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cmj-47-1.pdf
DOI: 10.4068/cmj.2011.47.1.1 Ⓒ Chonnam Medical Journal, 2011 Chonnam Med J 2011;47:1-131
Review Article
www.cmj.ac.kr
Tissue Engineering: Current Strategies and Future Directions Jennifer L. Olson, Anthony Atala and James J. Yoo*
Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, NC, USA
Novel therapies resulting from regenerative medicine and tissue engineering technol- ogy may offer new hope for patients with injuries, end-stage organ failure, or other clin- ical issues. Currently, patients with diseased and injured organs are often treated with transplanted organs. However, there is a shortage of donor organs that is worsening yearly as the population ages and as the number of new cases of organ failure increases. Scientists in the field of regenerative medicine and tissue engineering 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 tissues. In addition, the stem cell field is a rapidly advancing part of re- generative medicine, and new discoveries in this field create new options for this type of therapy. For example, new types of stem cells, such as amniotic fluid and placental stem cells that can circumvent the ethical issues associated with embryonic stem cells, have been discovered. 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 clinical setting, indicating that regenerative medicine holds much promise for the future.
Key Words: Biomaterials; Cell transplantation; Regenerative medicine; Stem cell; Tissue engineering
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Article History: received 31 March, 2011 accepted 8 April, 2011
Corresponding Author: James J. Yoo Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, 1834, Wake Forest Road, Winston-Salem, NC 27109, USA TEL: +1-336-713-7294 FAX: +1-336-713-7290 E-mail: [email protected]
INTRODUCTION
Patients suffering from diseased and injured organs are often treated with transplanted organs, and this treatment has been in use for over 50 years. In 1955, the kidney be- came the first entire organ to be replaced in a human, when Murray transplanted this organ between identical twins. Several years later, Murray performed an allogeneic kid- ney transplant from a non-genetically identical patient in- to another. This transplant, which overcame the immuno- logic barrier, marked a new era in medicine and opened the door for use of transplantation as a means of therapy for different organ systems. As modern medicine increases the human lifespan, the aging population grows, and the need for donor organs grows with it, because aging organs are generally more prone to failure. However, there is now a critical shortage of donor organs, and many patients in need of organs will
die while waiting for transplants. In addition, even if an or- gan becomes available, rejection of organs is still a major problem in transplant patients despite improvements in the methods used for immunosuppression following the transplant procedure. Even if rejection does not occur, the need for lifelong use of immunosuppressive medications leads to a number of complications in these patients. These problems have led physicians and scientists to look to new fields for alternatives to organ transplantation. In the 1960s, a natural evolution occurred in which re- searchers began to combine new devices and materials sci- ences with cell biology, and a new field that is now termed tissue engineering was born. As more scientists from differ- ent fields came together with the common goal of tissue re- placement, the field of tissue engineering became more for- mally established. Tissue engineering is now defined as “an interdisciplinary field which applies the principles of en- gineering and life sciences towards the development of bio-
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logical substitutes that aim to maintain, restore or improve tissue function.”1 Then, after the discovery of human stem cells by Thomson’s group in the early 1980s,2 the field of stem cell biology took shape and suggested that it may one day be possible to obtain and use donor stem cells in tissue engineering strategies, or perhaps even reactivate endoge- nous stem cells and use them to regenerate failing organs in adult patients. The fields of stem cells, cell transplantation, and tissue engineering all have one unifying concept-the regeneration of living tissues and organs. Thus, in 1999, William Haseltine, then the Scientific Founder and Chief Executive Officer of Human Genome Sciences, coined the term regenerative medicine, in effect bringing all these areas under one defin- ing field.3
In the past two decades, scientists have attempted to en- gineer virtually every tissue of the human body. This ar- ticle will review the basic techniques used in tissue en- gineering and discuss some of the progress that has been achieved in this field.
THE BASIC COMPONENTS OF REGENERATIVE MEDICINE STRATEGIES
The field of regenerative medicine encompasses various areas of technology, such as tissue engineering, stem cells, and cloning. Tissue engineering, one of the major areas of regenerative medicine, follows the principles of cell trans- plantation, materials science, and engineering toward the development of biological substitutes that can restore and maintain normal function. Tissue engineering strategies generally fall into two categories: the use of acellular scaf- folds, which depend on the body's natural ability to re- generate for proper orientation and direction of new tissue growth, and the use of scaffolds seeded with cells. Acellular scaffolds are usually prepared by manufacturing artificial scaffolds or by removing cellular components from tissues via mechanical and chemical manipulation to produce acel- lular, collagen-rich matrices.4-7 These matrices tend to slowly degrade on implantation and are generally replaced by the extracellular matrix (ECM) proteins that are se- creted by the in-growing cells. Cells can also be used for therapy via injection, either with carriers such as hydro- gels or alone.
1. Biomaterials for use in regenerative medicine In the past, synthetic materials were introduced to re- place or to rebuild diseased tissues or parts in the human body. The manufacture of new materials, such as tetra- fluoroethylene (Teflon) and silicone, opened a new field of research that led to the development of a wide array of de- vices that could be applied for human use. Although these devices could provide structural support or replacement, the functional component of the original tissue was not restored. However, studies in cell biology, molecular biol- ogy, and biochemistry allowed a better understanding of the ECM and its interaction with cells in the tissues of the
body, as well as interactions with growth factors and their ligands, and as a result, new biomaterials were designed with these interactions in mind. In tissue engineering, biomaterials replicate the bio- logical and mechanical function of the native ECM found in tissues in the body. Biomaterials provide a three-dimen- sional space in which cells can attach, grow, and form new tissues with appropriate structure and function. They also allow for the delivery of cells and appropriate bioactive fac- tors (e.g., cell adhesion peptides, growth factors) to desired sites in the body.8 Because most mammalian cell types are anchorage-dependent and will die if no cell-adhesion sub- strate is available, biomaterials provide this substrate while allowing delivery of cells with high loading efficiency. Biomaterials can also provide mechanical support against in vivo forces so that the predefined three-dimensional structure of a tissue-engineered organ is maintained dur- ing tissue development. The ideal biomaterial should be biodegradable and bio- resorbable to support the replacement of normal tissue without inducing inflammation. Incompatible materials are destined for an inflammatory or foreign-body response that eventually leads to rejection or necrosis. Because bio- materials provide temporary mechanical support while the cells undergo spatial reorganization into tissue, a prop- erly chosen biomaterial should allow the engineered tissue to maintain sufficient mechanical integrity to support it- self in early development, while in late development, it should have begun degradation such that it does not hinder further tissue growth.8 The degradation products, if pro- duced, should be removed from the body via metabolic path- ways at an adequate rate to ensure that the concentration of these degradation products in the tissues remains at a tolerable level.9
Generally, three classes of biomaterials have been uti- lized for engineering tissues: naturally derived materials (e.g., collagen and alginate),10-14 acellular tissue matrices (e.g., bladder submucosa and small intestinal submuco- sa),4-7 and synthetic polymers such as polyglycolic acid (PGA), polylactic acid (PLA), and poly (lactic-co-glycolic acid) (PLGA).15-18 These classes of biomaterials have been tested with respect to their biocompatibility.19,20 Naturally derived materials and acellular tissue matrices have the potential advantage of biological recognition. However, synthetic polymers can be produced reproducibly on a large scale with controlled properties such as strength, degrada- tion rate, and microstructure.
2. Cells for use in cell therapy and tissue engineering 1) Native cells: When native cells are used for tissue en- gineering, a small piece of donor tissue is dissociated into individual cells. These cells are expanded in culture and ei- ther injected directly back into the host or attached to a sup- port matrix and then reimplanted. The source of donor tis- sue can be heterologous (such as bovine), allogeneic (same species, different individual), or autologous. The preferred cells to use are autologous cells, where a biopsy of tissue
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is obtained from the host, the cells are dissociated and ex- panded in culture, and the expanded cells are implanted into the same host.5,21-28 The use of autologous cells, al- though it may cause an inflammatory response, avoids re- jection, and thus the deleterious side effects of immuno- suppressive medications can be avoided. Ideally, both structural and functional tissue replace- ment will occur with minimal complications when autolo- gous native cells are used. However, one of the limitations of applying cell-based regenerative medicine techniques to organ replacement has been the inherent difficulty of grow- ing specific cell types in large quantities. 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, one may be able to overcome the obstacles that limit cell expansion in vitro. For exam- ple, urothelial cells could be grown in the laboratory setting in the past, but only with limited expansion. Several proto- cols were developed over the past two decades that identi- fied the undifferentiated cells and kept them undifferen- tiated during their growth phase.27,29,30-32 With the use of these methods of cell culture, it is now possible to expand a urothelial strain from a single specimen that initially cov- ered 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.27 These studies indicated that it should be possible to collect autologous bladder cells from human patients, ex- pand them in culture, and return them to the donor in suffi- cient quantities for reconstructive purposes.27,30 Major ad- vances 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 for clinical application possible. Most current strategies for tissue engineering depend on 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 sit- uations, 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).
3. Stem cells for use in tissue engineering 1) Embryonic stem cells: Human embryonic stem (hES) cells exhibit two remarkable properties: the ability to pro- liferate in an undifferentiated but pluripotent state (self- renewal), and the ability to differentiate into many speci- alized cell types.36 They can be isolated by aspirating the inner cell mass from the embryo during the blastocyst stage (5 days post-fertilization) and are usually grown on feeder layers consisting of mouse embryonic fibroblasts or human
feeder cells.37 More recent reports have shown that these cells can be grown without the use of a feeder layer38 and thus avoid the exposure of these human cells to mouse vi- ruses and proteins. These cells have demonstrated lon- gevity in culture by maintaining their undifferentiated state for at least 80 passages when grown by use of current published protocols.2,39 In addition, hES cells are able to dif- ferentiate into cells from all three embryonic germ layers in vitro. Skin and neurons have been formed, indicating ec- todermal differentiation.40-43 Blood, cardiac cells, cartilage, endothelial cells, and muscle have been formed, indicating mesodermal differentiation.44-46 Pancreatic cells have been formed, indicating endodermal differentiation.47 In addi- tion, as further evidence of their pluripotency, embryonic stem cells can form embryoid bodies, which are cell ag- gregations that contain all three embryonic germ layers while in culture and can form teratomas in vivo.48 However, there are many ethical and religious concerns associated with hES cells because embryos are destroyed in order to obtain them. Thus, the use of these cells is currently banned in many countries. 2) Stem cells from somatic cell nuclear transfer: Stem cells for tissue engineering could also be generated through cloning procedures. There has been tremendous interest in the field of nuclear cloning since the birth of the cloned sheep Dolly in 1997, but actually, Dolly was not the first animal produced by using nuclear transfer. In fact, frogs were the first successfully cloned vertebrates derived from nuclear transfer.49 However, in the frog experiment, the nuclei used for cloning were derived from non-adult sources. In fact, live lambs were produced in 1996 by using nuclear transfer as well, but they were produced from differen- tiated epithelial cells derived from embryonic discs.50 The significance of Dolly was that she was the first mammal to be derived from an adult somatic cell by use of nuclear transfer.51 Since then, animals from several species have been grown by using nuclear transfer technology, inclu- ding cattle,52 goats,53 mice,54 and pigs.55-56
Two types of nuclear cloning, reproductive cloning and therapeutic cloning, have been described, and a better un- derstanding of the differences between the two types may help to alleviate some of the controversy that surrounds these technologies.57-58 Banned in most countries for hu- man applications, reproductive cloning is used to generate an embryo that has the identical genetic material as its cell source. This embryo can then be implanted into the uterus of a female to give rise to an infant that is a clone of the donor. On the other hand, therapeutic cloning is used to generate early stage embryos that are explanted in culture to produce embryonic stem cell lines whose genetic materi- al 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.59 Therefore, therapeutic cloning, which has also been called somatic cell nuclear transfer, may provide an alternative source of transplan- table cells. According to data from the Centers for Disease
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Control and Prevention, an estimated 3,000 Americans die every day of diseases that could have been treated with stem cell–derived tissues.60-61 With current allogeneic tis- sue transplantation protocols, rejection is a frequent com- plication because of immunologic incompatibility, and im- munosuppressive drugs are usually required.59 The use of transplantable tissue and organs derived from therapeutic cloning could lead to the avoidance of immune responses that typically are associated with transplantation of non- autologous tissues.60
While promising, somatic cell nuclear transfer technol- ogy has certain limitations that require further study be- fore 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 most em- bryos derived from the cloning process do not survive.62-64 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 acti- vation process that instructs the cloned oocytes to begin dividing. In addition, cell cycle synchronization between donor cells and recipient oocytes must be accomplished.65
3) Reprogramming and generation of iPS cells: Within the past few years, exciting reports of the successful trans- formation of adult somatic cells into pluripotent stem cells through genetic “reprogramming” have been published. Reprogramming is a technique that involves de-differen- tiation of adult somatic cells (such as fibroblasts) to produce patient-specific pluripotent stem cells. This process is es- pecially exciting because it allows pluripotent stem cells to be obtained without the use of embryos. Also, cells genera- ted by reprogramming are genetically identical to the so- matic cells used (and thus to the patient who donated these cells) and should not be rejected. Yamanaka was the first to discover that mouse embryonic fibroblasts (MEFs) and adult mouse fibroblasts could be reprogrammed into an “induced pluripotent state (iPS).”66 They examined 24 genes that were thought to be important for embryonic stem cells and identified 4 key genes that, when introduced into the reporter fibroblasts via retroviral vectors, resulted in drug-resistant cells. These were Oct3/4, Sox2, c-Myc, and Klf4. The resultant iPS cells possessed the immortal growth characteristics of self-renewing embryonic stem cells, expressed genes specific for embryonic stem 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 embryonic stem cells. Unlike embryonic stem 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 embryonic stem cells. In addition, the epigenetic state of the iPS cells was somewhere between that found in somatic cells and that found in embryonic stem cells, suggesting that the re-
programming was incomplete. These results were improved significantly by Wernig and Jaenisch in July 2007.67 Fibroblasts were infected with retroviral vectors and selected for the activation of endoge- nous 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 embryonic stem cells. Teratomas induced by these cells contained differentiated cell types representing all three embryonic germ layers. Most importantly, the re- programmed cells from this experiment could form viable chimeras and contribute to the germline-like embryonic stem cells, suggesting that these iPS cells were completely reprogrammed. Wernig et al observed that the number of reprogrammed colonies increased when drug selection was initiated later (day 20 rather than day 3 post-transduc- tion). This suggests that reprogramming is a slow and grad- ual process and may explain why previous attempts re- sulted in incomplete reprogramming. It has recently been shown that reprogramming of hu- man cells is possible.68-69 Yamanaka generated human iPS cells that are similar to hES cells in terms of morphology, proliferation, gene expression, surface markers, and ter- atoma formation. Thompson’s group showed that retro- viral transduction of the stem cell markers OCT4, SOX2, NANOG, and LIN28 could generate pluripotent stem cells. However, in both studies, the human iPS cells were similar but not identical to hES cells. Although reprogramming is an exciting phenomenon, our limited understanding of the mechanism underlying it currently limits the clinical ap- plicability of the technique, but the future potential of re- programming is quite exciting. 4) Amniotic fluid and placental stem cells: An alternate source of stem cells is the amniotic fluid and placenta. Amniotic fluid and the placenta are known to contain mul- tiple partially differentiated cell types derived from the de- veloping fetus. We isolated stem cell populations from these sources, called amniotic fluid and placental stem cells (AFPSC), that express embryonic and adult stem cell mar- kers.70 The undifferentiated stem cells expand extensively without feeders and double every 36 hours. Unlike hES cells, the AFPSC do not form tumors in vivo. Lines main- tained for over 250 population doublings retained long telo- meres and a normal karyotype. AFS cells are broadly multi- potent. 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. Examples of differen- tiated cells derived from AFS cells and displaying speciali- zed functions include neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein- gated inwardly rectifying potassium (GIRK) channels, hepatic lineage cells producing urea, and osteogenic line- age cells forming tissue engineered bone. The cells could
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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 and used without rejection, or they could be banked. A bank of 100,000 specimens could potentially supply 99% of the US population with a perfect genetic match for transplan- tation. Such a bank may be easier to create than with other cell sources, because there are approximately 4.5 million births per year in the USA.70
5) Adult stem cells: Adult stem cells, especially hemato- poietic stem cells, are the best understood cell type in stem cell biology.71 The presence of stem cells in the adult was first discerned by Till and McCulloch, who were investigat- ing the mechanisms by which the bone marrow could re- generate after exposure to radiation.72 However, adult stem cell research remains an area of intense study, because their potential for therapy may be applicable to a myriad of degenerative disorders. Within the past decade, adult stem cell populations have been found in many adult ti- ssues other than the bone marrow and the gastrointestinal tract, including the brain,73-74 skin,75 and muscle.76 Many other types of adult stem cells have been identified in or- gans all over the body and are thought to serve as the pri- mary repair entities for their corresponding organs.77 The discovery of such tissue-specific progenitors has opened up new avenues for research. A notable exception to the tissue-specificity of adult stem cells is the mesenchymal stem cell (MSC), also known as the multipotent adult progenitor cell. This cell type is de- rived from bone marrow stroma.78-79 Such cells can differ- entiate in vitro into numerous tissue types80-81 and can also differentiate developmentally if injected into a blastocyst. Multipotent adult progenitor cells can develop into a variety of tissues including neuronal,82 adipose,76 muscle,76,83 li- ver,84-85 lungs,86 spleen,87 and gut tissue,79 but notably not bone marrow or gonads. In addition, stem cells derived from adipose tissue may also be an autologous and self-renewing cell source. Adi- pose-derived stem cells (ADSCs) have been shown to diffe- rentiate into a variety of cell phenotypes, and since they are easily obtained, they show great promise for future types of reconstructive surgery based on tissue engineering and there have been several clinical trials using these cells. Wilson and Mizuno have both provided excellent, detailed reviews of these.88-89
Research into more differentiated types of adult stem cells has, however, progressed slowly, mainly because in- vestigators have had great difficulty in maintaining adult non-mesenchymal stem cells in culture. Some cells, such as those of the liver, pancreas, and nerve, have very low pro- liferative capacity in vitro, and the functionality of some cell types is reduced after the cells are cultivated. Isolation of cells has also been problematic, because stem cells are present in extremely low numbers in adult tissue.84,90 While the clinical utility of adult stem cells is currently limited, great potential exists for future use of such cells in tissue- specific regenerative therapies. The advantage of adult
stem cells is that they can be used in autologous therapies, thus avoiding any complications associated with immune rejection.
CELLULAR THERAPIES
The simplest regenerative medicine strategies are those that are based on the actions of cells, which can be implanted either alone or within a type of carrier material, such as a hydrogel. These cell therapies are designed to inject or im- plant healthy cells to replace populations of cells that are no longer functioning properly owing to disease or injury. The cells used in these therapies can be autologous cells de- rived from a tissue biopsy and expanded in culture, or they can be stem cells from various sources that can be guided to differentiate into appropriate cell types by using both en- dogenous and exogenous biochemical cues. For example, one area of intense study in regenerative medicine is the pancreas, because the ability to replace or regenerate the insulin-producing cells of this organ could lead to novel treatments or a cure for diabetes. In a series of exciting experiments, Zhou et al demonstrated that re- generation of the insulin-producing cells of the pancreas, the β-cells, may be possible by using cellular reprogram- ming techniques91 Using a mouse model, they showed that in vivo activation of a specific combination of three tran- scription factors (Ngn3, Pdx1, and Mafa) by use of adeno- viral vectors led to the reprogramming of adult differ- entiated pancreatic exocrine cells into cells that closely re- sembled β-cells. These cells were similar to native β-cells in size, shape, and ultrastructure, and they expressed genes that are specific to β-cells as well. Interestingly, these cells secreted insulin and expressed vascular endothelial growth factor (VEGF), which allowed them to remodel the local vasculature in a manner similar to native β-cells. In fact, these reprogrammed cells were able to partially ameliorate hyperglycemia in diabetic mice, suggesting that repro- gramming techniques for treating disease may one day be- come a reality. Degenerative muscle diseases such as Duchenne’s mus- cular dystrophy have devastating effects on quality of life. To date, these genetic disorders have no suitable treat- ment. Early enthusiasm for gene therapy interventions has been tempered by issues of vector toxicity and inade- quate gene transfer to target muscle cells in vivo. However, natural mechanisms of muscle repair have suggested that cell-based therapy could take advantage of natural homing mechanisms to direct cells to the proper location.92 Experi- ments using the mdx mouse model, in which the dystrophin gene is mutated, indicate that injection of normal muscle precursors and dermal fibroblasts into skeletal muscle can lead to increased expression of dystrophin and improved functional outcomes. However, this treatment option re- quires further studies before it can be widely applied in the clinic. Although many of these cell therapies are still in the ex- perimental stage, some are being translated to the clinic
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and clinical trials are being performed. Vesicoureteral re- flux (VUR; a condition in which urine flows backwards from the bladder into the ureter and kidney) and stress urinary incontinence are two urologic conditions that can result from dysfunction of a specific sphincter muscle. When se- vere, these conditions are repaired surgically. However, cell-based therapies for both VUR and incontinence would be an important alternative to surgical repair of these conditions. Ideally, such a therapy would be easily ad- ministered by injection and well tolerated by the patient. The injectable therapy should be non-antigenic, non-mi- gratory, volume stable, and safe for human use, and in addi- tion, it should be able to carry cells and serve as a matrix in vivo. Toward this goal, long-term studies were conducted to determine the effects of injectable chondrocytes for the treatment of VUR in vivo.93 Chondrocytes were chosen be- cause the use of autologous cartilage for the treatment of VUR in humans would satisfy all of the requirements for an ideal injectable cell-based therapy. Chondrocytes de- rived from an ear biopsy can be readily grown and expanded in culture. Neocartilage formation can be achieved in vitro and in vivo by using chondrocytes cultured on synthetic bio- degradable polymers. In the VUR experiments, chon- drocytes were suspended in an alginate matrix and injected around the vesicoureteral sphincter. In time, normal carti- lage replaced the alginate as the alginate slowly degraded. This system was then adapted for the treatment of VUR in a porcine model.94 These studies show that chondrocytes can be easily harvested and combined with alginate in vi- tro, that the suspension can be easily injected cystoscopi- cally, and that the elastic cartilage tissue formed can cor- rect the VUR without any evidence of obstruction. Two multicenter clinical trials were conducted by use of this engineered chondrocyte technology. First, patients with VUR were treated at 10 centers throughout the United States. The patients had a similar success rate as with oth- er injectable substances in terms of cure. Cartilage for- mation was not noted in patients with treatment failure. Patients who were cured probably had a biocompatible re- gion of engineered autologous tissue present.95 Secondly, patients with urinary incontinence were treated endo- scopically with injected chondrocytes at three different medical centers. Phase 1 trials showed an approximate success rate of 80% at 3 and 12 months postoperatively.96
TISSUE THERAPIES
Tissue engineering strategies are often referred to as “growing organs in the laboratory.” In these strategies, dif- ferentiated cells or stem cells are seeded onto a biomaterial scaffold and this construct is allowed to mature in vitro in a bioreactor for a short time before implantation in vivo. These constructs are designed to replace a malfunctioning organ in its entirety. In recent years, it has been shown that hollow organs, such as the urinary bladder, urethra, and blood vessels, can be successfully engineered in the labo-
ratory, and these successes are described below. The urethra can be repaired by using tissue-engineered grafts in several ways. It has been shown that various bio- materials without cells, such as PGA and acellular colla- gen-based matrices from small intestine and bladder, can be used experimentally (in animal models) for the regene- ration of urethral tissue.7,97-99 Acellular collagen matrices derived from bladder submucosa have been used experi- mentally and clinically. In animal studies, segments of the urethra were resected and replaced with acellular matrix grafts in an onlay fashion. Histological examination showed complete epithelialization and progressive vessel and mu- scle infiltration, and the animals were able to void through the neo-urethras.7 These results were confirmed in a clini- cal study of patients with hypospadias and urethral stric- ture disease.100 Decellularized cadaveric bladder submu- cosa was used as an onlay matrix for urethral repair in pa- tients with stricture disease and hypospadias. Patent, functional neo-urethras were noted in these patients with up to a 7-year follow-up. The use of an off-the-shelf matrix appears to be beneficial for patients with abnormal ure- thral conditions and obviates the need for obtaining autolo- gous grafts, thus decreasing operative time and eliminat- ing donor site morbidity. Unfortunately, the above techniques are not applicable for tubularized urethral repairs. The collagen matrices are able to replace urethral segments only when used in an on- lay fashion. However, if a tubularized repair is needed, the collagen matrices should be seeded with autologous cells to avoid the risk of stricture formation and poor tissue development.101 In addition, cell-seeded matrices must be used if the segment of urethra to be replaced is longer than about 1 cm.102 Recently, Raya-Rivera and colleagues used tissue-engineered urethras that had been created from pa- tients' own cells for tubularized urethral reconstruction. In this preliminary study, five boys who had urethral defects were treated. A tissue biopsy was taken from each patient, and the muscle and epithelial cells derived from the biopsy sample were expanded and seeded onto tubularized poly- glycolic acid:poly(lactide-co-glycolide acid) scaffolds to cre- ate neo-urethras for implantation. The patients then un- derwent urethral reconstruction with the engineered urethras. After surgery, these patients were followed for up to 6 years. All of the patients experienced an increase in urinary flow rate, and serial radiographic and endo- scopic studies showed that they maintained wide urethral calibers throughout the follow-up period and did not develop strictures. In addition, urethral biopsies were performed in these patients, and these revealed that by 3 months post- surgery, the engineered grafts had developed a normal appearing tissue architecture consisting of a urothelial layer surrounded by a muscular layer.103
In addition, similar techniques have been used to create tissue-engineered bladder constructs. Urothelial and mu- scle cells can be expanded in vitro, seeded onto polymer scaffolds, and allowed to attach and form sheets of cells.104 These principles were applied in several studies in which
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tissue-engineered bladders were used to repair subtotal cystectomies in beagle dogs.28,105-106 The first clinical ex- perience in which engineered bladder tissue for cystoplasty reconstruction in patients was conducted starting in 1999. A small pilot study of seven patients was reported, using a collagen scaffold seeded with cells either with or without omentum coverage, or a combined PGA-collagen scaffold seeded with cells and omental coverage. The patients re- constructed with the engineered bladder tissue created with the PGA-collagen cell-seeded scaffolds showed in- creased compliance, decreased end-filling pressures, in- creased capacities, and longer dry periods.107 Although the experience is promising in terms of showing that engi- neered tissues can be implanted safely, it is just a start in terms of accomplishing the goal of engineering fully func- tional bladders. Further experimental and clinical work is being conducted. Xenogenic or synthetic materials have been used as re- placement blood vessels for complex cardiovascular lesions. However, these materials typically lack growth potential, and may place the recipient at risk for complications such as stenosis, thromboembolization, or infection.108 Tissue- engineered vascular grafts have been constructed by using autologous cells and biodegradable scaffolds and have been applied in dog and lamb models.109-112 The key advantage of using these autografts is that they degrade in vivo and thus allow the new tissue to form without the long-term presence of foreign material.108 Translation of these techni- ques from the laboratory to the clinical setting has begun, with autologous vascular cells harvested, expanded, and seeded onto a biodegradable scaffold.113 The resultant auto- logous construct was used to replace a stenosed pulmonary artery that had been previously repaired. Seven months af- ter implantation, no evidence of graft occlusion or aneur- ysmal changes was noted in the recipient. In addition, an- other group created tissue-engineered blood vessels by us- ing the cell-sheet multilayer method and then used these constructs to successfully create vascular access points for hemodialysis in 10 patients.114 More recently, the same group completed a larger study on these engineered vessels for hemodialysis, which indicated that the 1-month and 6-month patency of the grafts was 78% and 60%, respec- tively, which meets the approved criteria for a high-risk pa- tient cohort.115
Finally, few treatment options are currently available for patients who suffer from severe congenital tracheal pa- thology, such as stenosis, atresia, and agenesis, due to the limited availability of autologous transplantable tissue in the neonatal period. Tissue engineering in the fetal period may be a viable alternative for the surgical treatment of these prenatally diagnosed congenital anomalies, because cells could be harvested and grown into transplantable ti- ssue in parallel with the remainder of gestation. Chondro- cytes from both elastic and hyaline cartilage specimens have been harvested from fetal lambs, expanded in vitro, and then dynamically seeded onto biodegradable scaffolds.116 The constructs were then implanted as replacement tra-
cheal tissue in fetal lambs. The resultant tissue-engineered cartilage was noted to undergo engraftment and epithelial- ization, while maintaining its structural support and pa- tency. Recently, Martin Birchall’s group moved this technology into a human patient with end-stage airway disease.117 This group was able to remove the cellular material and MHC antigens from a human donor trachea and, using a speci- alized bioreactor, seed this acellular matrix with chon- drocytes and epithelial cells derived from the patient to re- ceive the graft. This construct was then used to replace the patient’s left main bronchus. There were no perioperative complications, and the left lung ventilated normally as soon as the graft was placed. At 3 months after surgery, the patient’s lung function was in the normal range for her age and sex, and she was able to function normally. Although longer follow-up and larger study populations are needed, this report indicates that tissue engineering may be a new option for patients with airway disease. However, whereas there has been exciting progress with tissue engineering techniques for hollow organs, the develop- ment of methods to generate larger, solid organs with more complex histological structure has been much more diffi- cult. A number of issues must be addressed before fully func- tional, engineered organs such as liver and kidney can be prepared in the laboratory. First, these organs contain ex- tremely complex internal structures made up of numerous cell types arranged in very specific ways, and simple cell- seeding techniques may not be sufficient for reconstructing these structures. In addition, the large size of these organs dictates that the delivery of oxygen and nutrients to each part of the organ will be a challenge, unless a method for engineering a functional vascular network within the or- gan can be found. However, despite the challenges, there have been some encouraging results from several studies. For example, the kidney contains multiple cell types and a complex functional anatomy that renders it one of the most difficult to reconstruct,21,118 yet we were able to create a rudimentary form of this organ that appeared to have at least the filtration properties of the native kidney. We applied the principles of both tissue engineering and therapeutic cloning in an effort to produce genetically iden- tical renal tissue in a large animal model, the cow (Bos tau- rus).119 Bovine skin fibroblasts from adult Holstein steers were obtained by ear notch, and single donor cells were iso- lated and microinjected into the perivitelline space of donor enucleated oocytes (nuclear transfer). The resulting blas- tocysts were implanted into progestin-synchronized recip- ients to allow for further in vivo growth. After 12 weeks, cloned renal cells were harvested and expanded in vitro. Next, the cloned renal cells were seeded on scaffolds con- sisting of three collagen-coated cylindrical silastic cathe- ters. The ends of the three membranes of each scaffold were connected to catheters that terminated into a collecting reservoir. This created a renal neo-organ with a mecha- nism for collecting the excreted urinary fluid. These scaf- folds with the collecting devices were transplanted sub-
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Tissue Engineering
cutaneously into the same steer from which the genetic ma- terial originated and then retrieved 12 weeks after implan- tation. At this time, a yellow urine-like fluid was observed col- lecting within the reservoir of the device. Chemical analysis of this fluid, including urea nitrogen and creatinine levels, electrolyte levels, specific gravity, and glucose concentra- tion, revealed that the implanted renal cells possessed fil- tration, reabsorption, and secretory capabilities. Histolo- gical examination of the retrieved implants revealed exten- sive vascularization and self-organization of the cells into glomeruli and tubule-like structures. A clear continuity be- tween the glomeruli, the tubules, and the silastic catheter was noted that allowed the passage of urine into the collect- ing reservoir. These studies demonstrated that cells de- rived from nuclear transfer can be successfully harvested, expanded in culture, and transplanted in vivo with the use of biodegradable scaffolds on which the single suspended cells can organize into tissue structures that are geneti- cally identical to those of the host. These studies were the first demonstration of the use of therapeutic cloning for re- generation of tissues in vivo. However, the size of this de- vice was small, and the challenge will be to create a larger device with functioning vasculature and innervations, so that it can replace all of the myriad metabolic functions of the kidney.
CHALLENGES AND FUTURE DIRECTIONS IN REGENERATIVE MEDICINE: TRANSLATING REGENERATIVE THERAPIES TO THE CLINIC
The experiences with urethral, bladder, blood vessel, and tracheal replacement using tissue engineering provide en- couragement for future efforts to engineer other organs in the laboratory. These experiences also cast light on unsolved problems. For example, innervation of tissues and organs is important for achieving full functionality. In the canine engineered bladder experiments, the observation of posi- tive S-100 staining was consistent with growth of neural structures into the neo-bladders, and bladder function was restored soon after implantation.28 Innervation of tissue- engineered constructs has been observed in other systems such as the small intestine.120 Not only is successful con- nection with the nervous system important for the func- tionality of neo-organs, but evidence suggests that it can enhance tissue regeneration.121-122 The controlled release of neurotrophic factors is one potential approach to promote peripheral nerve regeneration and synapse formation with engineered tissue.123 Direct electrical stimulation has pro- ven useful in muscle regeneration124 and may have broader applicability. An even more fundamental issue for the ultimate success of laboratory-grown organs, particularly those with com- plex three-dimensional structure, is the provision of ade- quate oxygen and the generation of new vasculature. It has been appreciated for some years that in metabolically ac- tive tissues, the distance over which oxygen typically must
diffuse from a capillary bed to reach a cell is about 0.1 mm, but that in clinical grafts, the distance from the edge to the center of the graft is likely to exceed that by at least 50- fold.125-126 Therefore, with few exceptions (e.g., cartilage), oxygen is rate-limiting for the viability of grafted cells, and thus for organ engineering. Neovascularization, an intri- cate morphogenetic process that allows the formation of ex- tensively branched vessels, even in an adult, must occur rapidly and efficiently for a grafted neo-organ to thrive after implantation.127 Moreover, special measures may be nece- ssary to ensure survival of grafted tissue during the initial period after implantation, until a functional vascular bed is in place. Currently, three types of strategies have been devised to solve the oxygen supply problem. The first strategy involves the use of mechanical or che- mical sources of oxygen that can support the construct be- fore and immediately after implantation, until the neo- vascularization process is completed and can provide the neo-organ with sufficient blood circulation. An intra-tissue perfusion system utilizing an array of micro-needles to de- liver oxygen and nutrients and eliminate waste enhances the viability and functionality of thick (1 mm) slices of liver tissue in vitro and might facilitate in vivo grafting.128 In ad- dition, the use of oxygen-carrying molecules such as per- fluorocarbons could promote the function of cells in culture and of encapsulated cells and organ constructs implanted into animals.129-130 Our laboratory recently showed that a PLGA film incorporating an oxygen-generating system (sodium percarbonate) could prevent the necrosis of ische- mic tissue over several days in vivo.131 We hope to develop such novel scaffold materials further to support the surviv- al of large, complex organ constructs in the initial period after implantation. Second, “prevascularization” strategies aim to generate neo-organs engineered with a preexisting channel struc- ture to facilitate the generation of a competent vascular network.130,132 To accomplish this, endothelial lineage cells can be pre-seeded into the channels or may be recruited in vivo by using biochemical signals that are embedded in or released by the scaffold. However, there is still the question of how to create channels in a way that will be interpreted as a natural vascular network by the body. One solution would be to employ decellularized tissue as the scaffold. A recent study demonstrated that perfusion of an entire heart with detergents yields an acellular structure in which the native vascular channels remain intact.133 We independently devised perfusion-decellularization tech- nology using liver tissue and have found that the vascular tree of the whole organ scaffold remains patent and can be repopulated with large numbers of endothelial cells.134 Alternatively, several technologies can be used to manu- facture scaffolds with preformed channels, potentially with cells incorporated, designed to promote neo-vascula- rization. For example, laser guided “writing” was used to pattern endothelial cells and promote their aggregation in- to tubular vessels.135 Similarly, ink-jet-based bioprinting of cells and biomaterials by thermal ink jet technology can
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provide remarkable control of the fine structure of engi- neered tissues, including the generation of intricate vessel networks.136 We have used layer-by-layer ink jet printing to produce three-dimensional constructs containing endo- thelial cells and showed that these develop functional mi- crovascularization when implanted in vivo, as assessed by magnetic resonance imaging.137 Electrospinning of living cells with biomaterials offers similar potential to fabricate organ structures with pre-patterned vessels.138 Mathe- matical modeling of scaffolds designed to contain a preex- isting arteriovenous loop shows how the provision of an oxygen source within the scaffold can dynamically support further neo-vascularization and tissue development.139
Third, it is well established that growth factors such as VEGF and FGF can promote vascularization in engineered tissues.140 Recent efforts have extended this approach by incorporating additional pro-angiogenic molecules into scaffolds, such as organ-specific ECM from liver to support sinusoidal endothelial cells.141 Synthetic biomaterials de- signed to provide signals normally presented by the ECM will complement, and may eventually supersede, the use of the native molecules.142
Finally, several of the clinical trials involving bioengi- neered products have been placed on hold because of the costs involved with the specific technology. With a bioengi- neered product, costs are usually high because of the bio- logical nature of the therapies involved, and as with any therapy, the cost that the medical health care system can allow for a specific technology is limited. Therefore, the costs of bioengineered products have to be lowered before they can have an impact clinically. This is currently being addressed for multiple tissue-engineered technologies. As the technologies advance over time, and the volume of the application is considered, costs will naturally decrease.
SUMMARY AND CONCLUSION
Regenerative medicine efforts are currently underway experimentally for virtually every type of tissue and organ within the human body. As regenerative medicine incorpo- rates the fields of tissue engineering, cell biology, nuclear transfer, and materials science, personnel who have mas- tered the techniques of cell harvest, culture, expansion, transplantation, and polymer design are essential for the successful application of these technologies to extend hu- man life. Various tissues are at different stages of develop- ment, with some already being used clinically, a few in pre- clinical trials, and some in the discovery stage. Recent prog- ress suggests that engineered tissues may have an expanded clinical applicability in the future and may represent a via- ble therapeutic option for those who would benefit from the life-extending benefits of tissue replacement or repair.
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