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 became 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 kidney transplant from a non-genetically identical patient into another. This transplant, which overcame the immunologic 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 organ 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 researchers began to combine new devices and materials sciences with cell biology, and a new field that is now termed tissue engineering was born. As more scientists from different fields came together with the common goal of tissue replacement, the field of tissue engineering became more formally established. Tissue engineering is now defined as "an interdisciplinary field which applies the principles of engineering and life sciences towards the development of biological 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 endogenous 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 defining field.3

In the past two decades, scientists have attempted to engineer virtually every tissue of the human body. This article will review the basic techniques used in tissue engineering and discuss some of the progress that has been achieved in this field.

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 transplantation, 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 scaffolds, which depend on the body's natural ability to regenerate 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 acellular, 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 secreted by the in-growing cells. Cells can also be used for therapy via injection, either with carriers such as hydrogels or alone.

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 implant 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 derived 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 endogenous 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 regeneration of the insulin-producing cells of the pancreas, the β-cells, may be possible by using cellular reprogramming techniques91 Using a mouse model, they showed that in vivo activation of a specific combination of three transcription factors (Ngn3, Pdx1, and Mafa) by use of adenoviral vectors led to the reprogramming of adult differentiated pancreatic exocrine cells into cells that closely resembled 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 reprogramming techniques for treating disease may one day become a reality.

Degenerative muscle diseases such as Duchenne's muscular dystrophy have devastating effects on quality of life. To date, these genetic disorders have no suitable treatment. Early enthusiasm for gene therapy interventions has been tempered by issues of vector toxicity and inadequate 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 Experiments 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 requires further studies before it can be widely applied in the clinic.

Although many of these cell therapies are still in the experimental stage, some are being translated to the clinic and clinical trials are being performed. Vesicoureteral reflux (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 severe, 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 administered by injection and well tolerated by the patient. The injectable therapy should be non-antigenic, non-migratory, volume stable, and safe for human use, and in addition, 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 because 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 derived 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 biodegradable polymers. In the VUR experiments, chondrocytes were suspended in an alginate matrix and injected around the vesicoureteral sphincter. In time, normal cartilage 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 vitro, that the suspension can be easily injected cystoscopically, and that the elastic cartilage tissue formed can correct 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 other injectable substances in terms of cure. Cartilage formation was not noted in patients with treatment failure. Patients who were cured probably had a biocompatible region of engineered autologous tissue present.95 Secondly, patients with urinary incontinence were treated endoscopically 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 engineering strategies are often referred to as "growing organs in the laboratory." In these strategies, differentiated 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 laboratory, 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 biomaterials without cells, such as PGA and acellular collagen-based matrices from small intestine and bladder, can be used experimentally (in animal models) for the regeneration of urethral tissue.7,97-99 Acellular collagen matrices derived from bladder submucosa have been used experimentally 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 muscle infiltration, and the animals were able to void through the neo-urethras.7 These results were confirmed in a clinical study of patients with hypospadias and urethral stricture disease.100 Decellularized cadaveric bladder submucosa was used as an onlay matrix for urethral repair in patients 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 urethral conditions and obviates the need for obtaining autologous grafts, thus decreasing operative time and eliminating 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 onlay 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 patients' 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 polyglycolic acid:poly(lactide-co-glycolide acid) scaffolds to create neo-urethras for implantation. The patients then underwent 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 endoscopic 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 muscle 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 tissue-engineered bladders were used to repair subtotal cystectomies in beagle dogs.28,105-106 The first clinical experience 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 reconstructed with the engineered bladder tissue created with the PGA-collagen cell-seeded scaffolds showed increased compliance, decreased end-filling pressures, increased capacities, and longer dry periods.107 Although the experience is promising in terms of showing that engineered tissues can be implanted safely, it is just a start in terms of accomplishing the goal of engineering fully functional bladders. Further experimental and clinical work is being conducted.

Xenogenic or synthetic materials have been used as replacement 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 techniques from the laboratory to the clinical setting has begun, with autologous vascular cells harvested, expanded, and seeded onto a biodegradable scaffold.113 The resultant autologous construct was used to replace a stenosed pulmonary artery that had been previously repaired. Seven months after implantation, no evidence of graft occlusion or aneurysmal changes was noted in the recipient. In addition, another group created tissue-engineered blood vessels by using 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%, respectively, which meets the approved criteria for a high-risk patient cohort.115

Finally, few treatment options are currently available for patients who suffer from severe congenital tracheal pathology, 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 tissue in parallel with the remainder of gestation. Chondrocytes 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 tracheal tissue in fetal lambs. The resultant tissue-engineered cartilage was noted to undergo engraftment and epithelialization, while maintaining its structural support and patency.

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 specialized bioreactor, seed this acellular matrix with chondrocytes and epithelial cells derived from the patient to receive 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 development of methods to generate larger, solid organs with more complex histological structure has been much more difficult. A number of issues must be addressed before fully functional, engineered organs such as liver and kidney can be prepared in the laboratory. First, these organs contain extremely 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 organ 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 identical renal tissue in a large animal model, the cow (Bos taurus).119 Bovine skin fibroblasts from adult Holstein steers were obtained by ear notch, and single donor cells were isolated and microinjected into the perivitelline space of donor enucleated oocytes (nuclear transfer). The resulting blastocysts were implanted into progestin-synchronized recipients 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 consisting of three collagen-coated cylindrical silastic catheters. 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 mechanism for collecting the excreted urinary fluid. These scaffolds with the collecting devices were transplanted subcutaneously into the same steer from which the genetic material originated and then retrieved 12 weeks after implantation.

At this time, a yellow urine-like fluid was observed collecting within the reservoir of the device. Chemical analysis of this fluid, including urea nitrogen and creatinine levels, electrolyte levels, specific gravity, and glucose concentration, revealed that the implanted renal cells possessed filtration, reabsorption, and secretory capabilities. Histological examination of the retrieved implants revealed extensive vascularization and self-organization of the cells into glomeruli and tubule-like structures. A clear continuity between the glomeruli, the tubules, and the silastic catheter was noted that allowed the passage of urine into the collecting reservoir. These studies demonstrated that cells derived 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 genetically identical to those of the host. These studies were the first demonstration of the use of therapeutic cloning for regeneration of tissues in vivo. However, the size of this device 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.

The experiences with urethral, bladder, blood vessel, and tracheal replacement using tissue engineering provide encouragement 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 positive 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 connection with the nervous system important for the functionality 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 proven 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 complex three-dimensional structure, is the provision of adequate oxygen and the generation of new vasculature. It has been appreciated for some years that in metabolically active 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 intricate morphogenetic process that allows the formation of extensively 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 necessary 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 chemical sources of oxygen that can support the construct before and immediately after implantation, until the neovascularization 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 deliver 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 addition, the use of oxygen-carrying molecules such as perfluorocarbons 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 ischemic tissue over several days in vivo.131 We hope to develop such novel scaffold materials further to support the survival of large, complex organ constructs in the initial period after implantation.

Second, "prevascularization" strategies aim to generate neo-organs engineered with a preexisting channel structure 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 technology 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 manufacture scaffolds with preformed channels, potentially with cells incorporated, designed to promote neo-vascularization. For example, laser guided "writing" was used to pattern endothelial cells and promote their aggregation into tubular vessels.135 Similarly, ink-jet-based bioprinting of cells and biomaterials by thermal ink jet technology can provide remarkable control of the fine structure of engineered tissues, including the generation of intricate vessel networks.136 We have used layer-by-layer ink jet printing to produce three-dimensional constructs containing endothelial cells and showed that these develop functional microvascularization 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 Mathematical modeling of scaffolds designed to contain a preexisting 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 designed 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 bioengineered products have been placed on hold because of the costs involved with the specific technology. With a bioengineered product, costs are usually high because of the biological 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.

Regenerative medicine efforts are currently underway experimentally for virtually every type of tissue and organ within the human body. As regenerative medicine incorporates the fields of tissue engineering, cell biology, nuclear transfer, and materials science, personnel who have mastered the techniques of cell harvest, culture, expansion, transplantation, and polymer design are essential for the successful application of these technologies to extend human life. Various tissues are at different stages of development, with some already being used clinically, a few in preclinical trials, and some 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 the life-extending benefits of tissue replacement or repair.