Christine E. Schmidt is assistant professor of chemical and biomedical engineering

Tissue Engineering Every year surgeons perform more than eight million surgical procedures in the United States to treat the millions of Americans who experience organ failure or tissue loss. Physicians treat these patients by transplanting organs from one individual to another, performing reconstructive surgery, or by using mechanical devices such as kidney dialyzers, prosthetic hip joints, or mechanical heart valves. Although these approaches have saved many lives, they are imperfect. The transplantation of organs such as the heart, liver, and kidney is so common that the limitation is not the surgical technique, but the declining availability of donor organs. For example, only about 3,000 donor livers are available for the roughly 30,000 people who may die from liver failure each year. For surgical reconstruction, tissue may be moved from one part of the patient to another part. These autografts (tissue grafts from the patient) include skin grafts for burns, blood vessel grafts for heart bypass surgeries, and nerve grafts for facial and hand reconstruction. The disadvantages of using autografts include the need for multiple surgeries and loss of function at the donor site. In addition, surgical reconstruction often involves using the body's tissues for purposes not originally intended and can result in long-term complications. Using a segment of the intestine to replace a diseased esophagus, for instance, is common and typically sufficient. In some cases, however, ulcers develop on the intestine surface due to its inability to adapt to its new environment. Mechanical devices such as kidney dialysis machines cannot perform all of the functions of a single organ and, therefore, do not completely prevent further patient deterioration. For other devices such as prosthetic hip joints or mechanical heart valves, the prosthesis is subject to mechanical failure, and long-term drug intervention is required to prevent blood-clotting. As a result of these drawbacks to current therapies, what is emerging as a new discipline is the science of tissue engineering. Its goals are to create tissues in culture for use not only as model systems in fundamental studies, but more importantly, for use as replacement tissues for damaged or diseased body parts. Although efforts to generate bioartificial tissues and organs for human therapies go back at least thirty years, such efforts have come closer to clinical success only in the last ten years. This has been made possible by major advances in molecular and cell biology, cell culture technologies, and materials science. The term "tissue engineering" is relatively recent and has been used more widely in the last five years to describe the interdisciplinary field that applies the principles of engineering and the life sciences toward the development of bioartificial tissues and organs. One of the major strategies adopted for the creation of new tissues is the growth of isolated cells on three-dimensional templates or scaffolds (matrices) under conditions that will coax the cells to develop into a functional tissue (Figure 1). When implanted, this bioartificial tissue should become structurally and functionally integrated into the body. The matrices can be fashioned from natural materials such as collagen or from synthetic polymers such as plastics. Ultimately, the scaffold material should be biodegradable over time and should simply serve as an initial three-dimensional template for tissue growth. As the cells grow and differentiate on the scaffold, they will produce various proteins needed to recreate a tissue. Degradation of the scaffold ensures that only natural tissue remains in the body. Cells from the patient (autologous cells) can usually be obtained from a small tissue biopsy, and these cells are desired as they avoid an immune response in the patient. The use of autologous cells is not always feasible, however, so cells from other sources can be used in combination with immunosuppressive drugs. Investigations are also underway to breed animals whose tissues would be immunologically accepted in humans. This raises ethical issues about breeding animals so that their organs can be harvested for humans and the possibility, although remote, of transmitting a virus from the animal population to humans. As an alternative, genetic engineering approaches are being explored to produce universal donor cells that would not be rejected by the body and that would not pose health risks. The major challenge in tissue engineering has been accurately imitating nature. To do this, scientists must understand: (1) the biology of the organ to be replaced; (2) the spatial organization of the organ; (3) the biology and physical limitations of the tissues surrounding the organ; (4) the limitations of in vitro (laboratory) culture techniques; (5) the chemistry of creating an appropriate scaffold; (6) the physical and biochemical interactions of isolated cells with the scaffold material; and (7) the required mechanical properties of the scaffold. Scientists involved with tissue engineering efforts must interact with many other scientists (biologists, biomechanical engineers, physicians, and polymer chemists) to accomplish the seemingly impossible feat of recreating what only nature has accomplished. Chemical engineers play an increasingly important role in tissue engineering and the development of cell transplant devices, as mass transport and materials synthesis-key elements of chemical engineering curricula-are critical issues in tissue engineering and device design (Figure 2). This article reviews some of the progress in the area of tissue engineering as a whole and reviews in more detail some of the activities in our research laboratories at The University of Texas at Austin. Skin Tissue Engineering. Researchers have attempted to engineer virtually every mammalian tissue, including skin, pancreas, liver, blood vessels, nerve, cartilage, tendon, bone, breast tissue, cornea, intestine, and heart valves. Some tissue engineering efforts are clinically successful, but most are at various stages of research and development (from conceptual ideas to early clinical testing of efficacy). To date, tissue engineered skin has been the most promising clinically. Approximately 10,000 people die each year in the United States because of burns. Patients with severe burns have lost their dermis, a layer about two millimeters thick that lies beneath the thin epidermis. Skin is usually removed from another portion of the patient to cover the injured area. For larger burns, in which a large enough donor piece of skin may not exist, the skin graft may be slit so it can be stretched, with scarring occurring at each slit. In other cases, a temporary covering is used to protect the damaged area while enough healthy skin grows to permit subsequent harvests. Skin from a corpse has been the traditional choice for this temporary protective covering, but because of the body's immune response, the covering has to be removed and replaced frequently. During this time, disfiguring scar tissue forms on the injured tissue, permanently restricting movement of the damaged area. These less than ideal approaches will soon be a thing of the past, however. Several types of bioartificial skin are either in development or available for patient use. In one case, the skin substitute consists of an upper layer of silicone (to prevent fluid loss) and a lower layer made from natural proteins from the body (collagen and chondroitin sulfate). The protein layer slowly induces new blood vessel and tissue growth while at the same time degrading. In essence, the patient develops a new dermis. About three weeks later, the silicone layer is removed and replaced with an autograft of the epidermis-a much less problematic procedure than grafting dermis, because epidermis is much thinner and is constantly being shed and regrown. In a refinement of the procedure, the second skin graft is eliminated by seeding the protein layer of the artificial graft with epidermal cells obtained from a small skin graft of the patient. Another promising approach involves the use of a biodegradable synthetic polymer (polyglycolic acid) mesh that has been seeded with human foreskin cells (obtained from the circumcision of a newborn). In deep injuries of the dermis and epidermis, this bioartificial graft would be covered with a thin graft of epidermis. Other promising approaches are also being pursued. Pancreas and Liver Tissues. Other tissues being heavily investigated by tissue engineers are the pancreas and liver. These organs are much more complex than skin, and research is still in its infancy. Each year more than 728,000 new cases of diabetes are diagnosed, and 150,000 Americans die from the disease and its complications. One type of diabetes is characterized by destruction of insulin-secreting islet cells in the pancreas, leading to an inability of the body to properly utilize glucose, its primary energy source. Tissue engineering approaches for the eventual treatment of diabetes have focused on transplanting healthy islets, usually encased in some protective membrane to prevent immune rejection. The encapsulation technique prevents the body's immune cells from attacking the implanted islets but allows the passage of oxygen, essential nutrients, and insulin across the membrane. Porcine (pig) islets are used in many studies, and genetically engineered cells which overproduce insulin are also being explored. When the liver fails, the toxins normally removed and metabolized by the liver must be eliminated using methods such as dialysis or hemoperfusion. However, these techniques do not perform all the functions of a healthy liver. Thus research is underway to create a bioartificial liver via the transplantation of hepatocytes (liver cells), by encapsulating them in membranes, placing them in a liquid suspension, or growing them on three-dimensional scaffolds. Although some function of the hepatocytes has been observed, efforts to tissue engineer a liver have been largely unsuccessful. Hepatocytes require an ample blood supply. The lack of a sufficient supply of blood is the primary reason for the cell death observed with hepatocyte transplantation. Fortunately, several tissue engineering approaches are underway to improve hepatocyte survival by enhancing cell contact with blood sources. In one case, the scaffold is implanted into the body first to permit the growth of blood vessels into the matrix. The hepatocytes are injected using a needle through the skin and into the scaffold only after it has been "prevascularized". In a second case, scaffolds are being engineered to have tiny channels for blood flow. The scaffold, seeded with hepatocytes, will be implanted in series with a blood vessel to provide an immediate supply of blood for the hepatocytes. Vascular System. One area of interest in our laboratories at The University of Texas is the vascular system. We have fabricated a model of a blood vessel for the purpose of understanding vascular development and regulation and for the designing of tissues suitable for use as vascular grafts in patients. Blood vessel substitutes, as needed in heart bypass surgery for example, are in high demand as more than 500,000 coronary artery bypass operations are performed annually in the United States alone. The best results are obtained if one of the patient's own vessels is grafted, but if an autologous vessel cannot be used, a prosthetic vessel may be implanted. Vein grafts require that a vein be removed from another part of the patient (usually the leg), requiring multiple surgical procedures. We know that 20 percent to 30 percent of all patients do not have usable veins for this procedure, usually due to previous vein harvest, amputation, or other medical conditions. In addition, as synthetic grafts are foreign to the body, they may pose long-term health risks. Although synthetic materials can be successfully used in the formation of large-diameter vessels, they are not as useful for the construction of the small-diameter grafts required for many heart bypass surgeries, as the grafts are more prone to clotting. Many researchers believe that modification of existing grafts with a lining mimicking the natural endothelial cell lining found on blood vessels in the body would minimize clotting by providing a barrier between blood-clotting components and the graft material. The use of endothelial cells derived from the patient would be ideal since an immune response would be avoided. However, harvesting endothelial cells from a patient for use on a vascular graft is clinically infeasible in emergencies. To overcome this limitation, we are studying the use of animal endothelial cells which are readily available but need to be modified to suppress the immune reaction. Animal endothelial cells are recognized as foreign and then rapidly rejected by the body's immune system primarily due to a single sugar group expressed on the surface of the animal cells, which is recognized by natural antibodies in humans. Genetic modification of the pathway leading to expression of this sugar group in other cells has previously been shown to successfully reduce the sugar group's surface display and to suppress the immune response. By exploiting a similar molecular strategy, we are taking steps toward the development of a universal endothelium that would not be rejected by the body and could be combined with a suitable scaffold to prevent clotting of a vascular graft (Figure 3). Nervous System. Our research in this area is aimed at gaining a fundamental understanding of nerve cell-biomaterial interactions, with the goal of engineering materials that will facilitate the regeneration of damaged central nerves (spinal cord injuries) and peripheral nerves. Nerve injuries have a significant impact on the quality of life. In the United States an estimated 235,000 individuals suffer from physically debilitating spinal cord injuries, with total associated costs of more than $350 billion. Prompt attention by regional spinal cord injury centers has decreased mortality, primarily by preventing secondary injury and complications. Despite this care, the inevitable final outcome is life-long disability, and only a small number of patients experience even modest recovery. Ideally, medical treatment would help to reverse the neurologic deficit, but acute spinal cord injury has been extraordinarily resistant to effective treatment. Peripheral nerve injuries, such as facial paralysis and nerve damage in limbs resulting from accidents, occur more frequently than spinal cord injuries. Peripheral nerve injury can result from mechanical, thermal, chemical, or pathological sources and may lead to loss of muscle function or to sensory loss and phantom sensations. Hand or facial reconstruction, either after an accident or to repair an existing defect, often requires that nerves be rerouted or replaced. In the case of facial paralysis, about one in sixty or seventy people will in their lifetime suffer from partial or complete facial paralysis, such as Bell's palsy, of which about 20 percent may require surgical grafting procedures. In the treatment of peripheral nerve damage, surgeons use a nerve graft from the patient (autograft), but regeneration is incomplete and inevitably some irreversible damage occurs. Additional disadvantages include loss of function at the donor site and the need for multiple surgeries which increase risk and cost to the patient. There is reason to believe that peripheral nerve regeneration can be improved and that eventually regeneration of the spinal cord will be possible, especially in light of recent evidence of spinal cord regeneration in the rat. In an effort to bypass limitations of current treatments for nerve damage, researchers are investigating the use of nerve guidance channels to bridge the gap between damaged nerve ends. Nerve guidance channels are tubular devices that help direct axons (nerve protrusions that guide regeneration) sprouting off the regenerating nerve end, provide a conduit for diffusion of growth-enhancing molecules secreted by the damaged nerve stumps, and minimize infiltrating scar tissue that may impede regeneration (Figure 4). Furthermore, nerve guidance channels can be fashioned to actively stimulate the nerve to regenerate, for example, by the release of specific biochemical factors. Although most research in this area has been applied to the development of alternatives to peripheral nerve grafts, studies are beginning to focus on creating guidance channels to promote regeneration of axons in the spinal cord. At The University of Texas we focus on using a locally applied electrical stimulus, in the form of a nerve guidance channel fashioned from the electrically conducting polymer, polypyrrole, to promote nerve regeneration. Past studies have shown that electrical fields enhance regeneration of some tissues such as bone and nerve. Our work demonstrates a further significant enhancement of axon extension (nerve growth) on polypyrrole films stimulated with low electrical current compared to controls. In addition, initial studies in rats show that polypyrrole does not induce a long-term immune response and can support nerve regeneration over a one centimeter defect. Eventually we hope to use polypyrrole to aid nerve regeneration in the body, but polypyrrole in its present form is not biodegradable. Degradable materials for implantation are desired since they pose fewer long-term health risks. Thus we are currently working on the synthesis of a biodegradable form of polypyrrole that we can use for nerve regeneration studies, and possibly for other areas of tissue engineering. Despite the considerable evidence that electrical charges significantly enhance nerve regeneration, the mechanism of this effect is unknown. Part of our research is geared toward understanding how and to what extent electrical conduction through polypyrrole enhances nerve regeneration. A fundamental understanding of this process will enable us to better engineer therapies to improve nerve regeneration. Summary. This article highlights just a few examples of the tissue engineering efforts taking place worldwide and at The University of Texas at Austin. Research is underway to create living tissue replacements for essentially every human tissue, including skin, pancreas, liver, blood vessels, nerve, cartilage, tendon, bone, breast tissue, cornea, intestine, and heart valves. In particular, this article emphasizes the work being conducted in our laboratories on blood vessels and nerve guidance channels. Although it is still too early to tell, the successes of bioartificial skin are evidence of the positive effect that this research will have on people's lives and well-being. Few areas of technology require more interdisciplinary research or have the potential to impact more positively the quality of life than tissue engineering. Dr. Christine E. Schmidt is assistant professor of chemical and biomedical engineering at The University of Texas at Austin. Her research interests include understanding the cellular mechanisms that give rise to tissue morphogenesis for application in nerve and vascular repair. Dr. Schmidt was a NIH postdoctoral research fellow in chemical engineering at Massachusetts Institute of Technology for eighteen months, during which time she worked in collaboration with surgeons at Harvard Medical School in the area of nerve regeneration. She received her B.S. in chemical engineering from UT Austin and Ph.D. from the University of Illinois at Urbana-Champaign. Dr. Schmidt can be reached at 512-471-1690 or schmidt@che.utexas.edu