrospinning of nanofibrous scaffolds used in tissue engineering applications. Moreover, this process can be easily scaled up making the transformation from small-scale laboratory research into large-batch processes feasible. (Greiner, 2008; Zhong, 2010) In addition to being used to fabricate nonwoven mats for wound dressings, there is currently much interest in making scaffolds for bladder tissue engineering. (Roth, 2010; Gelain, 2008) Baker et al used a 3D electrospun nanoscaffold of polystyrene to determine whether the phenotype of cells isolated from the stroma of human ureter specimens could be modulated by growing cells within such scaffold. (Baker, 2008) Non-aligned scaffolds made out of fibers with mean diameter of 200 nm were used in that study. Unfortunately the analysis of SM-MHC protein expression in cultures grown in 3D revealed that those cultures failed to undergo differentiation. It was explained that nanostructured polystyrene scaffolds have a high propensity to adsorb serum which maintained stromal cell cultures in a synthetic non-differentiated phenotype. In this case, the approach of growing cell cultures in a serum-free media on the 3D polystyrene scaffold did not bring positive results. It was found that despite of its promising advantages of electrospun scaffold, stromal cells did not differentiate primarily due to the high propensity of the scaffold to the absorption of proteins from the serum. However, serum-free media reduced the absorption of proteins but cells still showed a greatly reduced plating efficiency and cultures failed to survive.

Findings describing the interactions between human bladder urothelial cells (TEU-2) and a scaffold composite consisting of an electrospun fibrous matrix made of PCU and PLLA fused to a thin film for potential use in urothelial tissue regeneration. (Kundu, 2011) Their results suggested that when electrospun scaffolds alone (without the thin film) were used, their fibrous surface structure while allowing good cellular adhesion, inhibited the proliferation of urothelial cells, whereas cells seeded onto thin films with non-fibrous surfaces proliferated quickly. In addition, it was found that cells on electrospun fibers demonstrated numerous cellular extensions along the matrix fibers, rather than the substantial cell-cell integrations. It was explained that this finding may suggest that the fibrous nature of the electrospun materials do not facilitate the formation of an intact urothelium. The addition of thin films onto electrospun scaffolds, enhanced urothelial cell proliferation, surface coverage, cell-cell interactions, and multilayering in vitro. Electrospun scaffolds alone offer highly porous structures and deep cellular penetration, however they are unlikely to provide a sufficient barrier to water and solute transport when not embedded in a biomimetic synthetic polymer. Furthermore, the three-dimensional structure of such scaffolds may affect cell proliferation, differentiation or adhesion.

## 5. Conclusion

The task to create functional organs as a replacement therapy is indeed quite daunting. Tissue engineering studies during the late 1980s and early 1990s laid the groundwork for the current windfall of clinical applications that have been deployed for organ replacement. The continual evolution of materials science influenced materials combined with discoveries in the field of stem cell biology provides hope that goals of regenerative medicine can be achieved. Although there have been many significant advances with regard to urologic tissue engineering, the field is still in its infancy. There is still a great need to pursue basic and clinical science studies in order to benefit those afflicted with urological trauma or disease.

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Arun K. Sharma and Dorota I. Rozkiewicz (August 17th 2011). Aspects of Urological Tissue Engineering, Tissue Engineering for Tissue and Organ Regeneration, Daniel Eberli, IntechOpen, DOI: 10.5772/19863. Available from:

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