- Email: [email protected]
Tissue engineering of reproductive tissues and organs
Tissue engineering of reproductive tissues and organs Anthony Atala, M.D. Department of Urology, Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
Regenerative medicine and tissue engineering technology may soon offer new hope for patients with serious injuries and end-stage reproductive organ failure. Scientists are now applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that can restore and maintain normal function in diseased and injured reproductive tissues. In addition, the stem cell field is advancing, and new discoveries in this field will lead to new therapeutic strategies. For example, newly discovered types of stem cells have been retrieved from uterine tissues such as amniotic fluid and placental stem cells. The process of therapeutic cloning and the creation of induced pluripotent cells provide still other potential sources of stem cells for cell-based tissue engineering applications. Although stem cells are still in the research phase, some therapies arising from tissue engineering endeavors that make use of autologous adult cells have already entered the clinic. This article discusses these tissue engineering strategies for various organs in the male and female reproductive tract. (Fertil SterilÒ 2012;98:21–9. Ó2012 by American Society for Reproductive Medicine.) Key Words: Amniotic fluid stem cell, biomaterial, penis, testes, uterus, urethra, vagina
P
atients suffering from various congenital and acquired disorders of the reproductive tract are currently treated with surgical and hormonal therapies. However, these techniques are not ideal. For example, many current hormonal therapies do not provide physiologic levels of sex hormones, and this can lead to a number of problems. In addition, many surgical procedures, such as grafting to replace damaged areas of the male urethra, may eventually fail due to many factors. This has led scientists in the field of regenerative medicine and tissue engineering to apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that may eventually improve the quality of life for these patients in a number of ways, from providing physiologic hormone secretion to perhaps one day restoring fertility with tissue-engineered reproductive
organs. This article reviews recent advances in regenerative medicine and describes applications of these new technologies in reproductive medicine. The field of regenerative medicine encompasses various areas of technology, such as tissue engineering, stem cells, and cloning. Tissue engineering combines the principles of cell transplantation, materials science, and bioengineering to develop new biological substitutes that may restore and maintain normal organ function. Tissue engineering strategies generally fall into two categories: the use of acellular matrices, which depend on the body's natural ability to regenerate and serve as guides for proper orientation and direction of new tissue growth, and the use of matrices seeded with cells. Acellular tissue matrices are usually prepared by manufacturing artificial scaffolds, or by removing cellular com-
Received April 16, 2012; revised and accepted May 25, 2012. A.A. has nothing to disclose. Reprint requests: Anthony Atala, M.D., Director, Wake Forest Institute for Regenerative Medicine, The W. Boyce Professor and Chair, Urology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157 (E-mail: [email protected]). Fertility and Sterility® Vol. 98, No. 1, July 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.05.038 VOL. 98 NO. 1 / JULY 2012
ponents from tissues via mechanical and chemical manipulation to produce collagen-rich matrices (1–4). These matrices tend to slowly degrade after implantation and are generally replaced by the extracellular matrix (ECM) proteins that are secreted by host cells that infiltrate the matrix. Cells can also be used for therapy via injection, either with carriers such as hydrogels, or alone. In addition, cells can be used for matrix-based tissue engineering strategies. To do this, a small piece of donor tissue is dissociated into individual cells. These cells are either implanted directly into the host, or they are expanded in culture, attached to a support matrix, and then the cellmatrix construct is implanted into the host after expansion. The source of donor tissue can be heterologous (such as bovine), allogeneic (same species, different individual), or autologous. Ideally, both structural and functional tissue replacement will occur with minimal complications. Autologous cells derived from a biopsy of tissue are the preferred type to use. They are obtained from the host, dissociated from the tissue biopsy, and expanded in culture. Next, they are implanted back into the same host (2, 5–12). The use of 21
VIEWS AND REVIEWS autologous cells, while it may cause an initial inflammatory response, avoids rejection, and thus the deleterious side effects of immunosuppressive medications can be avoided. Thus, most current strategies for tissue engineering depend upon a sample of autologous cells from the diseased organ of the host. However, for many patients with extensive end-stage organ failure, a tissue biopsy may not yield enough normal cells for expansion and transplantation. In other instances, primary autologous human cells cannot be expanded from a particular organ such as the pancreas. In these situations, stem cells are envisioned as being an alternative source of cells from which the desired tissue can be derived. Stem cells can be derived from discarded human embryos (human embryonic stem cells), from fetal tissue, or from adult sources (bone marrow, fat, skin). Therapeutic cloning has also played a role in the development of the field of regenerative medicine. Recently, the findings of induced pluripotency in which cells such as fibroblasts are ‘‘reprogrammed’’ to behave like stem cells has been described, and it may also provide a new source of cells for tissue-engineering strategies.
BIOMATERIALS FOR USE IN TISSUE ENGINEERING In many cell-based tissue engineering methods, cells are obtained from a tissue, expanded in vitro, and then seeded onto a scaffold composed of an appropriate biomaterial. These biomaterials replicate the biologic and mechanical function of the native extracellular matrix (ECM) found in tissues in the body by serving as an artificial ECM. Biomaterials also provide a three-dimensional scaffold for the cells to adhere to and form new tissues with appropriate structure and function. They also allow for the delivery of cells and appropriate bioactive factors to desired sites in the body (13). Bioactive factors, such as cell-adhesion peptides and growth factors, can be loaded along with cells to help regulate cellular function. As the majority of mammalian cell types are anchorage dependent and will die if no cell-adhesion substrate is available, biomaterials provide a cell-adhesion substrate that can deliver cells to specific sites in the body with high loading efficiency. Biomaterials can also provide mechanical support against in vivo forces and ensure that the predefined threedimensional structure of an organ is maintained during tissue development. The ideal biomaterial should be biodegradable and bioresorbable to support the replacement of normal tissue without inducing inflammation. Incompatible materials that induce inflammatory or foreign-body responses eventually become necrotic and/or are rejected. Degradation products, if produced, should be removed from the body via metabolic pathways at an adequate rate to keep the concentration of these products in the tissues at a tolerable level (14). The biomaterial should also provide an environment in which appropriate regulation of cell behavior (adhesion, proliferation, migration, and differentiation) can occur such that functional tissue can form. Cell behavior in the newly formed tissue has been shown to be regulated by multiple interactions of the cells with their microenvironment, including interactions with 22
cell-adhesion ligands (15) and with soluble growth factors. Since biomaterials provide temporary mechanical support while the cells undergo spatial reorganization into tissue, the properly chosen biomaterial should allow the engineered tissue to maintain sufficient mechanical integrity to support itself in early development, while in late development, it should begin to degrade so that it does not hinder further tissue growth (13). Three broad classes of biomaterials have been utilized in tissue engineering studies: naturally derived materials (e.g., collagen and alginate), acellular tissue matrices (e.g., bladder submucosa and small intestinal submucosa), and synthetic polymers such as polyglycolic acid (PGA), polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA). Collagen is the most abundant and ubiquitous structural protein in the body, and may be readily purified from both animal and human tissues with an enzyme treatment and salt/acid extraction (16). Collagen contains cell adhesion domain sequences (e.g., RGD) that may assist in maintaining the phenotype and activity of many types of cells, including fibroblasts (17) and chondrocytes (18). Acellular tissue matrices are collagen-rich matrices prepared by removing cellular components from tissues. The matrices are often prepared by mechanical and chemical manipulation of a segment of tissue (1–4). These matrices slowly degrade upon implantation, and are replaced and remodeled by new ECM proteins synthesized and secreted by transplanted or ingrowing host cells. Commonly used acellular tissue matrices include bladder submucosa (BSM) and small intestinal submucosa (SIS). Polyesters of naturally occurring a-hydroxy acids, including PGA, PLA, and PLGA, are also widely used in tissue engineering. These polymers have gained FDA approval for human use in a variety of applications, including sutures (19). Because these polymers are thermoplastics, they can be easily formed into a three dimensional scaffold with a desired microstructure, gross shape, and dimension by various techniques including molding, extrusion, solvent casting (20), phase separation techniques, and gas foaming techniques (21). Recently, composite scaffolds consisting of collagen and the synthetic polymer polycaprolactone (PCL) have been formed using a novel electrospinning technique (22, 23). Many applications in tissue engineering often require a scaffold with high porosity and ratio of surface area to volume, and electrospinning allows these parameters to be varied quickly and easily. Other biodegradable synthetic polymers, including poly(anhydrides) and poly(ortho-esters), can also be used to fabricate scaffolds for tissue engineering with controlled properties (24).
CELLS FOR USE IN TISSUE ENGINEERING Embryonic Stem Cells Human embryonic stem cells (hESCs) exhibit two remarkable properties: the ability to proliferate in an undifferentiated but pluripotent state (self-renewal), and the ability to differentiate into almost all of the specialized cell types in the body (25). They can be isolated by aspirating the inner cell mass from the embryo during the blastocyst stage (5 days after fertilization). Human embryonic stem cells have been shown to VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility® differentiate into cells from all three embryonic germ layers in vitro. Skin and neurons have been formed, indicating ectodermal differentiation (26–29). Blood, cardiac cells, cartilage, endothelial cells, and muscle have been formed, indicating mesodermal differentiation (30–32). Pancreatic cells have been formed, indicating endodermal differentiation (33). In addition, as further evidence of their pluripotency, embryonic stem cells can form embryoid bodies, which are cell aggregations that contain all three embryonic germ layers while in culture, and can form teratomas in vivo (34). However, the tendency to form teratomas in vivo is also a limitation to widespread usage of hESCs in cell therapy and tissue engineering, as it is difficult if not impossible to control the tumorigenicity of these cells in vivo, and this leads to a number of safety concerns surrounding the usage of these cells for therapeutic benefit. In addition, the ethics considerations surrounding the destruction of human embryos to obtain hESCs are yet to be resolved. Finally, the use of human embryonic stem cells is banned in some countries, including the United States, and so their use in tissue engineering is infrequent in those countries at this time.
Laboratory-Generated Stem Cells In addition, stem cells for tissue engineering could be generated through ‘‘cloning’’ procedures. Somatic cell nuclear transfer (SCNT) is used to generate early-stage embryos that are explanted in culture to produce embryonic stem cell lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue and organ replacement applications (35). Therefore, somatic cell nuclear transfer may provide an alternative source of transplantable cells. While promising, somatic cell nuclear transfer technology has certain limitations that require further study before this technique can be applied widely in tissue or organ replacement therapy. First, the efficiency of the cloning process is very low, as evidenced by the fact that the majority of embryos derived from the cloning process do not survive (36–38). To improve cloning efficiency, further improvements are required in many of the complex steps of nuclear transfer, such as the enucleation process for oocytes, the actual transfer of a nucleus to this enucleated oocyte, and the activation process that instructs the cloned oocytes to begin dividing. In addition, cell cycle synchronization between donor cells and recipient oocytes must be accomplished (39). Other barriers to the frequent use of SCNT in human applications are the requirement for human oocytes, which are difficult to obtain in the quantities required, and, as with hESCs, there are ethical considerations surrounding the production and destruction of human embryonic tissue that remain to be resolved. Recently, exciting reports of the successful transformation of adult cells into pluripotent stem cells through a type of genetic ‘‘reprogramming’’ have been published. Reprogramming is a technique that involves de-differentiation of adult somatic cells to produce patient-specific pluripotent stem cells, without the use of embryos. Cells generated by VOL. 98 NO. 1 / JULY 2012
reprogramming would be genetically identical to the somatic cells (and thus the patient who donated these cells) and would not be rejected. Takahashi and Yamanaka (40) were the first to discover that mouse embryonic fibroblasts (MEFs) and adult mouse fibroblasts could be reprogrammed into an ‘‘induced pluripotent state’’ (iPS). This group used mouse embryonic fibroblasts (MEFs) engineered to express a neomycin resistance gene from the Fbx15 locus, a gene expressed only in embryonic stem (ES) cells. They examined 24 genes that were thought to be important for embryonic stem cells and identified four key genes that, when introduced into the reporter fibroblasts, resulted in drug-resistant cells. These were Oct3/4, Sox2, c-Myc, and Klf4. This experiment indicated that expression of the four genes in these transgenic MEFs led to expression of a gene specific for ES cells. Mouse embryonic fibroblasts and adult fibroblasts were cotransduced with retroviral vectors, each carrying one of the four genes, and transduced cells were selected via drug resistance. The resultant iPS cells possessed the immortal growth characteristics of self-renewing ES cells, expressed genes specific for ES cells, and generated embryoid bodies in vitro and teratomas in vivo. When iPS cells were injected into mouse blastocysts, they contributed to a variety of cell types. However, although iPS cells selected in this way were pluripotent, they were not identical to ES cells. Unlike ES cells, chimeras made from iPS cells did not result in full-term pregnancies. Gene expression profiles of the iPS cells showed that they possessed a distinct gene expression signature that was different from that of ES cells. In addition, the epigenetic state of the iPS cells was somewhere between that found in somatic cells and that found in ES cells, suggesting that the reprogramming was incomplete. These results were improved significantly by Wernig et al. (41) in 2007. Fibroblasts were infected with retroviral vectors and selected for the activation of endogenous Oct4 or Nanog genes. Results from this study showed that DNA methylation, gene expression profiles, and the chromatin state of the reprogrammed cells were similar to those of ES cells. Teratomas induced by these cells contained differentiated cell types representing all three embryonic germ layers. Most importantly, the reprogrammed cells from this experiment were able to form viable chimeras and contribute to the germ line like ES cells, suggesting that these iPS cells were completely reprogrammed. Wernig et al. (41) observed that the number of reprogrammed colonies increased when drug selection was initiated later (day 20 rather than day 3 post-transduction). This suggests that reprogramming is a slow and gradual process and may explain why previous attempts resulted in incomplete reprogramming. It has recently been shown that reprogramming of human cells is possible. Yakahashi et al. (42) showed that retrovirusmediated transfection of OCT3/4, SOX2, KLF4, and c-MYC generates human iPS cells that are similar to hES cells in terms of morphology, proliferation, gene expression, surface markers, and teratoma formation. Yu et al. (43) showed that retroviral transduction of OCT4, SOX2, NANOG, and LIN28 could generate pluripotent stem cells without introducing any oncogenes (c-MYC). Both studies showed that human iPS were similar but not identical to hES cells. 23
VIEWS AND REVIEWS Another concern is that these iPS cells contain three to six retroviral integrations (one for each factor) which may increase the risk of tumorigenesis. Okita et al. (44) studied the tumor formation in chimeric mice generated from NanogiPS cells and found 20% of the offspring developed tumors due to the retroviral expression of c-myc. An alternative approach would be to use a transient expression method, such as adenovirus-mediated system, since both Okita et al. (44) and Meissner et al. (45) showed strong silencing of the viralcontrolled transcripts in iPS cells. This indicates that these viral genes are only required for the induction, not the maintenance, of pluripotency. Another concern is the use of transgenic donor cells for reprogrammed cells in the mouse studies. Both studies used donor cells from transgenic mice harboring a drug resistance gene driven by Fbx15, Oct3/4, or Nanog promoters so that, if these ES cell-specific genes were activated, the resulting cells could be easily selected using neomycin. However, the use of genetically modified donors hinders clinical applicability for humans. To assess whether iPS cells can be derived from nontransgenic donor cells, wild type MEF and adult skin cells were retrovirally transduced with Oct3/4, Sox2, c-Myc, and Klf4 and ES-like colonies were isolated by morphology alone, without the use of drug selection for Oct4 or Nanog (45). IPS cells from wild type donor cells formed teratomas and generated live chimeras. This study suggests that transgenic donor cells are not necessary to generate IPS cells.
Amniotic Fluid Stem Cells An alternate source of stem cells is the amniotic fluid. Amniotic fluid is known to contain multiple partially differentiated cell types derived from the developing fetus. We isolated stem cell populations from these sources, called amniotic fluid stem cells (AFSCs), that express embryonic and adult stem cell markers (46). The undifferentiated stem cells expand extensively without a feeder layer, and the population doubles every 36 hours. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. The AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking can be induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal, and hepatic lineages. In this respect, they meet a commonly accepted criterion for pluripotent stem cells, without implying that they can generate every adult tissue. However, unlike human embryonic stem cells, AFSCs do not form tumors in vivo, which would be advantageous in clinical applications. The cells could be obtained either from amniocentesis or chorionic villous sampling in the developing fetus, or from the placenta at the time of birth. The cells could be preserved for self use, used without rejection, or they could be banked.
Adult Stem Cells and Native Progenitor Cells One of the limitations of applying cell-based regenerative medicine techniques to organ replacement has been the inherent difficulty of growing specific cell types in large quantities. 24
Even when some organs such as the liver have a high regenerative capacity in vivo, cell growth and expansion in vitro may be difficult. By studying the privileged sites for committed precursor cells in specific organs as well as exploring the conditions that promote differentiation, it may be able to overcome the factors that limit cell expansion in vitro. For example, in the past, urothelial (cells that line the urinary tract) cells could be grown in the laboratory setting in the past, but these cultures had only limited expansion capability. Several protocols have been developed over the past two decades that have overcome this difficulty. Studies have identified the undifferentiated cells in a urothelial culture, and culture techniques to keep them undifferentiated during their growth phase have been developed (11, 47–50). Using these methods of cell culture, it is now possible to expand a urothelial strain from a single specimen that initially covers a surface area of 1 cm2 to one covering a surface area of 4,202 m2 (the equivalent of one football field) within 8 weeks (11). These studies have indicated that it should be possible to collect autologous bladder cells from human patients, expand them in culture, and return them to the donor in sufficient quantities for reconstructive purposes (11, 48, 50, 51), and this is indeed the case. In fact, expanded urothelial cells have been used in several clinical studies, including an important study that showed that a tissue-engineered bladder could be implanted into select patients (52). Major advances have been achieved within the past decade on the possible expansion of a variety of primary human cells, with specific techniques that make the use of autologous cells possible for clinical application.
TISSUE ENGINEERING OF SPECIFIC STRUCTURES IN THE REPRODUCTIVE TRACT Investigators around the world, including our laboratory, have been working toward the development of several cell types, tissues, and organs for clinical application in the reproductive tract.
Urethra Various strategies have been proposed over the years for the regeneration of urethral tissue. Woven meshes of PGA without cells (53, 54) or with cells (55) were used to regenerate urethras in various animal models. Naturally derived collagen-based materials such as decellularized bladder submucosa (4) and acellular urethral submucosa (56) have also been tested in various animal models for urethral reconstruction. Bladder submucosa matrix (4) proved to be a suitable graft for repair of urethral defects in rabbits. The grafts demonstrated a normal urothelial luminal lining and organized muscle bundles on the outer portion of the graft. These results were confirmed clinically in a series of patients with a history of failed hypospadias reconstruction. The urethral defects in these patients were repaired with human bladder acellular collagen matrices (57). The neourethral tissue was created by anastomosing the matrix to the urethral plate in an onlay fashion. The size of the created neourethra VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility®
FIGURE 1
Tissue engineering of the urethra using a collagen matrix. (A) Representative case of a patient with a bulbar stricture. (B) During the urethral repair surgery, strictured tissue is excised, preserving the urethral plate on the left side, and matrix is anastomosed to the urethral plate in an onlay fashion on the right. The boxes in both photos indicate the area of interest, including the urethra, which appears white in the left photograph. In the left photograph, the arrow indicates the area of stricture in the urethra. On the right, the arrow indicates the repaired stricture. Note that the engineered tissue now obscures the native white urethral tissue in an onlay fashion in the right photograph. (C) Urethrogram 6 months after repair. (D) Cystoscopic view of urethra before surgery on the left side, and 4 months after repair on the right side. Atala. Tissue engineering of reproductive tissues and organs. Fertil Steril 2012.
ranged from 5 to 15 cm. After a 3-year follow-up, three of the four patients had a successful outcome in regard to cosmetic appearance and function. One patient who had a 15-cm neourethra created developed a subglanular fistula. Similar results were obtained in pediatric and adult patients with primary urethral stricture disease using the same collagen matrices (Fig. 1) (58). Another study in 30 patients with recurrent stricture disease showed that a healthy urethral bed (two or fewer prior urethral surgeries) was needed for successful urethral reconstruction using the acellular collagen-based grafts (59). More than 200 pediatric and adult patients with urethral disease have been successfully treated in an onlay manner with a bladder-derived collagen-based matrix. One advantage of this matrix over the traditional nongenital tissue grafts that have been used for urethroplasty is that the matrix material is ‘‘off the shelf.’’ This eliminates the necessity of additional surgical procedures for graft harvesting, which may decrease operative time as well as the potential morbidity due to the harvest procedure. These techniques, which employed acellular matrices that had not been reseeded with cells, were applied experimentally and clinically in a successful manner for onlay urethral repairs. However, further study indicated that when tubularized urethral repairs with unseeded matrices were attempted experimentally, adequate urethral tissue regeneration was not achieved, and complications such as graft contracture and stricture formation occurred (60). To determine if seeding the matrix with cells from the urinary tract could improve the results of tubularized urethral repairs, autologous rabbit bladder epithelial and smooth muscle cells were grown and VOL. 98 NO. 1 / JULY 2012
seeded onto preconfigured tubular matrices. Entire urethra segments were then resected and urethroplasties were performed with tubularized collagen matrices either seeded with cells or without cells. The tubularized collagen matrices seeded with autologous cells formed new tissue which was histologically similar to native urethra. The tubularized collagen matrices without cells lead to poor tissue development, fibrosis, and stricture formation. These findings were confirmed clinically when a trial using tubularized nonseeded SIS for urethral stricture repair was performed in eight evaluable patients. Two patients with short inflammatory strictures maintained urethral patency. Stricture recurrence developed in the other six patients within 3 months of surgery (61). Most recently, Raya-Rivera et al. (62) were able to show that synthetic biomaterials can also be used in urethral reconstruction when they are tubularized and seeded with autologous cells. This group used polygycolic acid:poly(lactidecoglycolic acid) scaffolds seeded with autologous cells derived from bladder biopsies taken from each patient. The seeded scaffolds were then used to repair urethral defects in five boys. Upon follow-up evaluation, it was found that most of the boys had excellent urinary flow rates postoperatively, and voiding cystourethrograms indicated that these patients maintained wide urethral calibers. Urethral biopsies revealed that the grafts had developed a normal appearing architecture consisting of urothelial and muscular tissue.
Male Reproductive Organs Reconstructive surgery is required for a wide variety of pathologic penile conditions, including penile carcinoma, trauma, severe erectile dysfunction, and congenital conditions such as 25
VIEWS AND REVIEWS ambiguous genitalia, hypospadias, and epispadias. One of the major limitations of phallic reconstructive surgery is the availability of sufficient autologous tissue, and while other materials are used in some cases, operative complications such as infection, graft failure, and donor site morbidity are not negligible. Phallic reconstruction with autologous tissue, derived from the patient's own cells, may be preferable in selected cases. One of the major components of the phallus is corporal smooth muscle. Initial experiments have shown that cultured human corporal smooth muscle cells may be used in conjunction with biodegradable polymers to create corpus cavernosum tissue de novo (63). In a subsequent study, human corporal smooth muscle cells and endothelial cells seeded on biodegradable polymer scaffolds were able to form vascularized cavernosal muscle when implanted in vivo (64). Later, to minimize any immune reactions that a synthetic biomaterial might cause, naturally derived acellular corporal tissue matrix with the same architecture as native corpora was developed. Acellular collagen matrices were derived from processed donor rabbit corpora using cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were derived from donor penile tissue, and the cells were expanded in vitro and seeded on the acellular matrices. The matrices were covered with the appropriate cell architecture 4 weeks after implantation (65).
To look at the functional parameters of the engineered corpora, acellular corporal collagen matrices were obtained from donor rabbit penis and autologous corpus cavernosal smooth muscle and endothelial cells were harvested, expanded, and seeded on the matrices (Fig. 2). An entire cross-sectional segment of protruding rabbit phallus was excised, leaving the urethra intact. Cell seeded matrices were interposed into the excised corporal space. Functional and structural parameters (cavernosography, cavernosometry, mating behavior, and sperm ejaculation) were followed, and histologic, immunocytochemical, and Western blot analyses were performed up to 6 months after implantation. The engineered corpora cavernosa achieved adequate structural and functional parameters (66, 67). This technology was further confirmed when the entire rabbit corpora was removed and replaced with the engineered scaffolds. It is most interesting that mating activity in the animals with the engineered corpora appeared normal by 1 month after implantation. The presence of sperm was confirmed during mating, and was present in all the rabbits with the engineered corpora. The female rabbits mated with the animals implanted with engineered corpora, and they conceived and delivered healthy pups. These studies demonstrate that penile corpora cavernosa tissue can be engineered. Patients with testicular dysfunction require androgen replacement for somatic development. Conventional treatment
FIGURE 2
(A) Cavernosometry shows that all rabbits implanted with the bioengineered corpora after complete pendular penile corporal excision had sufficient intracorporal pressure (ICP) to attain erection. The levels of ICP were comparable to native corpora. (B) Cavernosography shows a homogenous appearance of corpora in the bioengineered group, similar to the native corpora, and multiple filling defects in the negative control group. Atala. Tissue engineering of reproductive tissues and organs. Fertil Steril 2012.
26
VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility® for testicular dysfunction consists of periodic intramuscular injections of chemically modified testosterone or, more recently, skin patch applications. However, long-term nonpulsatile testosterone therapy is not optimal and can cause multiple problems, including excessive erythropoiesis and bone density changes. To address the problem, a system was designed in which Leydig cells, which produce most of the testosterone in the male, were microencapsulated in an alginatepoly-L-lysine solution and injected into castrated animals. Serum testosterone was measured serially; the animals were able to maintain testosterone levels in the long term (68). Other studies have shown that testicular prostheses created with chondrocytes in bioreactors could be loaded with testosterone, and that these prostheses could provide controlled testosterone release into the bloodstream when implanted. The prostheses were implanted in athymic mice with bilateral anorchia, and testosterone was released long term, maintaining the androgen level at a physiologic range (69). One could envision combining the Leydig cell technology described above with engineered prostheses for the long-term functional replacement of androgen levels.
Female Reproductive Organs Congenital malformations of the uterus may have profound implications clinically. Patients with cloacal exstrophy and intersex disorders may not have sufficient uterine tissue present for future reproduction. We investigated the possibility of engineering functional uterine tissue using autologous cells (70). Autologous rabbit uterine smooth muscle and epithelial
cells were harvested, then grown and expanded in culture. These cells were seeded onto preconfigured uterine-shaped biodegradable polymer scaffolds, which were then used for subtotal uterine tissue replacement in the corresponding autologous animals. Upon retrieval 6 months after implantation, histologic, immunocytochemical, and Western blot analyses confirmed the presence of normal uterine tissue components. Biomechanical analyses and organ bath studies showed that the functional characteristics of these tissues were similar to those of normal uterine tissue. Breeding studies using these engineered uteri are currently being performed. In addition, other types of studies involving uterine regeneration have been performed. For example, Campbell et. al. (71) performed another type of study in which the peritoneal cavity of rats and rabbits were used as in vivo bioreactors to produce uterine tissue grafts. In this study, biomaterial templates of the appropriate shape were implanted in the peritoneal cavities of rats or rabbits. After 2 to 3 weeks, the templates were removed, and the encapsulating myofibroblast-rich tissue that resulted from the foreign body response to the biomaterial was harvested. This tissue was then used to replace resected segments of the uterus in the same animals in which the tissue was grown. They reported that at 12 weeks after grafting, this novel uterine graft tissue thickened and developed the morphology of normal uterus. This structure included a lumen lined with endometrium, which was surrounded by several layers of smooth muscle cells (myometrium-like) interspersed with collagen. Importantly, these grafted uterine horns supported embryos to the late stages of gestation. In another study by Li et al.
FIGURE 3
Appearance of tissue-engineered neovaginas. (A) Tubular polymer scaffold after cell seeding and 1 week in vitro culture, prior to implantation in vivo. (B, D, F) Gross appearance and (C, E, G) vaginography of cell-seeded constructs 1, 3, and 6 months, after implantation, respectively. (H) Unseeded control scaffold before implantation. (I, K, L) Gross appearance of unseeded construct at 1, 3, and 6 months after implantation. (J) Vaginography of unseeded graft at 1 month. Atala. Tissue engineering of reproductive tissues and organs. Fertil Steril 2012.
VOL. 98 NO. 1 / JULY 2012
27
VIEWS AND REVIEWS (72), a collagen-targeting basic fibroblast growth factor (bFGF) delivery system was constructed. This bFGF delivery system was tested in a rat model of severe uterine damage. In this model, partial uterine horn excision was performed, and then the excised horn was reconstructed using the collagen-based delivery system. This study showed improved regeneration of both the endometrium and muscular cells, improved vascularization, and better pregnancy outcomes in the rats reconstructed with the system. The dysfunctional uterine cervix may also benefit from tissue engineering strategies. Spontaneous preterm birth is a frequent complication of pregnancy, and in some cases, abnormalities of the cervix have been implicated in this issue. House et al. (73) have shown that tissue engineering techniques can be used to create three-dimensional cervical-like tissue constructs. Cervical cells were isolated from two premenopausal women and seeded on porous silk scaffolds. These constructs were cultured under dynamic or static culture conditions. After an 8-week culture interval, cervical cells had proliferated in all three dimensions and had synthesized an extracellular matrix with biochemical constituents and morphology similar to native tissue, but this matrix was better formed under dynamic culture conditions. This study suggests that it may be possible to engineer cervical tissue for a variety of conditions. Similarly, several pathologic conditions, including congenital malformations and malignancy, can adversely affect normal vaginal development or anatomy. Vaginal reconstruction has traditionally been challenging due to the paucity of available native tissue. The feasibility of engineering vaginal tissue in vivo has been investigated (74). Vaginal epithelial and smooth muscle cells of female rabbits were harvested, grown, and expanded in culture (Fig. 3). These cells were seeded onto biodegradable polymer scaffolds, and the cell-seeded constructs were then implanted into nude mice for up to 6 weeks. Immunocytochemical, histologic, and Western blot analyses confirmed the presence of vaginal tissue phenotypes. Electrical field stimulation studies in the tissue-engineered constructs showed similar functional properties to those of normal vaginal tissue. When these constructs were used for autologous total vaginal replacement, patent vaginal structures were noted in the tissueengineered specimens, while the non-cell-seeded structures were noted to be stenotic (74).
Acknowledgment: The author thanks Jennifer L. Olson, Ph.D., for editorial assistance with this manuscript.
REFERENCES 1.
2. 3.
4. 5. 6.
7. 8. 9. 10. 11.
12.
13. 14.
15. 16.
17.
18. 19. 20.
CONCLUSION Regenerative medicine technologies for virtually every type of tissue and organ within the male and female reproductive system are currently being developed. Various tissues are at different stages of development. Some are already being used clinically, such as engineered urethral tissue, while a few others are in preclinical trials and many more are in the discovery stage. Recent progress suggests that engineered tissues may have an expanded clinical applicability in the future and may represent a viable therapeutic option for those who would benefit from benefits of reproductive tissue replacement or repair. 28
21. 22.
23.
24. 25.
Dahms SE, Piechota HJ, Dahiya R, Lue TF, Tanagho EA. Composition and biomechanical properties of the bladder acellular matrix graft: comparative analysis in rat, pig and human. Br J Urol 1998;82:411–9. Yoo JJ, Meng J, Oberpenning F, Atala A. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 1998;51:221–5. Piechota HJ, Dahms SE, Nunes LS, Dahiya R, Lue TF, Tanagho EA. In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft. J Urol 1998;159:1717–24. Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a possible ‘‘off the shelf’’ biomaterial for urethral repair. Urology 1999;54:407–10. Amiel GE, Atala A. Current and future modalities for functional renal replacement. Urol Clin North Am 1999;26:235–46. Amiel GE, Komura M, Shapira O, Yoo JJ, Yazdani S, Berry J, et al. Engineering of blood vessels from acellular collagen matrices coated with human endothelial cells. Tissue Eng 2006;12:2355–65. Yoo JJ, Park HJ, Lee I, Atala A. Autologous engineered cartilage rods for penile reconstruction. J Urol 1999;162:1119–21. Atala A. Autologous cell transplantation for urologic reconstruction. J Urol 1998;159:2–3. Atala A. Bladder regeneration by tissue engineering. BJU Int 2001;88: 765–70. Atala A. Creation of bladder tissue in vitro and in vivo: a system for organ replacement. Adv Exp Med Biol 1999;462:31–42. Cilento BG, Freeman MR, Schneck FX, Retik AB, Atala A. Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro. J Urol 1994;152:665–70. Oberpenning F, Meng J, Yoo JJ, Atala A. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999;17:149–55. Kim BS, Mooney DJ. Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol 1998;16:224–30. Bergsma JE, Rozema FR, Bos RR, Boering G, de Bruijn WC, Pennings AJ. In vivo degradation and biocompatibility study of in vitro pre-degraded aspolymerized polyactide particles. Biomaterials 1995;16:267–74. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11–25. Li ST. Biologic biomaterials: tissue derived biomaterials (collagen). In: Bronzino JD, editor. The biomedical engineering handbook. Boca Raton, FL: CRS Press; 1995:627–47. Silver FH, Pins G. Cell growth on collagen: a review of tissue engineering using scaffolds containing extracellular matrix. J Long Term Eff Med Implants 1992;2:67–80. Sams AE, Nixon AJ. Chondrocyte-laden collagen scaffolds for resurfacing extensive articular cartilage defects. Osteoarthritis Cartilage 1995;3:47–59. Gilding D. Biodegradable polymers. In: Williams D, editor. Biocompatibility of clinical implant materials. Boca Raton, FL: CRC Press; 1981:209–32. Mikos AG, Lyman MD, Freed LE, Langer R. Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials 1994; 15:55–8. Harris LD, Kim BS, Mooney DJ. Open pore biodegradable matrices formed with gas foaming. J Biomed Mater Res 1998;42:396–402. Lee SJ, Liu J, Oh SH, Soker S, Atala A, Yoo JJ. Development of a composite vascular scaffolding system that withstands physiological vascular conditions. Biomaterials 2008;29:2891–8. Lee SJ, Oh SH, Liu J, Soker S, Atala A, Yoo JJ. The use of thermal treatments to enhance the mechanical properties of electrospun poly(epsiloncaprolactone) scaffolds. Biomaterials 2008;29:1422–30. Peppas NA, Langer R. New challenges in biomaterials. Science 1994;263: 1715–20. Brivanlou AH, Gage FH, Jaenisch R, Jessell T, Melton D, Rossant J. Stem cells. Setting standards for human embryonic stem cells. Science 2003; 300:913–6. VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility® 26.
27.
28.
29.
30.
31.
32.
33. 34.
35.
36. 37. 38. 39. 40.
41.
42.
43.
44. 45.
46.
47. 48.
49.
Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19: 1134–40. Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovitz-Eldor J, Goldstein RS, et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–5. Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Nat Acad Sci USA 2000;97:11307–12. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–33. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Nat Acad Sci USA 2001;98:10716–21. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108:407–14. Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R. Endothelial cells derived from human embryonic stem cells. Proc Nat Acad Sci USA 2002;99: 4391–6. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–7. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000;6:88–95. Hochedlinger K, Rideout WM, Kyba M, Daley GQ, Blelloch R, Jaenisch R. Nuclear transplantation, embryonic stem cells and the potential for cell therapy. Hematol J 2004;5(Suppl 3):S114–7. Rideout WM 3rd, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome. Science 2001;293:1093–8. Solter D. Mammalian cloning: advances and limitations. Nat Rev Genet 2000;1:199–207. Hochedlinger K, Jaenisch R. Nuclear transplantation: lessons from frogs and mice. Curr Opin Cell Biol 2002;14:741–8. Dinnyes A, De Sousa P, King T, Wilmut I. Somatic cell nuclear transfer: recent progress and challenges. Cloning Stem Cells 2002;4:81–90. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126: 663–76. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007;448:318–24. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–72. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917–20. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells 1. Nature 2007;448:313–7. Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 2007;25: 1177–81. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100–6. Scriven SD, Booth C, Thomas DF, Trejdosiewicz LK, Southgate J. Reconstitution of human urothelium from monolayer cultures. J Urol 1997;158:1147–52. Liebert M, Hubbel A, Chung M, Wedemeyer G, Lomax MI, Hegeman A, et al. Expression of mal is associated with urothelial differentiation in vitro: identification by differential display reverse-transcriptase polymerase chain reaction. Differentiation 1997;61:177–85. Liebert M, Wedemeyer G, Abruzzo LV, Kunkel SL, Hammerberg C, Cooper KD, et al. Stimulated urothelial cells produce cytokines and express an activated cell surface antigenic phenotype. Semin Urol 1991;9:124–30.
VOL. 98 NO. 1 / JULY 2012
50. 51.
52. 53. 54.
55.
56.
57. 58. 59.
60. 61. 62.
63.
64.
65.
66. 67.
68. 69.
70.
71.
72.
73.
74.
Puthenveettil JA, Burger MS, Reznikoff CA. Replicative senescence in human uroepithelial cells. Adv Exp Med Biol 1999;462:83–91. Nguyen HT, Park JM, Peters CA, Adam RM, Orsola A, Atala A, et al. Cell-specific activation of the HB-EGF and ErbB1 genes by stretch in primary human bladder cells. In Vitro Cell Dev Biol Anim 1999;35:371–5. Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet 2006;367:1241–6. Bazeed MA, Thuroff JW, Schmidt RA, Tanagho EA. New treatment for urethral strictures. Urology 1983;21:53–7. Olsen L, Bowald S, Busch C, Carlsten J, Eriksson I. Urethral reconstruction with a new synthetic absorbable device: an experimental study. Scand J Urol Nephrol 1992;26:323–6. Atala A, Vacanti JP, Peters CA, Mandell J, Retik AB, Freeman MR. Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vitro. J Urol 1992;148:658–62. Sievert KD, Bakircioglu ME, Nunes L, Tu R, Dahiya R, Tanagho EA. Homologous acellular matrix graft for urethral reconstruction in the rabbit: histological and functional evaluation. J Urol 2000;163:1958–65. Atala A, Guzman L, Retik AB. A novel inert collagen matrix for hypospadias repair. J Urol 1999;162:1148–51. El-Kassaby AW, Retik AB, Yoo JJ, Atala A. Urethral stricture repair with an off-the-shelf collagen matrix. J Urol 2003;169:170–3. El Kassaby A, Aboushwareb T, Atala A. Randomized comparative study between buccal mucosal and acellular bladder matrix grafts in complex anterior urethral strictures. J Urol 2008;179:1432–6. De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol 2002;168:1789–92. le Roux PJ. Endoscopic urethroplasty with unseeded small intestinal submucosa collagen matrix grafts: a pilot study. J Urol 2005;173:140–3. Raya-Rivera A, Esquiliano DR, Yoo JJ, Lopez-Bayghen E, Soker S, Atala A. Tissue-engineered autologous urethras for patients who need reconstruction: an observational study. Lancet 2011;377:1175–82. Kershen RT, Yoo JJ, Moreland RB, Krane RJ, Atala A. Reconstitution of human corpus cavernosum smooth muscle in vitro and in vivo. Tissue Eng 2002;8:515–24. Park HJ, Yoo JJ, Kershen RT, Moreland R, Atala A. Reconstitution of human corporal smooth muscle and endothelial cells in vivo. J Urol 1999;162: 1106–9. Falke G, Yoo JJ, Kwon TG, Moreland R, Atala A. Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices. Tissue Eng 2003;9:871–9. Kwon TG, Yoo JJ, Atala A. Autologous penile corpora cavernosa replacement using tissue engineering techniques. J Urol 2002;168:1754–8. Chen KL, Eberli D, Yoo JJ, Atala A. Bioengineered corporal tissue for structural and functional restoration of the penis. Proc Natl Acad Sci USA 2010;107:3346–50. Machluf M, Atala A. Emerging concepts for tissue and organ transplantation. Graft 1998;1:31–7. Raya-Rivera AM, Baez C, Atala A, Yoo JJ. Tissue engineered testicular prostheses with prolonged testosterone release. World J Urol 2008;26: 307–14. Wang T, Koh C, Yoo JJ. Creation of an engineered uterus for surgical reconstruction. American Academy of Pediatrics Section on Urology; New Orleans, 2003. Campbell GR, Turnbull G, Xiang L, Haines M, Armstrong S, Rolfe BE, et al. The peritoneal cavity as a bioreactor for tissue engineering visceral organs: bladder, uterus and vas deferens. J Tissue Eng Regen Med 2008; 2:50–60. Li X, Sun H, Lin N, Hou X, Wang J, Zhou B, et al. Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor. Biomaterials 2011;32:8172–81. House M, Sanchez CC, Rice WL, Socrate S, Kaplan DL. Cervical tissue engineering using silk scaffolds and human cervical cells. Tissue Eng Part A 2010; 16:2101–12. De Filippo RE, Yoo JJ, Atala A. Engineering of vaginal tissue in vivo. Tissue Eng 2003;9:301–6.
29
Regenerative medicine and tissue engineering technology may soon offer new hope for patients with serious injuries and end-stage reproductive organ failure. Scientists are now applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that can restore and maintain normal function in diseased and injured reproductive tissues. In addition, the stem cell field is advancing, and new discoveries in this field will lead to new therapeutic strategies. For example, newly discovered types of stem cells have been retrieved from uterine tissues such as amniotic fluid and placental stem cells. The process of therapeutic cloning and the creation of induced pluripotent cells provide still other potential sources of stem cells for cell-based tissue engineering applications. Although stem cells are still in the research phase, some therapies arising from tissue engineering endeavors that make use of autologous adult cells have already entered the clinic. This article discusses these tissue engineering strategies for various organs in the male and female reproductive tract. (Fertil SterilÒ 2012;98:21–9. Ó2012 by American Society for Reproductive Medicine.) Key Words: Amniotic fluid stem cell, biomaterial, penis, testes, uterus, urethra, vagina
P
atients suffering from various congenital and acquired disorders of the reproductive tract are currently treated with surgical and hormonal therapies. However, these techniques are not ideal. For example, many current hormonal therapies do not provide physiologic levels of sex hormones, and this can lead to a number of problems. In addition, many surgical procedures, such as grafting to replace damaged areas of the male urethra, may eventually fail due to many factors. This has led scientists in the field of regenerative medicine and tissue engineering to apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that may eventually improve the quality of life for these patients in a number of ways, from providing physiologic hormone secretion to perhaps one day restoring fertility with tissue-engineered reproductive
organs. This article reviews recent advances in regenerative medicine and describes applications of these new technologies in reproductive medicine. The field of regenerative medicine encompasses various areas of technology, such as tissue engineering, stem cells, and cloning. Tissue engineering combines the principles of cell transplantation, materials science, and bioengineering to develop new biological substitutes that may restore and maintain normal organ function. Tissue engineering strategies generally fall into two categories: the use of acellular matrices, which depend on the body's natural ability to regenerate and serve as guides for proper orientation and direction of new tissue growth, and the use of matrices seeded with cells. Acellular tissue matrices are usually prepared by manufacturing artificial scaffolds, or by removing cellular com-
Received April 16, 2012; revised and accepted May 25, 2012. A.A. has nothing to disclose. Reprint requests: Anthony Atala, M.D., Director, Wake Forest Institute for Regenerative Medicine, The W. Boyce Professor and Chair, Urology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157 (E-mail: [email protected]). Fertility and Sterility® Vol. 98, No. 1, July 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.05.038 VOL. 98 NO. 1 / JULY 2012
ponents from tissues via mechanical and chemical manipulation to produce collagen-rich matrices (1–4). These matrices tend to slowly degrade after implantation and are generally replaced by the extracellular matrix (ECM) proteins that are secreted by host cells that infiltrate the matrix. Cells can also be used for therapy via injection, either with carriers such as hydrogels, or alone. In addition, cells can be used for matrix-based tissue engineering strategies. To do this, a small piece of donor tissue is dissociated into individual cells. These cells are either implanted directly into the host, or they are expanded in culture, attached to a support matrix, and then the cellmatrix construct is implanted into the host after expansion. The source of donor tissue can be heterologous (such as bovine), allogeneic (same species, different individual), or autologous. Ideally, both structural and functional tissue replacement will occur with minimal complications. Autologous cells derived from a biopsy of tissue are the preferred type to use. They are obtained from the host, dissociated from the tissue biopsy, and expanded in culture. Next, they are implanted back into the same host (2, 5–12). The use of 21
VIEWS AND REVIEWS autologous cells, while it may cause an initial inflammatory response, avoids rejection, and thus the deleterious side effects of immunosuppressive medications can be avoided. Thus, most current strategies for tissue engineering depend upon a sample of autologous cells from the diseased organ of the host. However, for many patients with extensive end-stage organ failure, a tissue biopsy may not yield enough normal cells for expansion and transplantation. In other instances, primary autologous human cells cannot be expanded from a particular organ such as the pancreas. In these situations, stem cells are envisioned as being an alternative source of cells from which the desired tissue can be derived. Stem cells can be derived from discarded human embryos (human embryonic stem cells), from fetal tissue, or from adult sources (bone marrow, fat, skin). Therapeutic cloning has also played a role in the development of the field of regenerative medicine. Recently, the findings of induced pluripotency in which cells such as fibroblasts are ‘‘reprogrammed’’ to behave like stem cells has been described, and it may also provide a new source of cells for tissue-engineering strategies.
BIOMATERIALS FOR USE IN TISSUE ENGINEERING In many cell-based tissue engineering methods, cells are obtained from a tissue, expanded in vitro, and then seeded onto a scaffold composed of an appropriate biomaterial. These biomaterials replicate the biologic and mechanical function of the native extracellular matrix (ECM) found in tissues in the body by serving as an artificial ECM. Biomaterials also provide a three-dimensional scaffold for the cells to adhere to and form new tissues with appropriate structure and function. They also allow for the delivery of cells and appropriate bioactive factors to desired sites in the body (13). Bioactive factors, such as cell-adhesion peptides and growth factors, can be loaded along with cells to help regulate cellular function. As the majority of mammalian cell types are anchorage dependent and will die if no cell-adhesion substrate is available, biomaterials provide a cell-adhesion substrate that can deliver cells to specific sites in the body with high loading efficiency. Biomaterials can also provide mechanical support against in vivo forces and ensure that the predefined threedimensional structure of an organ is maintained during tissue development. The ideal biomaterial should be biodegradable and bioresorbable to support the replacement of normal tissue without inducing inflammation. Incompatible materials that induce inflammatory or foreign-body responses eventually become necrotic and/or are rejected. Degradation products, if produced, should be removed from the body via metabolic pathways at an adequate rate to keep the concentration of these products in the tissues at a tolerable level (14). The biomaterial should also provide an environment in which appropriate regulation of cell behavior (adhesion, proliferation, migration, and differentiation) can occur such that functional tissue can form. Cell behavior in the newly formed tissue has been shown to be regulated by multiple interactions of the cells with their microenvironment, including interactions with 22
cell-adhesion ligands (15) and with soluble growth factors. Since biomaterials provide temporary mechanical support while the cells undergo spatial reorganization into tissue, the properly chosen biomaterial should allow the engineered tissue to maintain sufficient mechanical integrity to support itself in early development, while in late development, it should begin to degrade so that it does not hinder further tissue growth (13). Three broad classes of biomaterials have been utilized in tissue engineering studies: naturally derived materials (e.g., collagen and alginate), acellular tissue matrices (e.g., bladder submucosa and small intestinal submucosa), and synthetic polymers such as polyglycolic acid (PGA), polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA). Collagen is the most abundant and ubiquitous structural protein in the body, and may be readily purified from both animal and human tissues with an enzyme treatment and salt/acid extraction (16). Collagen contains cell adhesion domain sequences (e.g., RGD) that may assist in maintaining the phenotype and activity of many types of cells, including fibroblasts (17) and chondrocytes (18). Acellular tissue matrices are collagen-rich matrices prepared by removing cellular components from tissues. The matrices are often prepared by mechanical and chemical manipulation of a segment of tissue (1–4). These matrices slowly degrade upon implantation, and are replaced and remodeled by new ECM proteins synthesized and secreted by transplanted or ingrowing host cells. Commonly used acellular tissue matrices include bladder submucosa (BSM) and small intestinal submucosa (SIS). Polyesters of naturally occurring a-hydroxy acids, including PGA, PLA, and PLGA, are also widely used in tissue engineering. These polymers have gained FDA approval for human use in a variety of applications, including sutures (19). Because these polymers are thermoplastics, they can be easily formed into a three dimensional scaffold with a desired microstructure, gross shape, and dimension by various techniques including molding, extrusion, solvent casting (20), phase separation techniques, and gas foaming techniques (21). Recently, composite scaffolds consisting of collagen and the synthetic polymer polycaprolactone (PCL) have been formed using a novel electrospinning technique (22, 23). Many applications in tissue engineering often require a scaffold with high porosity and ratio of surface area to volume, and electrospinning allows these parameters to be varied quickly and easily. Other biodegradable synthetic polymers, including poly(anhydrides) and poly(ortho-esters), can also be used to fabricate scaffolds for tissue engineering with controlled properties (24).
CELLS FOR USE IN TISSUE ENGINEERING Embryonic Stem Cells Human embryonic stem cells (hESCs) exhibit two remarkable properties: the ability to proliferate in an undifferentiated but pluripotent state (self-renewal), and the ability to differentiate into almost all of the specialized cell types in the body (25). They can be isolated by aspirating the inner cell mass from the embryo during the blastocyst stage (5 days after fertilization). Human embryonic stem cells have been shown to VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility® differentiate into cells from all three embryonic germ layers in vitro. Skin and neurons have been formed, indicating ectodermal differentiation (26–29). Blood, cardiac cells, cartilage, endothelial cells, and muscle have been formed, indicating mesodermal differentiation (30–32). Pancreatic cells have been formed, indicating endodermal differentiation (33). In addition, as further evidence of their pluripotency, embryonic stem cells can form embryoid bodies, which are cell aggregations that contain all three embryonic germ layers while in culture, and can form teratomas in vivo (34). However, the tendency to form teratomas in vivo is also a limitation to widespread usage of hESCs in cell therapy and tissue engineering, as it is difficult if not impossible to control the tumorigenicity of these cells in vivo, and this leads to a number of safety concerns surrounding the usage of these cells for therapeutic benefit. In addition, the ethics considerations surrounding the destruction of human embryos to obtain hESCs are yet to be resolved. Finally, the use of human embryonic stem cells is banned in some countries, including the United States, and so their use in tissue engineering is infrequent in those countries at this time.
Laboratory-Generated Stem Cells In addition, stem cells for tissue engineering could be generated through ‘‘cloning’’ procedures. Somatic cell nuclear transfer (SCNT) is used to generate early-stage embryos that are explanted in culture to produce embryonic stem cell lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue and organ replacement applications (35). Therefore, somatic cell nuclear transfer may provide an alternative source of transplantable cells. While promising, somatic cell nuclear transfer technology has certain limitations that require further study before this technique can be applied widely in tissue or organ replacement therapy. First, the efficiency of the cloning process is very low, as evidenced by the fact that the majority of embryos derived from the cloning process do not survive (36–38). To improve cloning efficiency, further improvements are required in many of the complex steps of nuclear transfer, such as the enucleation process for oocytes, the actual transfer of a nucleus to this enucleated oocyte, and the activation process that instructs the cloned oocytes to begin dividing. In addition, cell cycle synchronization between donor cells and recipient oocytes must be accomplished (39). Other barriers to the frequent use of SCNT in human applications are the requirement for human oocytes, which are difficult to obtain in the quantities required, and, as with hESCs, there are ethical considerations surrounding the production and destruction of human embryonic tissue that remain to be resolved. Recently, exciting reports of the successful transformation of adult cells into pluripotent stem cells through a type of genetic ‘‘reprogramming’’ have been published. Reprogramming is a technique that involves de-differentiation of adult somatic cells to produce patient-specific pluripotent stem cells, without the use of embryos. Cells generated by VOL. 98 NO. 1 / JULY 2012
reprogramming would be genetically identical to the somatic cells (and thus the patient who donated these cells) and would not be rejected. Takahashi and Yamanaka (40) were the first to discover that mouse embryonic fibroblasts (MEFs) and adult mouse fibroblasts could be reprogrammed into an ‘‘induced pluripotent state’’ (iPS). This group used mouse embryonic fibroblasts (MEFs) engineered to express a neomycin resistance gene from the Fbx15 locus, a gene expressed only in embryonic stem (ES) cells. They examined 24 genes that were thought to be important for embryonic stem cells and identified four key genes that, when introduced into the reporter fibroblasts, resulted in drug-resistant cells. These were Oct3/4, Sox2, c-Myc, and Klf4. This experiment indicated that expression of the four genes in these transgenic MEFs led to expression of a gene specific for ES cells. Mouse embryonic fibroblasts and adult fibroblasts were cotransduced with retroviral vectors, each carrying one of the four genes, and transduced cells were selected via drug resistance. The resultant iPS cells possessed the immortal growth characteristics of self-renewing ES cells, expressed genes specific for ES cells, and generated embryoid bodies in vitro and teratomas in vivo. When iPS cells were injected into mouse blastocysts, they contributed to a variety of cell types. However, although iPS cells selected in this way were pluripotent, they were not identical to ES cells. Unlike ES cells, chimeras made from iPS cells did not result in full-term pregnancies. Gene expression profiles of the iPS cells showed that they possessed a distinct gene expression signature that was different from that of ES cells. In addition, the epigenetic state of the iPS cells was somewhere between that found in somatic cells and that found in ES cells, suggesting that the reprogramming was incomplete. These results were improved significantly by Wernig et al. (41) in 2007. Fibroblasts were infected with retroviral vectors and selected for the activation of endogenous Oct4 or Nanog genes. Results from this study showed that DNA methylation, gene expression profiles, and the chromatin state of the reprogrammed cells were similar to those of ES cells. Teratomas induced by these cells contained differentiated cell types representing all three embryonic germ layers. Most importantly, the reprogrammed cells from this experiment were able to form viable chimeras and contribute to the germ line like ES cells, suggesting that these iPS cells were completely reprogrammed. Wernig et al. (41) observed that the number of reprogrammed colonies increased when drug selection was initiated later (day 20 rather than day 3 post-transduction). This suggests that reprogramming is a slow and gradual process and may explain why previous attempts resulted in incomplete reprogramming. It has recently been shown that reprogramming of human cells is possible. Yakahashi et al. (42) showed that retrovirusmediated transfection of OCT3/4, SOX2, KLF4, and c-MYC generates human iPS cells that are similar to hES cells in terms of morphology, proliferation, gene expression, surface markers, and teratoma formation. Yu et al. (43) showed that retroviral transduction of OCT4, SOX2, NANOG, and LIN28 could generate pluripotent stem cells without introducing any oncogenes (c-MYC). Both studies showed that human iPS were similar but not identical to hES cells. 23
VIEWS AND REVIEWS Another concern is that these iPS cells contain three to six retroviral integrations (one for each factor) which may increase the risk of tumorigenesis. Okita et al. (44) studied the tumor formation in chimeric mice generated from NanogiPS cells and found 20% of the offspring developed tumors due to the retroviral expression of c-myc. An alternative approach would be to use a transient expression method, such as adenovirus-mediated system, since both Okita et al. (44) and Meissner et al. (45) showed strong silencing of the viralcontrolled transcripts in iPS cells. This indicates that these viral genes are only required for the induction, not the maintenance, of pluripotency. Another concern is the use of transgenic donor cells for reprogrammed cells in the mouse studies. Both studies used donor cells from transgenic mice harboring a drug resistance gene driven by Fbx15, Oct3/4, or Nanog promoters so that, if these ES cell-specific genes were activated, the resulting cells could be easily selected using neomycin. However, the use of genetically modified donors hinders clinical applicability for humans. To assess whether iPS cells can be derived from nontransgenic donor cells, wild type MEF and adult skin cells were retrovirally transduced with Oct3/4, Sox2, c-Myc, and Klf4 and ES-like colonies were isolated by morphology alone, without the use of drug selection for Oct4 or Nanog (45). IPS cells from wild type donor cells formed teratomas and generated live chimeras. This study suggests that transgenic donor cells are not necessary to generate IPS cells.
Amniotic Fluid Stem Cells An alternate source of stem cells is the amniotic fluid. Amniotic fluid is known to contain multiple partially differentiated cell types derived from the developing fetus. We isolated stem cell populations from these sources, called amniotic fluid stem cells (AFSCs), that express embryonic and adult stem cell markers (46). The undifferentiated stem cells expand extensively without a feeder layer, and the population doubles every 36 hours. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. The AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking can be induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal, and hepatic lineages. In this respect, they meet a commonly accepted criterion for pluripotent stem cells, without implying that they can generate every adult tissue. However, unlike human embryonic stem cells, AFSCs do not form tumors in vivo, which would be advantageous in clinical applications. The cells could be obtained either from amniocentesis or chorionic villous sampling in the developing fetus, or from the placenta at the time of birth. The cells could be preserved for self use, used without rejection, or they could be banked.
Adult Stem Cells and Native Progenitor Cells One of the limitations of applying cell-based regenerative medicine techniques to organ replacement has been the inherent difficulty of growing specific cell types in large quantities. 24
Even when some organs such as the liver have a high regenerative capacity in vivo, cell growth and expansion in vitro may be difficult. By studying the privileged sites for committed precursor cells in specific organs as well as exploring the conditions that promote differentiation, it may be able to overcome the factors that limit cell expansion in vitro. For example, in the past, urothelial (cells that line the urinary tract) cells could be grown in the laboratory setting in the past, but these cultures had only limited expansion capability. Several protocols have been developed over the past two decades that have overcome this difficulty. Studies have identified the undifferentiated cells in a urothelial culture, and culture techniques to keep them undifferentiated during their growth phase have been developed (11, 47–50). Using these methods of cell culture, it is now possible to expand a urothelial strain from a single specimen that initially covers a surface area of 1 cm2 to one covering a surface area of 4,202 m2 (the equivalent of one football field) within 8 weeks (11). These studies have indicated that it should be possible to collect autologous bladder cells from human patients, expand them in culture, and return them to the donor in sufficient quantities for reconstructive purposes (11, 48, 50, 51), and this is indeed the case. In fact, expanded urothelial cells have been used in several clinical studies, including an important study that showed that a tissue-engineered bladder could be implanted into select patients (52). Major advances have been achieved within the past decade on the possible expansion of a variety of primary human cells, with specific techniques that make the use of autologous cells possible for clinical application.
TISSUE ENGINEERING OF SPECIFIC STRUCTURES IN THE REPRODUCTIVE TRACT Investigators around the world, including our laboratory, have been working toward the development of several cell types, tissues, and organs for clinical application in the reproductive tract.
Urethra Various strategies have been proposed over the years for the regeneration of urethral tissue. Woven meshes of PGA without cells (53, 54) or with cells (55) were used to regenerate urethras in various animal models. Naturally derived collagen-based materials such as decellularized bladder submucosa (4) and acellular urethral submucosa (56) have also been tested in various animal models for urethral reconstruction. Bladder submucosa matrix (4) proved to be a suitable graft for repair of urethral defects in rabbits. The grafts demonstrated a normal urothelial luminal lining and organized muscle bundles on the outer portion of the graft. These results were confirmed clinically in a series of patients with a history of failed hypospadias reconstruction. The urethral defects in these patients were repaired with human bladder acellular collagen matrices (57). The neourethral tissue was created by anastomosing the matrix to the urethral plate in an onlay fashion. The size of the created neourethra VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility®
FIGURE 1
Tissue engineering of the urethra using a collagen matrix. (A) Representative case of a patient with a bulbar stricture. (B) During the urethral repair surgery, strictured tissue is excised, preserving the urethral plate on the left side, and matrix is anastomosed to the urethral plate in an onlay fashion on the right. The boxes in both photos indicate the area of interest, including the urethra, which appears white in the left photograph. In the left photograph, the arrow indicates the area of stricture in the urethra. On the right, the arrow indicates the repaired stricture. Note that the engineered tissue now obscures the native white urethral tissue in an onlay fashion in the right photograph. (C) Urethrogram 6 months after repair. (D) Cystoscopic view of urethra before surgery on the left side, and 4 months after repair on the right side. Atala. Tissue engineering of reproductive tissues and organs. Fertil Steril 2012.
ranged from 5 to 15 cm. After a 3-year follow-up, three of the four patients had a successful outcome in regard to cosmetic appearance and function. One patient who had a 15-cm neourethra created developed a subglanular fistula. Similar results were obtained in pediatric and adult patients with primary urethral stricture disease using the same collagen matrices (Fig. 1) (58). Another study in 30 patients with recurrent stricture disease showed that a healthy urethral bed (two or fewer prior urethral surgeries) was needed for successful urethral reconstruction using the acellular collagen-based grafts (59). More than 200 pediatric and adult patients with urethral disease have been successfully treated in an onlay manner with a bladder-derived collagen-based matrix. One advantage of this matrix over the traditional nongenital tissue grafts that have been used for urethroplasty is that the matrix material is ‘‘off the shelf.’’ This eliminates the necessity of additional surgical procedures for graft harvesting, which may decrease operative time as well as the potential morbidity due to the harvest procedure. These techniques, which employed acellular matrices that had not been reseeded with cells, were applied experimentally and clinically in a successful manner for onlay urethral repairs. However, further study indicated that when tubularized urethral repairs with unseeded matrices were attempted experimentally, adequate urethral tissue regeneration was not achieved, and complications such as graft contracture and stricture formation occurred (60). To determine if seeding the matrix with cells from the urinary tract could improve the results of tubularized urethral repairs, autologous rabbit bladder epithelial and smooth muscle cells were grown and VOL. 98 NO. 1 / JULY 2012
seeded onto preconfigured tubular matrices. Entire urethra segments were then resected and urethroplasties were performed with tubularized collagen matrices either seeded with cells or without cells. The tubularized collagen matrices seeded with autologous cells formed new tissue which was histologically similar to native urethra. The tubularized collagen matrices without cells lead to poor tissue development, fibrosis, and stricture formation. These findings were confirmed clinically when a trial using tubularized nonseeded SIS for urethral stricture repair was performed in eight evaluable patients. Two patients with short inflammatory strictures maintained urethral patency. Stricture recurrence developed in the other six patients within 3 months of surgery (61). Most recently, Raya-Rivera et al. (62) were able to show that synthetic biomaterials can also be used in urethral reconstruction when they are tubularized and seeded with autologous cells. This group used polygycolic acid:poly(lactidecoglycolic acid) scaffolds seeded with autologous cells derived from bladder biopsies taken from each patient. The seeded scaffolds were then used to repair urethral defects in five boys. Upon follow-up evaluation, it was found that most of the boys had excellent urinary flow rates postoperatively, and voiding cystourethrograms indicated that these patients maintained wide urethral calibers. Urethral biopsies revealed that the grafts had developed a normal appearing architecture consisting of urothelial and muscular tissue.
Male Reproductive Organs Reconstructive surgery is required for a wide variety of pathologic penile conditions, including penile carcinoma, trauma, severe erectile dysfunction, and congenital conditions such as 25
VIEWS AND REVIEWS ambiguous genitalia, hypospadias, and epispadias. One of the major limitations of phallic reconstructive surgery is the availability of sufficient autologous tissue, and while other materials are used in some cases, operative complications such as infection, graft failure, and donor site morbidity are not negligible. Phallic reconstruction with autologous tissue, derived from the patient's own cells, may be preferable in selected cases. One of the major components of the phallus is corporal smooth muscle. Initial experiments have shown that cultured human corporal smooth muscle cells may be used in conjunction with biodegradable polymers to create corpus cavernosum tissue de novo (63). In a subsequent study, human corporal smooth muscle cells and endothelial cells seeded on biodegradable polymer scaffolds were able to form vascularized cavernosal muscle when implanted in vivo (64). Later, to minimize any immune reactions that a synthetic biomaterial might cause, naturally derived acellular corporal tissue matrix with the same architecture as native corpora was developed. Acellular collagen matrices were derived from processed donor rabbit corpora using cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were derived from donor penile tissue, and the cells were expanded in vitro and seeded on the acellular matrices. The matrices were covered with the appropriate cell architecture 4 weeks after implantation (65).
To look at the functional parameters of the engineered corpora, acellular corporal collagen matrices were obtained from donor rabbit penis and autologous corpus cavernosal smooth muscle and endothelial cells were harvested, expanded, and seeded on the matrices (Fig. 2). An entire cross-sectional segment of protruding rabbit phallus was excised, leaving the urethra intact. Cell seeded matrices were interposed into the excised corporal space. Functional and structural parameters (cavernosography, cavernosometry, mating behavior, and sperm ejaculation) were followed, and histologic, immunocytochemical, and Western blot analyses were performed up to 6 months after implantation. The engineered corpora cavernosa achieved adequate structural and functional parameters (66, 67). This technology was further confirmed when the entire rabbit corpora was removed and replaced with the engineered scaffolds. It is most interesting that mating activity in the animals with the engineered corpora appeared normal by 1 month after implantation. The presence of sperm was confirmed during mating, and was present in all the rabbits with the engineered corpora. The female rabbits mated with the animals implanted with engineered corpora, and they conceived and delivered healthy pups. These studies demonstrate that penile corpora cavernosa tissue can be engineered. Patients with testicular dysfunction require androgen replacement for somatic development. Conventional treatment
FIGURE 2
(A) Cavernosometry shows that all rabbits implanted with the bioengineered corpora after complete pendular penile corporal excision had sufficient intracorporal pressure (ICP) to attain erection. The levels of ICP were comparable to native corpora. (B) Cavernosography shows a homogenous appearance of corpora in the bioengineered group, similar to the native corpora, and multiple filling defects in the negative control group. Atala. Tissue engineering of reproductive tissues and organs. Fertil Steril 2012.
26
VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility® for testicular dysfunction consists of periodic intramuscular injections of chemically modified testosterone or, more recently, skin patch applications. However, long-term nonpulsatile testosterone therapy is not optimal and can cause multiple problems, including excessive erythropoiesis and bone density changes. To address the problem, a system was designed in which Leydig cells, which produce most of the testosterone in the male, were microencapsulated in an alginatepoly-L-lysine solution and injected into castrated animals. Serum testosterone was measured serially; the animals were able to maintain testosterone levels in the long term (68). Other studies have shown that testicular prostheses created with chondrocytes in bioreactors could be loaded with testosterone, and that these prostheses could provide controlled testosterone release into the bloodstream when implanted. The prostheses were implanted in athymic mice with bilateral anorchia, and testosterone was released long term, maintaining the androgen level at a physiologic range (69). One could envision combining the Leydig cell technology described above with engineered prostheses for the long-term functional replacement of androgen levels.
Female Reproductive Organs Congenital malformations of the uterus may have profound implications clinically. Patients with cloacal exstrophy and intersex disorders may not have sufficient uterine tissue present for future reproduction. We investigated the possibility of engineering functional uterine tissue using autologous cells (70). Autologous rabbit uterine smooth muscle and epithelial
cells were harvested, then grown and expanded in culture. These cells were seeded onto preconfigured uterine-shaped biodegradable polymer scaffolds, which were then used for subtotal uterine tissue replacement in the corresponding autologous animals. Upon retrieval 6 months after implantation, histologic, immunocytochemical, and Western blot analyses confirmed the presence of normal uterine tissue components. Biomechanical analyses and organ bath studies showed that the functional characteristics of these tissues were similar to those of normal uterine tissue. Breeding studies using these engineered uteri are currently being performed. In addition, other types of studies involving uterine regeneration have been performed. For example, Campbell et. al. (71) performed another type of study in which the peritoneal cavity of rats and rabbits were used as in vivo bioreactors to produce uterine tissue grafts. In this study, biomaterial templates of the appropriate shape were implanted in the peritoneal cavities of rats or rabbits. After 2 to 3 weeks, the templates were removed, and the encapsulating myofibroblast-rich tissue that resulted from the foreign body response to the biomaterial was harvested. This tissue was then used to replace resected segments of the uterus in the same animals in which the tissue was grown. They reported that at 12 weeks after grafting, this novel uterine graft tissue thickened and developed the morphology of normal uterus. This structure included a lumen lined with endometrium, which was surrounded by several layers of smooth muscle cells (myometrium-like) interspersed with collagen. Importantly, these grafted uterine horns supported embryos to the late stages of gestation. In another study by Li et al.
FIGURE 3
Appearance of tissue-engineered neovaginas. (A) Tubular polymer scaffold after cell seeding and 1 week in vitro culture, prior to implantation in vivo. (B, D, F) Gross appearance and (C, E, G) vaginography of cell-seeded constructs 1, 3, and 6 months, after implantation, respectively. (H) Unseeded control scaffold before implantation. (I, K, L) Gross appearance of unseeded construct at 1, 3, and 6 months after implantation. (J) Vaginography of unseeded graft at 1 month. Atala. Tissue engineering of reproductive tissues and organs. Fertil Steril 2012.
VOL. 98 NO. 1 / JULY 2012
27
VIEWS AND REVIEWS (72), a collagen-targeting basic fibroblast growth factor (bFGF) delivery system was constructed. This bFGF delivery system was tested in a rat model of severe uterine damage. In this model, partial uterine horn excision was performed, and then the excised horn was reconstructed using the collagen-based delivery system. This study showed improved regeneration of both the endometrium and muscular cells, improved vascularization, and better pregnancy outcomes in the rats reconstructed with the system. The dysfunctional uterine cervix may also benefit from tissue engineering strategies. Spontaneous preterm birth is a frequent complication of pregnancy, and in some cases, abnormalities of the cervix have been implicated in this issue. House et al. (73) have shown that tissue engineering techniques can be used to create three-dimensional cervical-like tissue constructs. Cervical cells were isolated from two premenopausal women and seeded on porous silk scaffolds. These constructs were cultured under dynamic or static culture conditions. After an 8-week culture interval, cervical cells had proliferated in all three dimensions and had synthesized an extracellular matrix with biochemical constituents and morphology similar to native tissue, but this matrix was better formed under dynamic culture conditions. This study suggests that it may be possible to engineer cervical tissue for a variety of conditions. Similarly, several pathologic conditions, including congenital malformations and malignancy, can adversely affect normal vaginal development or anatomy. Vaginal reconstruction has traditionally been challenging due to the paucity of available native tissue. The feasibility of engineering vaginal tissue in vivo has been investigated (74). Vaginal epithelial and smooth muscle cells of female rabbits were harvested, grown, and expanded in culture (Fig. 3). These cells were seeded onto biodegradable polymer scaffolds, and the cell-seeded constructs were then implanted into nude mice for up to 6 weeks. Immunocytochemical, histologic, and Western blot analyses confirmed the presence of vaginal tissue phenotypes. Electrical field stimulation studies in the tissue-engineered constructs showed similar functional properties to those of normal vaginal tissue. When these constructs were used for autologous total vaginal replacement, patent vaginal structures were noted in the tissueengineered specimens, while the non-cell-seeded structures were noted to be stenotic (74).
Acknowledgment: The author thanks Jennifer L. Olson, Ph.D., for editorial assistance with this manuscript.
REFERENCES 1.
2. 3.
4. 5. 6.
7. 8. 9. 10. 11.
12.
13. 14.
15. 16.
17.
18. 19. 20.
CONCLUSION Regenerative medicine technologies for virtually every type of tissue and organ within the male and female reproductive system are currently being developed. Various tissues are at different stages of development. Some are already being used clinically, such as engineered urethral tissue, while a few others are in preclinical trials and many more are in the discovery stage. Recent progress suggests that engineered tissues may have an expanded clinical applicability in the future and may represent a viable therapeutic option for those who would benefit from benefits of reproductive tissue replacement or repair. 28
21. 22.
23.
24. 25.
Dahms SE, Piechota HJ, Dahiya R, Lue TF, Tanagho EA. Composition and biomechanical properties of the bladder acellular matrix graft: comparative analysis in rat, pig and human. Br J Urol 1998;82:411–9. Yoo JJ, Meng J, Oberpenning F, Atala A. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 1998;51:221–5. Piechota HJ, Dahms SE, Nunes LS, Dahiya R, Lue TF, Tanagho EA. In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft. J Urol 1998;159:1717–24. Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a possible ‘‘off the shelf’’ biomaterial for urethral repair. Urology 1999;54:407–10. Amiel GE, Atala A. Current and future modalities for functional renal replacement. Urol Clin North Am 1999;26:235–46. Amiel GE, Komura M, Shapira O, Yoo JJ, Yazdani S, Berry J, et al. Engineering of blood vessels from acellular collagen matrices coated with human endothelial cells. Tissue Eng 2006;12:2355–65. Yoo JJ, Park HJ, Lee I, Atala A. Autologous engineered cartilage rods for penile reconstruction. J Urol 1999;162:1119–21. Atala A. Autologous cell transplantation for urologic reconstruction. J Urol 1998;159:2–3. Atala A. Bladder regeneration by tissue engineering. BJU Int 2001;88: 765–70. Atala A. Creation of bladder tissue in vitro and in vivo: a system for organ replacement. Adv Exp Med Biol 1999;462:31–42. Cilento BG, Freeman MR, Schneck FX, Retik AB, Atala A. Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro. J Urol 1994;152:665–70. Oberpenning F, Meng J, Yoo JJ, Atala A. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999;17:149–55. Kim BS, Mooney DJ. Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol 1998;16:224–30. Bergsma JE, Rozema FR, Bos RR, Boering G, de Bruijn WC, Pennings AJ. In vivo degradation and biocompatibility study of in vitro pre-degraded aspolymerized polyactide particles. Biomaterials 1995;16:267–74. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11–25. Li ST. Biologic biomaterials: tissue derived biomaterials (collagen). In: Bronzino JD, editor. The biomedical engineering handbook. Boca Raton, FL: CRS Press; 1995:627–47. Silver FH, Pins G. Cell growth on collagen: a review of tissue engineering using scaffolds containing extracellular matrix. J Long Term Eff Med Implants 1992;2:67–80. Sams AE, Nixon AJ. Chondrocyte-laden collagen scaffolds for resurfacing extensive articular cartilage defects. Osteoarthritis Cartilage 1995;3:47–59. Gilding D. Biodegradable polymers. In: Williams D, editor. Biocompatibility of clinical implant materials. Boca Raton, FL: CRC Press; 1981:209–32. Mikos AG, Lyman MD, Freed LE, Langer R. Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials 1994; 15:55–8. Harris LD, Kim BS, Mooney DJ. Open pore biodegradable matrices formed with gas foaming. J Biomed Mater Res 1998;42:396–402. Lee SJ, Liu J, Oh SH, Soker S, Atala A, Yoo JJ. Development of a composite vascular scaffolding system that withstands physiological vascular conditions. Biomaterials 2008;29:2891–8. Lee SJ, Oh SH, Liu J, Soker S, Atala A, Yoo JJ. The use of thermal treatments to enhance the mechanical properties of electrospun poly(epsiloncaprolactone) scaffolds. Biomaterials 2008;29:1422–30. Peppas NA, Langer R. New challenges in biomaterials. Science 1994;263: 1715–20. Brivanlou AH, Gage FH, Jaenisch R, Jessell T, Melton D, Rossant J. Stem cells. Setting standards for human embryonic stem cells. Science 2003; 300:913–6. VOL. 98 NO. 1 / JULY 2012
Fertility and Sterility® 26.
27.
28.
29.
30.
31.
32.
33. 34.
35.
36. 37. 38. 39. 40.
41.
42.
43.
44. 45.
46.
47. 48.
49.
Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19: 1134–40. Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovitz-Eldor J, Goldstein RS, et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–5. Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Nat Acad Sci USA 2000;97:11307–12. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–33. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Nat Acad Sci USA 2001;98:10716–21. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108:407–14. Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R. Endothelial cells derived from human embryonic stem cells. Proc Nat Acad Sci USA 2002;99: 4391–6. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–7. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000;6:88–95. Hochedlinger K, Rideout WM, Kyba M, Daley GQ, Blelloch R, Jaenisch R. Nuclear transplantation, embryonic stem cells and the potential for cell therapy. Hematol J 2004;5(Suppl 3):S114–7. Rideout WM 3rd, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome. Science 2001;293:1093–8. Solter D. Mammalian cloning: advances and limitations. Nat Rev Genet 2000;1:199–207. Hochedlinger K, Jaenisch R. Nuclear transplantation: lessons from frogs and mice. Curr Opin Cell Biol 2002;14:741–8. Dinnyes A, De Sousa P, King T, Wilmut I. Somatic cell nuclear transfer: recent progress and challenges. Cloning Stem Cells 2002;4:81–90. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126: 663–76. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007;448:318–24. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–72. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917–20. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells 1. Nature 2007;448:313–7. Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 2007;25: 1177–81. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100–6. Scriven SD, Booth C, Thomas DF, Trejdosiewicz LK, Southgate J. Reconstitution of human urothelium from monolayer cultures. J Urol 1997;158:1147–52. Liebert M, Hubbel A, Chung M, Wedemeyer G, Lomax MI, Hegeman A, et al. Expression of mal is associated with urothelial differentiation in vitro: identification by differential display reverse-transcriptase polymerase chain reaction. Differentiation 1997;61:177–85. Liebert M, Wedemeyer G, Abruzzo LV, Kunkel SL, Hammerberg C, Cooper KD, et al. Stimulated urothelial cells produce cytokines and express an activated cell surface antigenic phenotype. Semin Urol 1991;9:124–30.
VOL. 98 NO. 1 / JULY 2012
50. 51.
52. 53. 54.
55.
56.
57. 58. 59.
60. 61. 62.
63.
64.
65.
66. 67.
68. 69.
70.
71.
72.
73.
74.
Puthenveettil JA, Burger MS, Reznikoff CA. Replicative senescence in human uroepithelial cells. Adv Exp Med Biol 1999;462:83–91. Nguyen HT, Park JM, Peters CA, Adam RM, Orsola A, Atala A, et al. Cell-specific activation of the HB-EGF and ErbB1 genes by stretch in primary human bladder cells. In Vitro Cell Dev Biol Anim 1999;35:371–5. Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet 2006;367:1241–6. Bazeed MA, Thuroff JW, Schmidt RA, Tanagho EA. New treatment for urethral strictures. Urology 1983;21:53–7. Olsen L, Bowald S, Busch C, Carlsten J, Eriksson I. Urethral reconstruction with a new synthetic absorbable device: an experimental study. Scand J Urol Nephrol 1992;26:323–6. Atala A, Vacanti JP, Peters CA, Mandell J, Retik AB, Freeman MR. Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vitro. J Urol 1992;148:658–62. Sievert KD, Bakircioglu ME, Nunes L, Tu R, Dahiya R, Tanagho EA. Homologous acellular matrix graft for urethral reconstruction in the rabbit: histological and functional evaluation. J Urol 2000;163:1958–65. Atala A, Guzman L, Retik AB. A novel inert collagen matrix for hypospadias repair. J Urol 1999;162:1148–51. El-Kassaby AW, Retik AB, Yoo JJ, Atala A. Urethral stricture repair with an off-the-shelf collagen matrix. J Urol 2003;169:170–3. El Kassaby A, Aboushwareb T, Atala A. Randomized comparative study between buccal mucosal and acellular bladder matrix grafts in complex anterior urethral strictures. J Urol 2008;179:1432–6. De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol 2002;168:1789–92. le Roux PJ. Endoscopic urethroplasty with unseeded small intestinal submucosa collagen matrix grafts: a pilot study. J Urol 2005;173:140–3. Raya-Rivera A, Esquiliano DR, Yoo JJ, Lopez-Bayghen E, Soker S, Atala A. Tissue-engineered autologous urethras for patients who need reconstruction: an observational study. Lancet 2011;377:1175–82. Kershen RT, Yoo JJ, Moreland RB, Krane RJ, Atala A. Reconstitution of human corpus cavernosum smooth muscle in vitro and in vivo. Tissue Eng 2002;8:515–24. Park HJ, Yoo JJ, Kershen RT, Moreland R, Atala A. Reconstitution of human corporal smooth muscle and endothelial cells in vivo. J Urol 1999;162: 1106–9. Falke G, Yoo JJ, Kwon TG, Moreland R, Atala A. Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices. Tissue Eng 2003;9:871–9. Kwon TG, Yoo JJ, Atala A. Autologous penile corpora cavernosa replacement using tissue engineering techniques. J Urol 2002;168:1754–8. Chen KL, Eberli D, Yoo JJ, Atala A. Bioengineered corporal tissue for structural and functional restoration of the penis. Proc Natl Acad Sci USA 2010;107:3346–50. Machluf M, Atala A. Emerging concepts for tissue and organ transplantation. Graft 1998;1:31–7. Raya-Rivera AM, Baez C, Atala A, Yoo JJ. Tissue engineered testicular prostheses with prolonged testosterone release. World J Urol 2008;26: 307–14. Wang T, Koh C, Yoo JJ. Creation of an engineered uterus for surgical reconstruction. American Academy of Pediatrics Section on Urology; New Orleans, 2003. Campbell GR, Turnbull G, Xiang L, Haines M, Armstrong S, Rolfe BE, et al. The peritoneal cavity as a bioreactor for tissue engineering visceral organs: bladder, uterus and vas deferens. J Tissue Eng Regen Med 2008; 2:50–60. Li X, Sun H, Lin N, Hou X, Wang J, Zhou B, et al. Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor. Biomaterials 2011;32:8172–81. House M, Sanchez CC, Rice WL, Socrate S, Kaplan DL. Cervical tissue engineering using silk scaffolds and human cervical cells. Tissue Eng Part A 2010; 16:2101–12. De Filippo RE, Yoo JJ, Atala A. Engineering of vaginal tissue in vivo. Tissue Eng 2003;9:301–6.
29