PRECLINICAL AND CLINICAL ACCOMPLISHMENTS
Conductive approaches. An excellent example of a conductive (or passive) approach to tissue engineering is the dental implant. This is a relatively simple application because the devices used do not include either living cells or diffusible biological signals. Although the idea of replacing lost teeth dates back to antiquity, it was not until the middle-to-late 20th century that reproducible and predictable clinical success in using dental implants was achieved.15 Today, the use of implants in dentistry is widespread and is considered a standard treatment option in conjunction with prosthetic rehabilitation for replacing multiple and single teeth. Another relatively simple example of a conductive approach to tissue engineering that is widely used in dentistry is guided-tissue regeneration. This is used most often to regenerate the periodontal supporting structures and uses a material barrier to create a protected compartment for selective wound healing.16,17
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.
The most successful application of cell transplantation involves the development of a tissue-engineered skin equivalent. For example, 250,000 square feet of skin tissue can be manufactured from a one-square-inch sample of starting tissue.25
Skin tissue is needed to treat burn victims and patients with diabetic ulcers. This need led to early research on the engineering of skin tissues, and resulted in the first FDA-approved tissue-engineered products for clinical use.26,27
Many cell sources and materials are used to engineer skin tissue. However, a common theme is the growth of cultured cells on biodegradable polymer scaffolds and formation of the new skin tissue in the laboratory. This skin tissue then can be packaged and stored for future use. The polymer scaffold degrades and/or is remodeled ultimately by host and transplanted cells after placement, resulting in a completely natural tissue. A similar approach has also been developed for replacement of oral mucosa, although this procedure has not yet been marketed.28,29
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.