Principles of Tissue Engineering

Engineering

The History and Scope of Tissue Engineering

Joseph Vacanti, Charles A. Vacanti

I. Introduction

II. Scientific Challenges

III. Cells

IV. Materials

V. General Scientific Issues

VI. Social Challenges

VII. References

I INTRODUCTION

The dream is as old as humankind. Injury, disease, and congenital malformation have always been part of the human experience. If only damaged bodies could be restored, life could go on for loved ones as though tragedy had not intervened. In recorded history, this possibility first was manifested through myth and magic, as in the Greek legend of Prometheus and eternal liver regeneration. Then legend produced miracles, as in the creation of Eve in Genesis or the miraculous transplantation of a limb by the saints Cosmos and Damien. With the introduction of the scientific method came new understanding of the natural world. The methodical unraveling of the secrets of biology was coupled with the scientific understanding of disease and trauma. Artificial or prosthetic materials for replacing limbs, teeth, and other tissues resulted in the partial restoration of lost function. Also, the concept of using one tissue as a replacement for another was developed. In the 16th century, Tagliacozzi of Bologna, Italy, reported in his work Decusorum Chirurgia per Insitionem a description of a nose replacement that he constructed from a forearm flap. With the 19th-century scientific understanding of the germ theory of disease and the introduction of sterile technique, modern surgery emerged. The advent of anesthesia by the mid-19th century enabled the rapid evolution of many surgical techniques. With patients anesthetized, innovative and courageous surgeons could save lives by examining and treating internal areas of the body: the thorax, the abdomen, the brain, and the heart. Initially the surgical techniques were primarily extirpative, for example, removal of tumors, bypass of the bowel in the case of intestinal obstruction, and repair of life-threatening injuries. Maintenance of life without regard to the crippling effects of tissue loss or the psychosocial impact of disfigurement, however, was not an acceptable end goal. Techniques that resulted in the restoration of function through structural replacement became integral to the advancement of human therapy.

Now whole fields of reconstructive surgery have emerged to improve the quality of life by replacing missing function through rebuilding body structures. In our current era, modern techniques of transplanting tissue and organs from one individual into another have been revolutionary and lifesaving. The molecular and cellular events of the immune response have been elucidated sufficiently to suppress the response in the clinical setting of transplantation and to produce prolonged graft survival and function in patients. In a sense, transplantation can be viewed as the most extreme form of reconstructive surgery, transferring tissue from one individual into another.

As with any successful undertaking, new problems have emerged. Techniques using implantable foreign body materials have produced dislodgment, infection at the foreign body/tissue interface, fracture, and migration over time. Techniques moving tissue from one position to another have produced biologic changes because of the abnormal interaction of the tissue at its new location. For example, diverting urine into the colon can produce fatal colon cancers 20–30 years later. Making esophageal tubes from the skin can result in skin tumors 30 years later. Using intestine for urinary tract replacement can result in severe scarring and obstruction over time.

Transplantation from one individual into another, although very successful, has severe constraints. The major problem is accessing enough tissue and organs for all of the patients who need them. Currently, 92,587 people are on transplant waiting lists in the United States, and many will die waiting for available organs. Also, problems with the immune system produce chronic rejection and destruction over time. Creating an imbalance of immune surveillance from immunosuppression can cause new tumor formation. The constraints have produced a need for new solutions to provide needed tissue.

It is within this context that the field of tissue engineering has emerged. In essence, new and functional living tissue is fabricated using living cells, which are usually associated, in one way or another, with a matrix or scaffolding to guide tissue development. New sources of cells, including many types of stem cells, have been identified in the past several years, igniting new interest in the field. In fact, the emergence of stem cell biology has led to a new term, regenerative medicine. Scaffolds can be natural, man-made, or a composite of both. Living cells can migrate into the implant after implantation or can be associated with the matrix in cell culture before implantation. Such cells can be isolated as fully differentiated cells of the tissue they are hoped to recreate, or they can be manipulated to produce the desired function when isolated from other tissues or stem cell sources. Conceptually, the application of this new discipline to human health care can be thought of as a refinement of previously defined principles of medicine. The physician has historically treated certain disease processes by supporting nutrition, minimizing hostile factors, and optimizing the environment so that the body can heal itself. In the field of tissue engineering, the same thing is accomplished on a cellular level. The harmful tissue is eliminated; the cells necessary for repair are then introduced in a configuration optimizing survival of the cells in an environment that will permit the body to heal itself. Tissue engineering offers an advantage over cell transplantation alone in that organized three-dimensional functional tissue is designed and developed. This chapter summarizes some of the challenges that must be resolved before tissue engineering can become part of the therapeutic armamentarium of physicians and surgeons. Broadly speaking, the challenges are scientific and social.

II SCIENTIFIC CHALLENGES

As a field, tissue engineering has been defined only since the mid-1980s. As in any new undertaking, its roots are firmly implanted in what went before. Any discussion of when the field began is inherently fuzzy. Much still needs to be learned and developed to provide a firm scientific basis for therapeutic application. To date, much of the progress in this field has been related to the development of model systems, which have suggested a variety of approaches. Also, certain principles of cell biology and tissue development have been delineated. The field can draw heavily on the explosion of new knowledge from several interrelated, well-established disciplines and in turn may promote the coalescence of relatively new, related fields to achieve their potential. The rate of new understanding of complex living systems has been explosive since the 1970s. Tissue engineering can draw on the knowledge gained in the fields of cell and stem cell biology, biochemistry, and molecular biology and apply it to the engineering of new tissues. Likewise, advances in materials science, chemical engineering, and bioengineering allow the rational application of engineering principles to living systems. Yet another branch of related knowledge is the area of human therapy as applied by surgeons and physicians. In addition, the fields of genetic engineering, cloning, and stem cell biology may ultimately develop hand in hand with the field of tissue engineering in the treatment of human disease, each discipline depending on developments in the others.

We are in the midst of a biologic renaissance. Interactions of the various scientific disciplines can elucidate not only the potential direction of each field of study, but also the right questions to address. The scientific challenge in tissue engineering lies both in understanding cells and their mass transfer requirements and the fabrication of materials to provide scaffolding and templates.

III CELLS

If we postulate that living cells are required to fabricate new tissue substitutes, much needs to be learned with regard to their behavior in two normal circumstances: normal development in morphogenesis and normal wound healing. In both of these circumstances, cells create or recreate functional structures using preprogrammed information and signaling. Some approaches to tissue engineering rely on guided regeneration of tissue using materials that serve as templates for ingrowth of host cells and tissue. Other approaches rely on cells that have been implanted as part of an engineered device. As we gain understanding of normal developmental and wound-healing gene programs and cell behavior, we can use them to our advantage in the rational design of living tissues.

Acquiring cells for creation of body structures is a major challenge, the solution of which continues to evolve. The ultimate goal in this regard — the large-scale fabrication of structures — may be to create large cell banks composed of universal cells that would be immunologically transparent to an individual. These universal cells could be differentiated cell types that could be accepted by an individual or could be stem cell reservoirs, which could respond to signals to differentiate into differing lineages for specific structural applications. Much is already known about stem cells and cell lineages in the bone marrow and blood. Studies suggest that progenitor cells for many differentiated tissues exist within the marrow and blood and may very well be ubiquitous. Our knowledge of the existence and behavior of such cells in various mesenchymal tissues (muscle, bone, and cartilage), endodermally derived tissues (intestine and liver), or ectodermally derived tissues (nerves, pancreas, and skin) expands on a daily basis. A new area of stem cell biology involving embryonic stem cells holds promise for tissue engineering. The calling to the scientific community is to understand the principles of stem and progenitor cell biology and then to apply that understanding to tissue engineering. The development of immunologically inert universal cells may come from advances in genetic manipulation as well as stem cell biology.

As intermediate steps, tissue can be harvested as allograft, autograft, or xenograft. The tissues can then be dissociated and placed into cell culture, where proliferation of cells can be initiated. After expansion to the appropriate cell number, the cells can then be transferred to templates, where further remodeling can occur. Which of these strategies are practical and possibly applicable in humans remains to be explored.

Large masses of cells for tissue engineering need to be kept alive, not only in vitro but also in vivo. The design of systems to accomplish this, including in vitro flow bioreactors and in vivo strategies for maintenance of cell mass, presents an enormous challenge, in which significant advances have been made. The fundamental biophysical constraint of mass transfer of living tissue needs to be understood and dealt with on an individual basis as we move toward human application.

IV MATERIALS

There are so many potential applications to tissue engineering that the overall scale of the undertaking is enormous. The field is ripe for expansion and requires training of a generation of materials scientists and chemical engineers.

The optimal chemical and physical configurations of new biomaterials as they interact with living cells to produce tissue-engineered constructs are under study by many research groups. These biomaterials can be permanent or biodegradable. They can be naturally occurring materials, synthetic materials, or hybrid materials. They need to be developed to be compatible with living systems or with living cells in vitro and in vivo. Their interface with the cells and the implant site must be clearly understood so that the interface can be optimized. Their design characteristics are major challenges for the field and should be considered at a molecular chemical level. Systems can be closed, semipermeable, or open. Each design should factor into the specific replacement therapy considered. Design of biomaterials can also incorporate the biologic signaling that the materials may offer. Examples include release of growth and differentiation factors, design of specific receptors and anchorage sites, and three-dimensional site specificity using computer-assisted design and manufacture techniques. New nanotechnologies have been incorporated to design systems of extreme precision. Combining computational models with nanofabrication can produce microfluidic circulations to nourish and oxygenate new tissues.

V GENERAL SCIENTIFIC ISSUES

As new scientific knowledge is gained, many conceptual issues need to be addressed. Related to mass transfer is the fundamental problem associated with nourishing tissue of large mass as opposed to tissue with a relatively high ratio of surface area to mass. Also, functional tissue equivalents necessitate the creation of composites containing different cell types. For example, all tubes in the body are laminated tubes composed of a vascularized mesodermal element, such as smooth muscle, cartilage, or fibrous tissue. The inner lining of the tube, however, is specific to the organ system. Urinary tubes have a stratified transitional epithelium. The trachea has a pseudostratified columnar epithelium. The esophagus has an epithelium that changes along the gradient from mouth to stomach. The intestine has an enormous, convoluted surface area of columnar epithelial cells that migrate from a crypt to the tip of the villus. The colonic epithelium is, again, different for the purposes of water absorption and storage.

Even the well-developed manufacture of tissue-engineered skin used only the cellular elements of the dermis for a long period of time. Attention is now focusing on creating new skin consisting of both the dermis and its associated fibroblasts as well as the epithelial layer, consisting of keratinocytes. Obviously, this is a significant advance. But for truly normal skin to be engineered, all of the cellular elements should be contained so that the specialized appendages can be generated as well. These simple composites will indeed prove to be quite complex and require intricate designs. Thicker structures with relatively high ratios of surface area to mass, such as liver, kidney, heart, breast, and the central nervous system, will offer engineering challenges.

Currently, studies for developing and designing materials in three-dimensional space are being developed utilizing both naturally occurring and synthetic molecules. The applications of computer-assisted design and manufacture techniques to the design of these matrices are critically important. Transformation of digital information obtained from magnetic resonance scanning or computerized tomography scanning can then be developed to provide appropriate templates. Some tissues can be designed as universal tissues that will be suitable for any individual, or they may be custom-developed tissues specific to one patient. An important area for future study is the entire field of neural regeneration, neural ingrowth, and neural function toward end organ tissues such as skeletal or smooth muscle. Putting aside the complex architectural structure of these tissues, the cells contained in them have a very high metabolic requirement. As such, it is exceedingly difficult to isolate a large number of viable cells. An alternate approach may be the use of less mature progenitor cells, or stem cells, which not only would have a higher rate of survival as a result of their lower metabolic demand but also would be more able to survive the insult and hypoxic environment of transplantation. As stem cells develop and require more oxygen, their differentiation may stimulate the development of a vascular complex to nourish them. The understanding of and solutions to these problems are fundamentally important to the success of any replacement tissue that needs ongoing neural interaction for maintenance and function.

It has been shown that some tissues can be driven to completion in vitro in bioreactors. However, optimal incubation times will vary from tissue to tissue. Even so, the new tissue will require an intact blood supply at the time of implantation for successful engraftment and function.

Finally, all of these characteristics need to be understood in the fourth dimension, time. If tissues are implanted in a growing individual, will the tissues grow at the same rate? Will cells taken from an older individual perform as young cells in their new optimal environment? How will the biochemical characteristics change over time after implantation? Can the strength of structural support tissues such as bone, cartilage, and ligaments be improved in a bioreactor in which force vectors can be applied? When is the optimal timing of this transformation? When does tissue strength take over the biochemical characteristics as the material degrades?

VI SOCIAL CHALLENGES

If tissue engineering is to play an important role in human therapy, in addition to scientific issues, fundamental issues that are economic, social, and ethical in nature will arise. Something as simple as a new vocabulary will need to be developed and uniformly applied. A universal problem is funding. Can philanthropic dollars be accessed for the purpose of potential new human therapies? Will industry recognize the potential for commercialization and invest heavily? If this occurs, will the focus be changed from that of a purely academic endeavor? What role will governmental agencies play as the field develops? How will the field be regulated to ensure its safety and efficacy prior to human application? Is the new tissue to be considered transplanted tissue and, therefore, not be subject to regulation, or is it a pharmaceutical that must be subjected to the closest scrutiny by regulatory agencies? If lifesaving, should the track be accelerated toward human trials?

There are legal ramifications of this emerging technology as new knowledge is gained. What becomes proprietary through patents? Who owns the cells that will be sourced to provide the living part of the tissue fabrication?

In summary, one can see from this brief overview that the challenges in the field of tissue engineering remain significant. All can be encouraged by the progress that has been made in the past few years, but much discovery lies ahead. Ultimate success will rely on the dedication, creativity, and enthusiasm of those who have chosen to work in this exciting but still unproved field. Quoting from the epilogue of the previous edition: At any given instant in time, humanity has never known so much about the physical world and will never again know so little.

VII. REFERENCES

Langer R., Vacanti J.P. Tissue engineering. Science. 1993;260:920-926.

p. 929Lanza R.P., Langer R., Vacanti J.P. Principles of Tissue Engineering. 2nd ed. 2000.

Nerem R.M. Tissue engineering: the hope, the hype and the future. Tissue Eng.. 2006;12:1143-1150.

Vacanti C.A. History of tissue engineering and a glimpse into its future. Tissue Eng.. 2006;12:1137-1142.

Vacanti J.P., Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet. 1999;354:S132-S134.

The Challenge of Imitating Nature

Robert M. Nerem

I. Introduction

II. Cell Technology

III. Construct Technology

IV. Integration into the Living System

V. Concluding Discussion

VI. Acknowledgments

VII. References

I INTRODUCTION

Tissue engineering, through the imitation of nature, has the potential to confront the transplantation crisis caused by the shortage of donor tissues and organs and also to address other important, but yet unmet, patient needs. If we are to be successful in this, a number of challenges need to be faced. In the area of cell technology, these include cell sourcing, the manipulation of cell function, and the effective use of stem cell technology. Next are those issues that are part of what is called here construct technology. These include the design and engineering of tissuelike constructs and/or delivery vehicles and the manufacturing technology required to provide off-the-shelf availability to the clinician. Finally, there are those issues associated with the integration of cells or a construct into the living system, where the most critical issue may be the engineering of immune acceptance. Only if we can meet the challenges presented by these issues and only if we can ultimately address the tissue engineering of the most vital of organs will it be possible to achieve success in confronting the crisis in transplantation.

An underlying premise of this is that the utilization of the natural biology of the system will allow for greater success in developing therapeutic strategies aimed at the replacement, maintenance, and/or repair of tissue and organ function. Another way of saying this is that, just maybe, the great creator, in whatever form one believes he or she exists, knows something that we mere mortals do not, and if we can only tap into a small part of this knowledge base, if we can only imitate nature in some small way, then we will be able to achieve greater success in our efforts to address patient needs in this area. It is this challenge of imitating nature that has been accepted by those who are providing leadership to this new area of technology called tissue engineering (Langer and Vacanti, 1993; Nerem and Sambanis, 1995). To imitate nature requires that we first understand the basic biology of the tissues and organs of interest, including developmental biology; with this we then can develop methods for the control of these biologic processes; and based on the ability to control, we finally can develop strategies either for the engineering of living tissue substitutes or for the fostering of tissue repair or regeneration.

The initial successes have been for the most part substitutes for skin, a relatively simple tissue, at least by comparison with most other targets of opportunity. In the long term, however, tissue engineering has the potential for creating vital organs, such as the kidney, the liver, and the pancreas. Some even believe it will be possible to tissue engineer an entire heart. In addressing the repair, replacement, and/or regeneration of such vital organs, tissue engineering has the potential literally to confront the transplantation crisis, i.e., the shortage of donor tissues and organs available for transplantation. It also has the potential to develop strategies for the regeneration of nerves, another important and unmet patient need.

Although research in what we now call tissue engineering started more than a quarter of a century ago, the term tissue engineering was not coined until 1987, when Professor Y. C. Fung, from the University of California, San Diego, suggested this name at a National Science Foundation meeting. This led to the first meeting called tissue engineering, held in early 1988 at Lake Tahoe, California (Skalak and Fox, 1998). More recently the term regenerative medicine has come into use. For some this is a code word for stem cell technology, while for others regenerative medicine is the broader term, with tissue engineering representing only replacement, not repair or regeneration. Still others use the terms tissue engineering and regenerative medicine interchangeably. What is important is that it is a more biologic approach that has the potential to lead to new patient therapies and treatments, where in some cases none is currently available.

It should be noted that the concept of a more biologic approach dates back to 1938 (Carrel and Lindbergh, 1938). Since then there has been a large expansion in research efforts in this field and a considerable recognition of the enormous potential that exists. With this hope, there also has been a lot of hype; however, the future long term remains bright (Nerem, 2006). As the technology has become further developed, an industry has begun to emerge. This industry is still very much a fledgling one, with only a few companies possessing product income streams (Ahsan and Nerem, 2005). A study based on 2002 data documents a total of 89 companies active in the field, with $500 million annually in industrial research and development taking place (Lysaght and Hazlehurst, 2004). Although this study will soon be updated, based on the 2002 data, 80% of the new firms were in the stem cell area and 40% were located outside of the United States.

Tissue engineering is literally at the interface of the traditional medical implant industry and the biological revolution (Galletti et al., 1995). By harnessing the advances of this revolution, we can create an entirely new generation of tissue and organ implants as well as strategies for repair and regeneration. Already we are seeing increased investments in this field by the large medical device companies. A part of this is a convergence of biologics and devices, which is recognized by the medical implant industry. It is from this that the short-term successes in tissue engineering will come; however, long term it is the potential for a literal revolution in medicine and in the medical device/implant industry that must be realized.

This revolution will only occur, however, if we successfully meet the challenge of imitating nature. Thus, in the remainder of this chapter the critical issues involved in this are addressed. This is done by first discussing those issues associated with cell technology, i.e., issues important in cell sourcing and in the achievement of the functional characteristics required of the cells to be employed. Next to be discussed are those issues associated with construct technology. These include the organization of cells into a threedimensional architecture that functionally mimics tissue, the development of vehicles for the delivery of genes, cells, and proteins, and the technologies required to manufacture such products and provide them off the shelf to the clinician. Finally, issues involved in the integration of a living cell construct into, or the fostering of remodeling within, the living system is discussed. These range from the use of appropriate animal models to the issues of biocompatibility and immune acceptance. Success in tissue engineering will only be achieved if issues at these three different levels, i.e., cell technology, construct technology, and the technology for integration into the living system, can be addressed.

II CELL TECHNOLOGY

The starting point for any attempt to engineer a tissue or organ substitute is a consideration of the cells to be employed. Not only will one need to have a supply of sufficient quantity and one that can be ensured to be free of pathogens and contamination of any type whatsoever, but one will need to decide whether the source to be employed is to be autologous, allogeneic, or xenogeneic. As indicated in Table 2.1, each of these has both advantages and disadvantages; however, it should be noted that one important consideration for any product or treatment strategy is its off-the-shelf availability. This is obviously required for surgeries that must be carried out on short notice. However, even when the time for surgery is elective, it is only with off-the-shelf availability that the product and strategy will be used for the wide variety of patients who are in need and who are being treated throughout the entire health care system, including those in community hospitals.

Table 2.1 Cell source

With regard to the use of autologous cells, there are a number of potential sources. These include both differenti ated cells and adult stem/progenitor cells. It is only, however, if we can recruit the host’s own cells, e.g., to an acellular implant, that we can have off-the-shelf availability, and it is only by moving to off-the-shelf availability for the clinician that routine use becomes possible.

The skin substitutes developed by Organogenesis (Canton, MA) and Advanced Tissue Sciences (La Jolla, CA) represented the first living-cell, tissue-engineered products, and these in fact use allogeneic cells. The Organogenesis product, Apligraf™, is a bilayer model of skin involving fibroblasts and keratinocytes that are obtained from donated human foreskin (Parenteau, 1999). Apligraf™ is approved by the Food and Drug Administration (FDA); however, the first tissue-engineered products approved by the FDA were acellular. These included Integra™, based on a polymeric template approach (Yannas et al., 1982), and the Advanced Tissue Sciences product, TransCyte™. Approved initially for third-degree burns, TransCyte™ is made by seeding dermal fibroblasts in a polymeric scaffold; however, once cryopreserved it becomes a nonliving wound covering. Advanced Tissue Sciences also has a living-cell product, called Dermagraft™. It is a dermis model, also with dermal fibroblasts obtained from donated human foreskin (Naughton, 1999). Even though the cells employed by both Organogenesis and Advanced Tissue Sciences are allogeneic, immune acceptance did not have to be engineered because both the fibroblast and the keratinocyte do not constitutively express major histocompatibility complex (MHC) II antigens.

The next generation of tissue-engineered products will involve other cell types, and the immune acceptance of allogeneic cells will be a critical issue in many cases. As an example, consider a blood vessel substitute that employs both endothelial cells and smooth muscle cells. Although there is some unpublished data that suggest allogeneic smooth muscle cells may be immune acceptable, allogeneic endothelial cells certainly would not be. Thus, for the latter, one either uses autologous cells or else engineers the immune acceptance of allogeneic cells, as is discussed in a later section. Undoubtedly the first human clinical trials will be done using autologous endothelial cells; however, it appears that the use of such cells would severely limit the availability of a blood vessel substitute, unless the host’s own endothelial cells are recruited.

Once one has selected the cell type(s) to be employed, then the next issue relates to the manipulation of the functional characteristics of a cell so as to achieve the behavior desired. This can be done either by (1) manipulating a cell’s microenvironment, e.g., its matrix, the mechanical stresses to which it is exposed, or its biochemical environment, or by (2) manipulating a cell’s genetic program. With regard to the latter, the manipulation of a cell’s genetic program could be used as an ally to tissue engineering in a variety of ways. A partial list of possibilities includes the alteration of matrix synthesis; inhibition of the immune response; enhancement of nonthrombogenicity, e.g., through increased synthesis of antithrombotic agents; engineering the secretion of specific biologically active molecules, e.g., a specific insulin secretion rate in response to a specific glucose concentration; and the alteration of cell proliferation.

Much of the foregoing is in the context of creating a cell-seeded construct that can be implanted as a tissue or organ substitute; however, the fostering of the repair or remodeling of tissue also represents tissue engineering as defined here. Here a critical issue is how to deliver the necessary biologic cues in a spatially and temporally controlled fashion so as to achieve a healing environment. In the repair and/or regeneration of tissue, the use of genetic engineering might take a form that is more what we would call gene therapy. An example of this would be the introduction of growth factors to foster the repair of bone defects. In using a gene therapy approach to tissue engineering it should be recognized that in many cases only a transient expression will be required. Because of this, the use of gene therapy as a strategy in tissue engineering may become viable prior to its employment in treating genetically related diseases.

Returning to the issue of cell selection, there is considerable interest in the use of stem cells as a primary source for cell-based therapies, ones ranging from replacement to repair and/or regeneration. This interest includes both adult stem cells and progenitor cells as well as embryonic stem cells (Ahsan and Nerem, in press; Vats et al., 2005). With regard to the latter, the excitement about stem cells reached a new height in the late 1990s with articles reporting the isolation of the first lines of human embryonic stem cells (Thomson et al., 1998; Solter and Gearhart, 1999; Vogel, 1999). Since then considerable progress has been made; however, the hype continues to outpace the progress. This reached an unfortunate crescendo in the latter part of 2005 with the revelation that the major advances reported by the Korean scientist Woo Suk Hwang were based on the fabrication of results (Normile and Vogel, 2005; Normile et al., 2005, 2006). This was compounded by ethical issues and by the inclusion of Dr. Gerald Schatten from the University of Pittsburgh as a senior author (Guterman, 2006). Korea must be credited with launching a full investigation that led to Dr. Hwang’s losing his position. The University of Pittsburgh also conducted an investigation and found Dr. Schatten guilty of research misbehavior, a term not fully understood by the scientific community (Holden, 2006). The unfortunate thing is that this all happened at a time of considerable ethical and political controversy surrounding human embryonic stem cell research. From this we must all learn (Cho et al., 2006), and, in spite of this setback in the public arena, research in the human embryonic stem cell area continues to hold considerable promise for the future.

There is in fact a variety of different stem cells, and several comprehensive reviews of a general nature have recently appeared (Vats et al., 2005; Ahsan and Nerem, in press). It is the adult stem cells and progenitor cells that are being and will be used first clinically; however, long term there is considerable interest in embryonic stem cells. These cells are pluripotent, i.e., capable of differentiating into many cell types, even totipotent, i.e., capable of developing into all cell types. Although we are quite a long way from being able to use embryonic stem cells, a number of companies are working with stem cells in the context of tissue engineering and regenerative medicine. It needs to be recognized, however, that immunogenicity issues may be associated with the use of embryonic stem cells. Furthermore, different embryonic stem cell lines, even when in a totally undifferentiated state, can be significantly different. This is illustrated by the results of Rao et al. (2004) in a comparison of the transcriptional profile of two different embryonic stem cell lines. This difference should not be considered surprising, since the lines were derived from different embryos and undoubtedly cultured under different conditions.

To take full advantage of stem cell technology, however, it will be necessary to understand more fully how a stem cell differentiates into a tissue-specific cell. This requires knowledge not just about the molecular pathways of differentiation, but, even more importantly, about the identification of the combination of signals leading to a stem cell’s becoming a specific type of differentiated tissue cell. As an example, with the recognized differences between large-vessel endothelial cells and valvular endothelial cells (Butcher et al., 2004), what are the signals that will drive the differentiation toward one type of endothelial cell versus the other? Only with this type of knowledge will we be able to realize the full potential of stem cells. In addition, however, we will need to develop the technologies necessary to expand a cell population to the number necessary for clinical application, to do this in a controlled, reproducible manner, and to deliver cells at the right place and at the time required.

III CONSTRUCT TECHNOLOGY

With the selection of a source of cells, the next challenge in imitating nature is to develop an organized threedimensional architecture (with functional characteristics such that a specific tissue is mimicked) and/or a delivery vehicle for the cells. In this it is important to recognize the importance of a cell’s microenvironment in determining its function. In vivo a cell’s function is orchestrated by a symphony of signals. This symphony includes soluble molecules, the mechanical environment, i.e., physical forces, to which the cell is exposed, and the extracellular matrix. These are all part of the symphony. And if we want the end result to replicate the characteristics of native tissue, attention must be given to each of these components of a cell’s microenvironment.

The design and engineering of a tissuelike substitute are challenges in their own right. If the approach is to seed cells into a scaffold, then a basic issue is the type of scaffold that will allow the cells to make their own matrix. There are, of course, many possible approaches. One of these is a cell-seeded polymeric scaffold, an approach pioneered by Langer and his collaborators (Langer and Vacanti, 1993; Cima et al., 1991). This is the technology that was used by Advanced Tissue Sciences, and many consider this the classic tissue-engineering approach. There are other approaches, however, with one of these being a cell-seeded collagen gel. This approach was pioneered by Bell in the late 1970s and early 1980s (Bell et al., 1979; Weinberg and Bell, 1986), and this is used by Organogenesis in their skin substitute, Apligraf™.

A rather intriguing approach is that of Auger and his group in Quebec, Canada (Auger et al., 1995; Heureux et al., 1998). Auger refers to this as cell self-assembly, and it involves a layer of cells secreting their own matrix, which over a period of time becomes a sheet. Originally developed as part of the research on skin substitutes by Auger’s group, it has been extended to other applications. For example, the blood vessel substitute developed in Quebec involves rolling up one of these cell self-assembled sheets into a tube. One can in fact make tubes of multiple layers so as to mimic the architecture of a normal blood vessel.

An equally intriguing approach is that pioneered by the Campbells in Australia and their collaborators (Chue et al., 2004). In this they literally use the peritoneal cavity as an in vivo bioreactor to grow a blood vessel substitute. The concept is that a free-floating body in the peritoneal cavity initiates an inflammatory response and becomes encapsulated with cells. This is an autologous-cell approach, and it is also one where the cells make their own matrix.

Any discussion of different approaches to the creation of a three-dimensional, functional tissue equivalent would be remiss if acellular approaches were not included. Although in tissue engineering the end result should include functional cells, there are those who are employing a strategy whereby the implant is without cells, i.e., acellular, and the cells are then recruited from the recipient or host. A number of laboratories and companies are developing this approach. Examples include the products Integra™ and TransCyte™, already noted, and the development of SIS, i.e., small intestine submucosa (Badylak et al., 1999; Lindberg and Badylak, 2001). One result of this approach, in effect, is to bypass the cell-sourcing issue and replace this with the issue of cell recruitment, i.e., the recruiting of cells from the host in order to populate the construct. Because these are the patient’s own cells, there is no need for any engineering of immune acceptance.

Whatever is done, an objective in imitating nature must be to create a healing environment, one that will foster remodeling and ultimately repair. To do this requires delivering the appropriate, necessary cues in a controlled spatial and temporal fashion. This is needed whether the goal is replacement or repair or regeneration. Whatever the approach, the engineering of an architecture and of func tional characteristics that allow one to mimic a specific tissue is critical to achieving any success and to meeting the challenge of imitating nature. In fact, because of the interrelationship of structure and function in cells and tissues, it would be unlikely to have the appropriate functional characteristics without the appropriate three-dimensional architecture. Thus, many of the chapters in this book describe in some detail the approach being taken in the design and engineering of constructs for specific tissues and organs, and any further discussion of this is left to those chapters.

The challenge of imitating nature, however, does not stop with the design and engineering of a specific tissuelike substitute or a delivery vehicle. This is because the patient need that exists cannot be met by making one construct at a time on a benchtop in some research laboratory. Accepting the challenge of imitating nature must include the development of cost-effective manufacturing processes. These must allow for a scale-up from making one at a time to a production quantity of 100 or 1000 per week. Anything significantly less would not be cost effective; and if a product cannot be manufactured in large quantities and cost effectively, then it will not be widely available for routine use.

Much of the research on manufacturing technology has focused on bioreactor technology. A bioreactor simply represents a controlled environment — both chemically and mechanically — in which a tissuelike construct can be grown (Freed et al., 1993; Neitzel et al., 1998; Saini and Wick, 2003). The design of a bioreactor involves a number of critical issues. The list starts with the configuration of the bioreactor, its mass transport characteristics, and its scaleability. Then, if it is to be used in a manufacturing process, it is desirable to minimize the number of asceptic operations while maximizing automation. Reliability and reproducibility obviously will be critical, and it needs to be user friendly.

Although it is generally recognized that a construct, once implanted in the living system, will undergo remodeling, it is equally true that the environment of a bioreactor can be tailored to induce the in vitro remodeling of a construct so as to enhance characteristics critical to the success to be achieved when it is implanted (Seliktar et al., 1998). Thus, the manufacturing process can be used to influence directly the final product and is part of the overall process leading to the imitation of nature. An important issue in developing a substitute for replacement, however, is how much of the maturation of a substitute is done in vitro in a bioreactor as compared to what is done in vivo through the remodeling that takes place within the body itself, i.e., in the body’s own bioreactor environment. As pointed out by Dr. Frederick Schoen (private communication), in this one needs to recognize that the rate at which remodeling in vivo takes place will be extremely different from individual to individual. It is equally true that the extent of remodeling also will be different. Thus, the degree of maturation that occurs in vivo will be highly variable, depending on the host response.

Once a product is manufactured, a critical issue will be how it is delivered and made available to the clinician. The Organogenesis product, Apligraf™, is delivered fresh and originally had a 5-day shelf life at room temperature (Parenteau, 1999), although recently this has been extended. On the other hand, Dermagraft™, the skin substitute developed by Advanced Tissue Sciences, is cryopreserved and shipped and stored at −70°C (Naughton, 1999). This provides for a much more extended shelf life but introduces other issues that one must address. Ultimately, the clinician will want off-the-shelf availability, and one way or another this will need to be provided if a tissue-engineered product or strategy is to have wide use. Although cryobiology is a relatively old field and most cell types can be cryopreserved, there is much that still needs to be learned if we are successfully to cryopreserve three-dimensional tissue-engineered products.

IV INTEGRATION INTO THE LIVING SYSTEM

The final challenge to imitating nature is presented by moving a tissue-engineering concept into the living system. Here one starts with animal experiments, and there is a lack of good animal models for use in the evaluation of a tissue-engineered implant or strategy. This is despite the fact that a variety of animal models have been developed for the study of different diseases. Unfortunately, these models are still somewhat unproved, at least in many cases, when it comes to their use in evaluating the success of a tissue-engineering concept.

In addition, there is a significant need for the development of methods to evaluate quantitatively the performance of an implant, and a number of concepts are being advanced (Guldberg et al., 2003; Stabler et al., 2005). This is not only the case for animal studies, but is equally true for human clinical trials. With regard to the latter, it may not be enough to show efficacy and long-term patency; it may also be necessary to demonstrate the mechanism(s) that lead to the success of the strategy. Furthermore, it is not just clinical trials that have a need for more quantitative tools for assessment; it also would be desirable to have available the technologies necessary to assess periodically the continued viability and functionality of a tissue substitute or strategy after implantation into a patient.

Also, one cannot state that one has successfully met the challenge of imitating nature unless the implanted construct is biocompatible. Even if the implant is immune acceptable, there can still be an inflammatory response. This response can be considered separate from the immune response, although obviously interactions between these two might occur. In addition to any inflammatory response, for some types of tissue-engineered substitutes thrombosis will be an issue. This is certainly an important part of the biocompatibility of a blood vessel substitute.

Finally, important to the success of any tissueengineering approach is the immune response and that it be immune acceptable. This comes naturally with the use of autologous cells; however, if one moves to nonautologous cell systems (as this author believes we must, at least in many cases, if we are to make the products of tissue engineering widely available for routine use), then the challenge of engineering immune acceptance is critical to our achieving success in the imitation of nature. Today we have immunosuppressive drugs, e.g., cyclosporine; however, transplant patients treated this way face a lifetime where their entire immune system is affected, placing them at risk of infection and other problems.

It should be recognized that the issues surrounding the immune acceptance of an allogeneic cell-seeded implant are no different than those associated with a transplanted human tissue or organ. Both represent allogeneic cell transplantation, and this means that much of what is being learned in the field of transplant immunology can help us understand implant immunology and the engineering of immune acceptance for tissue-engineered substitutes. For example, it is now known that to have immune rejection there must not only be a recognition by the host of a foreign body, but there also must be present what is called the costimulatory signal, or sometimes simply signal 2. It has been demonstrated that, with donated allogeneic tissue, if one can block the costimulatory signal, one can extend survival of the transplant considerably (Larsen et al., 1996). There also is the chimeric approach, where one transplants into the patient from the donor both the specific tissue/organ and bone marrow. This suggests that perhaps in the future one will be able to use a stem cell-based chimeric approach. As an example, if one were to differentiate an embryonic stem cell both into the tissue-specific cells needed and into the cells required for implantation into the bone marrow, then from a single cell source one would create the chimerism desired.

Another approach is that of therapeutic cloning. Here a patient’s DNA is transferred into an embryonic stem cell, which in turn is differentiated into the cells needed for a particular tissue-engineering approach. As attractive as this approach appears, many think it is unrealistic, simply because of the scarcity of eggs and embryonic stem cells. Furthermore, as our knowledge of immunology continues to advance, other approaches might make the need for therapeutic cloning disappear (Brown, 2006). Thus, strategies are under development, and these may provide greater opportunities in the future for the use of allogeneic cells.

V CONCLUDING DISCUSSION

If we are to meet the challenge of imitating nature, there are a variety of issues. These have been divided here into three different categories. The issue of cell technology includes cell sourcing, the manipulation of cell function, and the use of stem cell technology. Construct technology includes the engineering of a tissuelike construct as a substitute or delivery vehicle and the manufacturing technology required to provide the product and ensure its off-the-shelf availability. Finally is the issue of integration into living systems. This has several important facets, with the most critical one being the engineering of immune acceptance.

Much of the discussion here has focused on the challenge of engineering tissuelike constructs for implantation. As noted earlier, however, equally important to tissue engineering are strategies for the fostering of remodeling and ultimately the repair and enhancement of function. As the field moves to the more complex biological tissues, e.g., ones that require innervation and vascularization, it may well be that a strategy of repair and/or regeneration is preferable to one of replacement.

As one example, consider a damaged, failing heart. Should the approach be to tissue engineer an entire heart, or should the strategy be to foster the repair of the myocardium? In this latter case, it may be possible to return the heart to relatively normal function through the implantation of a myocardial patch or even through the introduction of growth factors, angiogenic factors, or other biologically active molecules. Which strategy has the highest potential for success? Which approach will have the greatest public acceptance?

Even though short-term successes in tissue engineering may come from the convergence of biologics and devices, long term it is the generation of totally biologic products and strategies that must be envisioned. These will result in advances that include, for example, the following: in vitro models for the study of basic biology and for use in drug discovery; blood cells derived from stem cells and expanded in vitro, thus reducing the need for blood donors; an insulin-secreting, glucose-responsive bioartificial pancreas; and heart valves that when implanted into an infant grow as the child grows. In addition, the repair/regeneration of the central nervous system will become a reality. Furthermore, as one thinks about the future, medicine will move to being more predictive, more personalized, and, where possible, more preventive. It is entirely possible that we will be able to diagnose disease at a preclinical stage. In that event, the concept of inducing biological repair prior to the appearance of the clinical manifestations of disease becomes even more attractive.

Thus, the strategy being evolved in Atlanta, Georgia, by the Georgia Tech/Emory Center for the Engineering of Living Tissues, an engineering research center funded by the National Science Foundation, is one that more and more is placing the emphasis on repair and/or regeneration. It is moving beyond replacement that may provide the best opportunity to meet the challenge of imitating nature. Fundamental to this is understanding the basic biology, including developmental biology, even though the biological mechanisms involved in adult tissue repair/regeneration are far different from those involved in fetal development. Furthermore, to translate a basic biological understanding into a technology that reaches the patient bedside will require a multidisciplinary, even an interdisciplinary, effort, one involving life scientists, engineers, and clinicians. Only with such teams will we be able to meet the challenge of imitating nature, and only then can the existing patient need be addressed and will we as a community be able to confront the transplantation crisis.

VI ACKNOWLEDGMENTS

The author acknowledges with thanks the support by the National Science Foundation of the Georgia Tech/Emory Center for the Engineering of Living Tissues and the many discussions with GTEC’s faculty and student colleagues and with the representatives of the center’s industrial partners.

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Moving into the Clinic

Alan J. Russell, Trimothy Bertram

I. Introduction

II. History of Clinical Tissue Engineering

III. Strategies to Advance Toward the Clinic

IV. Bringing Technology Platforms to the Clinical Setting

V. Transition to Clinical Testing

VI. Establishing a Regulatory Pathway

VII. Conclusions

VIII. Acknowledgments

IX. References

I INTRODUCTION

In the early 1930s Charles Lindbergh, who was better known for his aerial activities, went to Rockefeller University and began to study the culture of organs. After the publication of his book about the culturing of organs ex vivo in order to repair or replace damaged or diseased organs, the field lay dormant for many years. Indeed, delivering respite to failing organs with devices or total replacement (transplant) became far more fashionable. Transplantation medicine has been a dramatic success. But in the late 1980s scientists, engineers, and clinicians began to conceptualize how de novo tissue generation might be used to address the tragic shortage of donated organs. The approach they proposed was as simple as it was dramatic. Biodegradable materials would be seeded with cells and cultured outside the body for a period of time before exchanging this artificial bioreactor for a natural bioreactor by implanting the seeded material into a patient. These early pioneers believed that the cells would degrade the material, and after implantation the cell-material construct would become a vascularized native tissue. Tissue engineering, as this approach came to be known, can be accomplished once we understand which materials and cells to use, how to culture these together ex vivo, and how to integrate the resulting construct into the body.

Most major medical advances take decades to progress from the laboratory to broad clinical implementation. Tissue engineering was such a compelling concept that the process moved much faster. As we discuss later, the speed at which tissue-engineering solutions can be implemented is inherently faster than traditional drug development strategies. For this reason, coupled with what was probably unexplained exuberance, business investors saw an immediate role for industry in delivering tissue-engineered products to patients. Traditionally, new fields are seeded with foundational research, development, and engineering prior to implementation, but an apparent alignment of interests caused many to believe that companies could deliver products immediately and that the traditional foundational aspects could wait.

The race to clinical implementation of a tissue-engineered medical product began with the incorporation of Advanced Tissue Science (ATS) in 1987. ATS and Organogenesis, an early competitor, began their quest by focusing on seeding biodegradable matrices with human foreskin fibroblasts. In the early days, Organogenesis, Integra, and Ortec focused on bovine-derived scaffolds, while ATS focused on human-derived scaffolds. Other companies focused on developing tissue-engineering products using scaffold alone or cells alone. The path to implementation has been very different for each class of company, as is summarized later.

With hindsight, one might say that the choice of living-skin equivalents as a first commercial product was probably driven by the willingness of the FDA to regulate them as Class III devices rather than biologics. This attractive feature of the products was supplemented by large predicted market sizes and the ease of culturing skin cells. As these products moved from the laboratory to the clinic, development issues such as which cells, materials, and bioreactors were supplanted with industrial challenges such as scale-up and immunocompatibility.

At the same time that ATS and Organogenesis were rapidly growing, the view emerged that delivery of tissue-engineered products to patients would require an allogeneic off-the-shelf solution having ease of use and long storage life in the United States — and persists today. This business-driven decision predicated development of allogeneic, cell-based therapies. Interestingly, in a study sponsored by the National Science Foundation, the World Technology Evaluation Center discovered that non-U.S. investors were focused on autologous cell therapies resulting from a belief that allogeneic therapy would be unsuccessful because of the need to suppress the patient’s immune system. This difference in emphasis between U.S. and non-U.S. investors continues today. Both approaches have genuine advantages and disadvantages. However, once it became clear that cell-seeded scaffolds could trigger dramatic changes in natural wound healing, thereby inducing de novo tissue formation and function, clinical implementation through industry progressed from skin to a wide array of tissues. In addition, pockets of excellence arose at major medical centers, where new innovations were tested clinically in relatively small numbers of patients.

So one is left with several questions: What have we learned from these early adoptions of tissue engineering? How can these lessons drive sustainable innovation that will both heal and generate a return on investment? Is broad clinical implementation of tissue engineering limited by the nature and structure of regulatory bodies? This chapter seeks to answer these questions by looking historically at selected high-profile clinical tissue-engineering programs and looking forward with a suggested generic approach to rapid clinical translation in this new era of advanced medical therapies.

II HISTORY OF CLINICAL TISSUE ENGINEERING

What Is Clinical Tissue Engineering?

As mentioned earlier, in the early 1990s the term tissue engineering was generally used to describe the combination of biomaterials and cells ex vivo to provide benefit once implanted in vivo. What emerged over the next decade, however, were biomaterials designed to alter the natural wound-healing response and cell-only therapies. Lessons learned in the development of each led to the fusion of these tools under the