For a more in-depth review of this field, we recommend several articles that make up a special report,² as well as recent texts on the subject.^3,8,9

	TISSUE ENGINEERING TAKES MANY FORMS

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Clinical problems relating to the loss and/or failure of tissues extend beyond dentistry to all fields of medicine, and are estimated to account for approximately one-half of all medical-related problems in the United States each year.¹⁰ Currently, the replacement of lost or deficient tissues involves prosthetic materials, drug therapies, and tissue and organ transplantation. However, all of these have limitations, including the inability of synthetic prostheses to replace any but the simplest structural functions of a tissue. An extreme shortage of organs and tissues for transplantation exists. Fewer than 10,000 organs are available for transplantation each year in the United States, while more than 50,000 patients are registered on transplantation waiting lists.¹¹ Such problems have motivated the development of tissue engineering, which can be defined as a "combination of the principles and methods of the life sciences with those of engineering to develop materials and methods to repair damaged or diseased tissues, and to create entire tissue replacements."¹²

Many strategies have evolved to engineer new tissues and organs, but virtually all combine a material with either bioactive molecules that induce tissue formation or cells grown in the laboratory. The bioactive molecules are frequently growth factor proteins that are involved in natural tissue formation and remodeling. The basic hypothesis underlying this approach is that the local delivery of an appropriate factor at a correct dose for a defined period of time can lead to the recruitment, proliferation and differentiation of a patient’s cells from adjacent sites. These cells can then participate in tissue repair and/or regeneration at the required anatomic locale.

The second general strategy uses cells grown in the laboratory and placed in a matrix at the site where new tissue or organ formation is desired. These transplanted cells usually are derived from a small tissue biopsy specimen and have been expanded in the laboratory to allow a large organ or tissue mass to be engineered. Typically, the new tissue will be formed in part from these transplanted cells.

With both approaches, specific materials deliver the molecules or cells to the appropriate anatomic site and provide mechanical support to the forming tissue by acting as a scaffold to guide new tissue formation.¹³ Currently, most tissue engineering efforts use biomaterials already approved for medical indications by the U.S. Food and Drug Administration, or FDA. The most widely used synthetic materials are polymers of lactide and glycolide (Figure 1¹⁴; see page 310), since these are commonly used for biodegradable sutures. Both polymers have a long track record for human use and are considered biocompatible, and their physical properties (for example, degradation rate, mechanical strength) can be readily manipulated. A natural polymer—type 1 collagen—is often used because of its relative biocompatibility and ability to be remodeled by cells. Other polymers familiar to dentistry, including alginate, are also being used.

View larger version (92K): [in this window] [in a new window]   Figure 1. Chemical structures and typical physical forms for polymers used in tissue engineering. A. Chemical structures of biodegradable polyesters commonly used to fabricate tissue-engineering scaffolds. B. Photomicrograph of a polyglycolic acid fiber-based scaffold. C. Photomicrograph of a poly(lactic-co-glycolic acid) porous sponge. Size bars are shown on the photomicrographs. (Photomicrographs from Kim and colleagues.14)

	PRECLINICAL AND CLINICAL ACCOMPLISHMENTS

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Tissue induction. In contrast to passive tissue formation achieved with conductive approaches, a tissue-inductive approach activates cells near the tissue with specific signals. The impetus for this approach was the discovery of defined molecules—termed growth factors—that could lead to new bone (osteogenesis) and blood vessel (angiogenesis) formation. Urist¹⁸ first demonstrated that new bone could be formed at a nonmineralizing site after implantation of powdered bone. This led to the isolation of the active ingredients (specific growth-factor proteins) from the bone powder, the eventual cloning of the genes encoding these proteins, and their now large-scale production by a number of companies.¹⁹ These proteins—termed bone morphogenetic proteins, or BMPs—have been used in many clinical trials, including studies of non-healing long-bone fractures and periodontal tissue regeneration (see "A Look to the Future of Dentistry" below), and are in the early phase of FDA review.

The identification of proteins that promote new blood vessel formation, and their clinical applications, followed a parallel track to that of the BMPs. Judah Folkman first recognized that specific molecules regulate new blood vessel formation, and several are now known to either promote or inhibit this process.²⁰ These are being used in several applications, including induction of new vessels to bypass blocked arteries.

An alternative tissue-inductive approach to using diffusible growth factors involves placing specific extracellular matrix molecules on a scaffold support at a tissue site. These molecules have the ability to direct the function of cells already present at that site and, therefore, to promote the formation of a desired tissue type or structure. For example, a preparation of enamel proteins derived from pigs is used to promote new bone formation in periodontal defects,²¹ while the protein laminin is being tested for its ability to improve gingival adhesion to dental implants.²²

For tissue induction to be successful clinically, it is critical to deliver the appropriate biologically active factors to the desired site at the appropriate dose for the necessary time. Typically, many of these proteins have short half-lives in the body, yet they must be present for an extended period to be effective. Up until now, clinicians and researchers have addressed these concerns by delivering extremely large doses of protein at the sites of interest. Newer efforts involve the development of controlled-release systems.²³ A somewhat similar approach involves delivering a gene that encodes the inductive factor instead of delivering the protein itself. One of us (D.M.) recently demonstrated that cells will take up the released DNA and produce sufficient amounts of the protein-inductive factor to promote new tissue formation.²⁴

An unresolved issue in tissue engineering is whether multiple protein signals, perhaps presented in a specific sequence, may be necessary to develop fully functional tissues. Currently, most studies involve delivery of a single factor to trigger the cascade of events required for this process. Unfortunately, factors needed to fully promote the formation of many tissue types (for example, kidney, salivary gland) have not yet been identified.

Cell transplantation. The transplantation of cells grown in the laboratory provides another inductive means to engineer new tissues (Figure 2). Cell transplantation is extremely attractive when inductive factors are not known for a specific tissue, when a large tissue mass or organ is needed, or when tissue replacement must be immediate. However, this approach requires the needed cells to be expanded in the laboratory. For some cell types (for example, acinar cells in salivary glands or islet cells in the pancreas), this is not yet possible.

View larger version (53K): [in this window] [in a new window]   Figure 2. A tissue-engineering approach that uses cultured cells and biodegradable polymer scaffolds. Tissue-specific cells are isolated from a biopsy specimen, expanded in culture and combined with a porous biodegradable polymer scaffold. The cells adhere to the scaffold, proliferate and, over time, form a new tissue that can be returned to the tissue donor or to another patient.

Cartilage destruction is common with many diseases and after trauma. Cartilaginous tissue is limited in its ability to regenerate in vivo and, consequently, there has been widespread interest among researchers and manufacturers in developing cartilage cell transplantation methods. Chondrocytes can be readily obtained from various sites in the body, expanded in vitro and transplanted on different carrier substrates. Such chondrocytes are now used clinically to repair articular cartilaginous defects,³⁰ as well as to repair certain urologic dysfunctions.³¹ The design of polymer scaffolds with appropriate mechanical and degradative properties has allowed investigators to engineer new cartilaginous tissues in animal models, with precisely defined sizes and shapes (for example, nasal septum³² and ear³³), that are potentially useful for craniofacial reconstruction.

The most exciting and challenging application of cell transplantation is the engineering of complete organs. Efforts to engineer virtually every major internal organ are now under way. Limited replacement of liver function has been demonstrated,³⁴ and Oberpenning and colleagues³⁵ recently showed that a large portion of the bladder could be engineered using cell transplantation on polymer scaffolds. Critical to these applications is the development of vasculature to support the metabolic needs of the organs and integration of the engineered organ with the host. Two approaches for this are currently being studied. The first involves transplanting endothelial cells on the scaffold with the tissue cell type of interest. Transplanted endothelial cells can increase the vasculature in polymer scaffolds and integrate with in-growing host capillaries.³⁶ The second approach uses localized delivery of inductive angiogenic factors at the site of the engineered tissue²³ (see "Tissue Induction" above).

Gene therapy. Generally, gene therapy is not considered to be an example of tissue engineering. However, gene transfer to well-differentiated cells arguably can be viewed as a way to engineer a tissue. Gene transfer in clinical settings has been used for about 10 years and began with the treatment of two children suffering from a severe combined immunodeficiency resulting from an inherited reduction in the enzyme adenosine deaminase, or ADA.³⁷ These patients were treated with a procedure termed ex vivo gene therapy. The ADA gene was transferred into their own lymphocytes in the laboratory, followed by the return of these cells to the patients. To date, both patients have survived, although it is impossible to conclude that their survival was the result of gene transfer because conventional therapy was administered along with the genetically modified cells. Indeed, there is still no published report of any clinical condition being corrected solely as a result of gene therapy.

IT IS REASONABLE TO EXPECT THAT CLINICAL GENE TRANSFER WILL BE ROUTINE, FOR BOTH PRIMARY AND ADJUNCTIVE THERAPIES, WITHIN THE NEXT 10 TO 20 YEARS.

Hundreds of clinical research protocols have been approved worldwide for gene transfer in a range of conditions, including cystic fibrosis, muscular dystrophy and numerous malignancies. Many of these studies have shown promise and have yielded partial efficacy. Although gene therapy is based on a solid foundation of fundamental science and has made enormous progress, the widespread clinical applications originally conceived have yet to be achieved.³⁸ The principal shortcoming in the field is the lack of adequate gene transfer vectors to deliver foreign genes to host cells. Most often, modified viruses are used, but all common viruses present drawbacks.^39,40 However, there is considerable research activity in this field. New vectors, both nonviral and viral, are being developed and are likely to offer advantages over current gene delivery systems. It is reasonable to expect that clinical gene transfer will be routine, for both primary and adjunctive therapies, within the next 10 to 20 years.

	A LOOK TO THE FUTURE OF DENTISTRY

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As described above, engineered skin tissue and cartilage are becoming available for certain medical applications, and strategies to engineer bony tissues are close to receiving FDA approval. We foresee dental applications of these engineered tissues within the next few years. However, reconstruction of complex tissue defects made up of multiple cell types has not yet been attempted in the craniofacial complex, even in preclinical trials. Such a goal (for example, engineering a complete and functional salivary gland) will likely take about 10 to 15 years.

Mineralized tissue defects. Tissue engineering is already being applied to the repair of periodontal defects, with the use of BMPs: the future is now. Indeed, considerable research activity is focused on applying tissue engineering principles to dental and craniofacial structures, probably because of the ease of access to these sites and the extent and nature of the clinical problems. Many problems managed by general dentists or specialists are prime candidates for tissue-engineering solutions, including fractures of bones and teeth, craniofacial skeletal defects, destruction of the pulp-dentin complex and periodontal disease.⁴¹

BMPs and other growth-factor–rich preparations are being applied with a variety of natural and synthetic scaffolds. The latter are particularly important considerations for many dental and craniofacial applications. Not only are biologically appropriate scaffolds required for the cells and inductive factors, but the scaffolds should not adversely affect patient appearance. In that regard, an advantage may be gained from polymers that are allowed to flow into a defined site, rather than those that are fixed or implanted. Such polymers are currently being developed by a number of research groups.^42,43

Gene therapy. There are several craniofacial examples of using gene therapy (see "Engineering Salivary Gland Function" below). The most substantial body of work uses gene-transfer techniques as either primary or adjunctive therapies for head and neck cancers.^44–46 Already several early-stage clinical studies have been conducted. Most of the focus has been on squamous-cell carcinoma, and some incremental progress has been achieved. In general, the cancer gene therapy effort is enormous, representing a large proportion of all gene therapy research. In the next decade, clinicians will likely be able to use gene-transfer technologies as part of their standard treatment of all neoplasias. Gene therapy also may offer a potentially novel approach to the treatment of severe chronic pain. Many studies have shown that genes can be readily transferred to cells in the central nervous system of animal models.^47,48 Finegold and colleagues⁴⁹ recently showed that viral-mediated transfer of the ß-endorphin gene leads to effective analgesia in a rat pain model.

Engineering salivary gland function. Although most work in engineering new organ growth has focused on tissues whose loss or failure will lead to the patient’s death (for example, the liver or endocrine pancreas), there are many circumstances involving tissue loss that are non–life-threatening, yet that markedly affect quality of life. Included in the latter group is the loss of salivary gland parenchyma and, thus, the ability to make saliva. For example, patients who receive ionizing radiation, or IR (> 52 gray) as part of their treatment for head and neck cancer experience irreversible salivary gland damage. In addition, patients with the autoimmune exocrinopathy Sjögren’s syndrome, or SS, also suffer the loss of salivary secretory tissue. Without saliva, these patients experience dysphagia, rampant caries, mucosal infections (for example, candidiasis), dysgeusia and considerable oral discomfort. Many patients receiving IR or those with SS experience complete gland destruction.

About two years ago, we initiated a pilot program to develop an artificial salivary gland for patients with little to no remaining secretory tissue. We have made substantial initial progress, proceeding from fairly rudimentary studies using a natural substratum (denuded trachea) to the use of engineered polymer scaffolds.^50,51 These efforts have focused on creating a rather simple device—a "blind-end" tube—suitable to engraft in the buccal mucosa of patients whose salivary parenchyma has been destroyed.⁵² The lumen of these tubes would be lined with compatible epithelial cells and be physiologically capable of unidirectional water movement (Figure 3; see page 314). We believe that there is a realistic opportunity to develop a first-generation artificial salivary gland suitable for initial clinical testing relatively soon (within about 10 years).

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic representation of a possible first-generation artificial salivary gland composed of a fluid-secreting blind-end tube. (Adapted with permission of the New York Academy of Sciences from Baum and colleagues.52)

We hypothesized that the major impediment to fluid flow from nonsecreting ductal cells was the absence of a pathway for water in their luminal membranes. Our strategy was to transfer the gene for a protein functioning as such a pathway—the water channel aquaporin-1—into radiation-surviving cells via a recombinant adenovirus. The virus, AdhAQP1, was tested in an irradiated rat model. After four months, rats exposed to 21 Gy had an approximate 65 percent reduction in salivary flow (Figure 4⁵³). Three days after being given AdhAQP1, these rats experienced an increase in fluid production to near-normal levels. Conversely, when irradiated rats were given a control virus, they exhibited no increase in flow.⁵⁴

View larger version (49K): [in this window] [in a new window]   Figure 4. Effect of AdhAQP1 infection on fluid secretion from irradiated rat submandibular glands. All animals were either sham-irradiated or irradiated with 21 gray to the salivary glands. After four months, the glands were infected with either a control virus (Addl312) or the experimental virus (AdhAQP1) encoding the water channel aquaporin-1. Three days later, animals were stimulated with pilocarpine to secrete saliva. (Data are from Delporte and colleagues.54) (Figure reprinted with permission of the International Association for Dental Research from Baum and O’Connell.53)

Using rodent models, we showed that both applications are possible in principle. For example, we examined the potentially life-threatening condition of azole-resistant mucosal candidiasis in immunosuppressed patients as a possible target disorder. After performing adenoviral-mediated gene transfer, we were able to express the human anticandidal peptide histatin 3 in rat salivary glands and show that the recombinant histatin 3 could kill fluconazole-resistant Candida albicans.⁵⁵ We also showed that after adenoviral-mediated gene transfer, both human alpha-1-antitrypsin and human growth hormone could be secreted into the bloodstream from rat salivary glands.^56,57

	CONSIDERATIONS

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We hope readers will appreciate that the term tissue engineering represents a spectrum of novel approaches to managing significant clinical problems. As a new field that is developing rapidly, it must surmount many challenges before any widespread clinical use can result. Most of these challenges are not unique to tissue-engineering approaches, but rather are variants of issues faced by previous (and now established) advances in biomedicine. It is beyond the scope of this review to address these concerns in detail. However, we wish to call attention to two areas of particular importance, one practical and the other philosophical. Interested readers can find additional information in several sources.^2,3,8,9

Manufacturing concerns. For tissue engineering to help alleviate clinical problems, it is necessary for tissue-engineered products to be manufactured reliably.^26,27 This need is almost self-evident, but worthy of emphasis. The goals of successful tissue-engineering research are all commercially applicable (that is, to develop products for patient use). This health-related use raises numerous concerns, including the following:

– feasibility of scaling up from research levels to industrial output;

– batch-to-batch repeatability in production;

– methods to achieve and maintain sterility;

– tissue procurement for cell preparations;

– optimal handling and storage methods.

THE TERM TISSUE ENGINEERING REPRESENTS A SPECTRUM OF NOVEL APPROACHES TO MANAGING SIGNIFICANT CLINICAL PROBLEMS.

These challenges already have been met for some tissue-engineered skin,^26,27 as well as for conventional pharmaceuticals. Although new tissue-engineered products likely will be similarly successful, these concerns must be addressed for each product individually.

An additional and important consideration in the application of these new technologies is the cost associated with each device. It is likely that the cost of producing inductive factors (that is, purified proteins) and cells will be high, and will contribute significantly to the end cost for the patient. For example, a commercially available autologous chondrocyte transplantation system for orthopedic use has a cost of about $10,000 per patient. Although it is likely that fewer cells would be required for localized dental applications of this or an analogous cellular therapy, the cost, without doubt, will be in this range.

This economic issue is one of several concerns leading many companies to develop cellular allograft products. Large-scale production of allogeneic cells (that is, cells not targeted to a specific person), in contrast to a relatively small-scale production of each patient’s cells for an autograft device, will greatly decrease cell production costs. While the cost of inductive proteins is likely to be less than that of cell-based products, the first product commercially available, a bone growth-factor therapy, is likely to add several thousand dollars to the cost of bone fracture treatments.

Ethical concerns. There is significant debate among researchers in the biomedical community about at least two major ethical concerns related to tissue-engineered products. The first, tissue procurement, also is a manufacturing concern (see "Manufacturing Concerns" above). For many tissue-engineered products (such as skin equivalents and bioartificial organs), viable cells are an essential component. Unless a patient’s own cells can be amplified in an adequate and timely manner, enabling them to be used in the tissue-engineered device (that is, a cell autograft), then cells must be derived from another tissue.

This situation raises a number of significant ethical issues. For example, should the tissue source be other people or can an animal tissue (that is, a xenograft) be used? If the source is to be other people (that is, a cell allograft), should they be paid for their tissue samples (such as skin, liver)? This may induce people in financial distress to "donate" their tissues. Since fetal tissues often have more growth potential than adult tissues, should fetal tissues be used as a cell source? If, as with organs for transplantation, there are not enough cellular sources to meet the demand for any particular tissue-engineered device, how does one decide who will get the products (on the basis of need, ability to pay)?

For several cell-based tissue-engineering products, the use of animal cells has been explored. Perhaps the most significant effort has been in the development of an artificial pancreas and the use of porcine cells. Recently, researchers have called for a moratorium on research using cellular xenografts, in large part because of a hypothetical risk.^58–60 This risk is that an animal (in this case, porcine) virus might successfully overcome the human species barrier, perhaps mutate, and result in a serious human disease. Although this circumstance, with respect to a porcine virus, is hypothetical, and there is no evidence that such an event could occur, there is recognition in the research community that the AIDS virus apparently had its origin in primates and "jumped species" through the human consumption of infected animals. A moratorium on xenograft research would recognize such potential societal implications and permit public and legislative discussion of xenograft use. Not surprisingly, there is no uniform agreement on this issue, although the dialogue has generally heightened awareness of ethical considerations in tissue engineering.^59–61

THE IMPACT OF TISSUE ENGINEERING LIKELY WILL BE MOST SIGNIFICANT WITH MINERALIZED TISSUES, ALREADY THE FOCUS OF SUBSTANTIAL RESEARCH EFFORTS.

	CONCLUSIONS

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New technology continually has had a major impact on dental practice, from the development of high-speed handpieces to modern restorative materials. Tissue engineering in the broadest sense unquestionably will affect dental practice significantly within the next 25 years. As an interdisciplinary endeavor, tissue engineering brings the power of modern biological, chemical and physical science to real clinical problems. The impact of tissue engineering likely will be most significant with mineralized tissues, already the focus of substantial research efforts. These efforts will yield numerous clinical dental benefits, including improved treatments for intraosseous periodontal defects, enhanced maxillary and mandibular grafting procedures, perhaps more biological methods to repair teeth after carious damage and possibly even regrowing lost teeth.

In addition, we expect to see a range of other tissue-engineering applications that may promote more rapid healing of oral wounds and ulcers, as well as the use of gene-transfer methods to manipulate salivary proteins and oral microbial colonization patterns. Less common, but still a treatment consideration for the dental profession, will be devices such as the artificial salivary gland and muscle (tongue) or mucosal grafts to replace tissues lost through surgery or trauma. This is an exciting time for biomedical science and its application. Clinical dental practice in 2025 will certainly be different.