Tissue Engineering, Stem Cells, and Cloning (2004)

Introduction to Tissue Engineering As one of the major components of regenerative medicine, tissue engineering follows the principles of cell transplantation, materials science, and engineering toward the development of biologic substitutes that can restore and maintain normal function. Tissue engineering strategies generally fall into two categories: acellular matrices, where matrices are used alone and depend on the body’s natural ability to regenerate for proper orientation and direction of new tissue growth, and matrices with cells. Acellular tissue matrices are usually prepared by removing cellular components from tissues via mechanical and chemical manipulation to produce collagen-rich matrices (1–4). These matrices tend to slowly degrade on implantation and are generally replaced by the ECM proteins that are secreted by the ingrowing cells. When cells are used for tissue engineering, a small piece of donor tissue is dissociated into individual cells. These cells are either implanted directly into the host or are expanded in culture, attached to a support matrix, and then reimplanted into the host after expansion. The source of donor tissue can be heterologous (such as bovine), allogeneic (same species, different individual), or autologous. Ideally, both structural and functional tissue replacements will occur with minimal complications. The most preferred cells to use are autologous cells, where a biopsy of tissue is obtained from the host, the cells are dissociated and expanded in culture, and the expanded cells are implanted into the same host (4–21). The use of autologous cells avoids rejection, and thus the deleterious side effects of immunosuppressive medications can be avoided. One of the limitations of applying cell-based regenerative medicine techniques toward organ replacement has been the inherent difficulty of growing specific cell types in large quantities. Even when some organs, such as the liver, have a high regenerative capacity in vivo, cell growth and expansion in vitro may be difficult. By studying the privileged sites for committed precursor cells in specific organs, as well as exploring the conditions that promote differentiation, one may be able to overcome the obstacles that could lead to cell expansion in vitro. For example, urothelial cells could be grown in the laboratory setting in the past, but only with limited expansion. Several protocols were developed over the last two decades that identified the undifferentiated cells, and kept them undifferentiated during their growth phase (12,22–24). By use of these methods of cell culture, it is now possible to expand a urothelial strain from a single specimen that initially covers a surface area of 1 cm2 to one covering a surface area of 4202 m2 (the equivalent area of one football field) within 8 wk (12). These studies indicated that it should be possible to collect autologous bladder cells from human patients, expand them in culture, and return them to the human donor in sufficient quantities for reconstructive purposes (12,13,23–32). Major advances have been achieved within the last decade on the possible expansion of a variety of primary human cells, with specific techniques that make the use of autologous cells possible for clinical application. For cell-based tissue engineering, the expanded cells are seeded onto a scaffold synthesized with the appropriate biomaterial. In tissue engineering, biomaterials replicate the biologic and mechanical function of the native ECM found in tissues in the body by serving as an artificial ECM. As a result, biomaterials provide a three-dimensional space for the cells to form into new tissues with appropriate structure and function, and also can allow for the delivery of cells and appropriate bioactive factors (e.g., cell adhesion peptides, growth factors), to desired sites in the body (33). Because the majority of mammalian cell types are anchorage dependent and will die if no cell-adhesion substrate is available, biomaterials provide a cell-adhesion substrate that can deliver cells to specific sites in the body with high loading efficiency. Biomaterials can also provide mechanical support against in vivo forces such that the predefined three-dimensional structure is maintained during tissue development. Furthermore, bioactive signals, such as cell-adhesion peptides and growth factors, can be loaded along with cells to help regulate cellular function. The ideal biomaterial should be biocompatible in that it is biodegradable and bioresorbable to support the replacement of normal tissue without inflammation. Incompatible materials are destined for an inflammatory or foreign-body response that eventually leads to rejection and/or necrosis. In addition, the degradation products, if produced, should be removed from the body via metabolic pathways at an adequate rate that keeps the concentration of these degradation products in the tissues at a tolerable level (34). Furthermore, the biomaterial should provide an environment in which appropriate regulation of cell behavior (e.g., adhesion, proliferation, migration, and differentiation) can occur such that functional tissue can form. Cell behavior in the newly formed tissue has been shown to be regulated by multiple interactions of the cells with their microenvironment, including interactions with cell-adhesion ligands (35) and with soluble growth factors (36). In addition, biomaterials provide temporary mechanical support that allows the tissue to grow in three dimensions while the cells undergo spatial tissue reorganization. The properly chosen biomaterial should allow the engineered tissue to maintain sufficient mechanical integrity to support itself in early development, whereas in late development, the properly chosen biomaterial should have begun degradation such that it does not hinder further tissue growth (33). Generally, three classes of biomaterials have been used for engineering tissues: naturally derived materials (e.g., collagen and alginate), acellular tissue matrices (e.g., bladder submucosa and small intestinal submucosa), and synthetic polymers (e.g., polyglycolic acid (PGA), polylactic acid (PLA), and poly(lactic-co-glycolic acid) (PLGA). These classes of biomaterials have been tested in respect to their biocompatibility (37,38). Naturally derived materials and acellular tissue matrices have the potential advantage of biologic recognition. However, synthetic polymers can be produced reproducibly on a large scale with controlled properties of their strength, degradation rate, and microstructure. Collagen is the most abundant and ubiquitous structural protein in the body, and may be readily purified from both animal and human tissues with an enzyme treatment and salt/acid extraction (39). Collagen implants degrade through a sequential attack by lysosomal enzymes. The in vivo resorption rate can be regulated by controlling the density of the implant and the extent of intermolecular cross-linking. The lower the density, the greater the interstitial space and generally the larger the pores for cell infiltration, leading to a higher rate of implant degradation. Collagen contains cell-adhesion domain sequences (e.g., RGD) that exhibit specific cellular interactions. This may assist to retain the phenotype and activity of many types of cells, including fibroblasts (40) and chondrocytes (41). Alginate, a polysaccharide isolated from seaweed, has been used as an injectable cell delivery vehicle (42) and a cell immobilization matrix (43) because of its gentle gelling properties in the presence of divalent ions such as calcium. Alginate is relatively biocompatible and approved by the US Food and Drug Administration (FDA) for human use as wound dressing material. Alginate is a family of copolymers of D-mannuronate and L-guluronate. The physical and mechanical properties of alginate gel are strongly correlated with the proportion and length of polyguluronate block in the alginate chains (42). Acellular tissue matrices are collagen-rich matrices prepared by removing cellular components from tissues. The matrices are often prepared by mechanical and chemical manipulation of a segment of tissue (1–4). The matrices slowly degrade upon implantation, and are replaced and remodeled by ECM proteins synthesized and secreted by transplanted or ingrowing cells. Polyesters of naturally occurring α-hydroxy acids, including PGA, PLA, and PLGA, are widely used in tissue engineering. These polymers have gained FDA approval for human use in a variety of applications, including sutures (44). The ester bonds in these polymers are hydrolytically labile, and these polymers degrade by nonenzymatic hydrolysis. The degradation products of PGA, PLA, and PLGA are nontoxic, natural metabolites and are eventually eliminated from the body in the form of carbon dioxide and water (44). The degradation rate of these polymers can be tailored from several weeks to several years by altering crystallinity, initial molecular weight, and the copolymer ratio of lactic to glycolic acid. Because these polymers are thermoplastics, they can be easily formed into a three-dimensional scaffold with a desired microstructure, gross shape, and dimension by various techniques, including molding, extrusion (45), solvent casting (46), phase separation techniques, and gas foaming techniques (47). Many applications in tissue engineering often require a scaffold with high porosity and ratio of surface area to volume. Other biodegradable synthetic polymers, including poly(anhydrides) and poly(ortho-esters), can also be used to fabricate scaffolds for tissue engineering with controlled properties (48). Stem Cells Most current strategies for tissue engineering depend upon a sample of autologous cells from the diseased organ of the host. However, for many patients with extensive end-stage organ failure, a tissue biopsy may not yield enough normal cells for expansion and transplantation. In other instances, primary autologous human cells can not be expanded from a particular organ, such as the pancreas. In these situations, pluripotent human embryonic stem cells are envisioned as a viable source of cells because they can serve as an alternative source of cells from which the desired tissue can be derived. Combining the techniques learned in tissue engineering over the past few decades with this potentially endless source of versatile cells could lead to novel sources of replacement organs. Embryonic stem cells exhibit two remarkable properties: the ability to proliferate in an undifferentiated but pluripotent state (self-renew), and the ability to differentiate into many specialized cell types (49). They can be isolated by immunosurgery from the inner cell mass of the embryo during the after and are usually grown on of embryonic fibroblasts or human cells have shown that these cells can be grown without the use of a and thus the of these human cells to and These cells have in by their undifferentiated state for at when grown current protocols embryonic stem cells have been shown to differentiate into cells from three embryonic in vitro. and have been cells, cells, and have been cells have been In addition, as further of their embryonic stem cells can form which are cell that three embryonic while in culture, and can form in vivo which has also been and the of a from a donor cell into an to an embryo with a to that of the has been in the of the of in the over by and which were the derived from were by in but the were derived from In the past advances in have been the of the not the to be produced from in were produced in and cells derived from embryonic The of that the to be derived from an cell from several have been grown including and types of and have been and a of the the two types may help to some of the that these in most for human applications, is used to an embryo that has the as its cell This embryo can then be implanted into the of a to to an that is a of the the other is used to early that are in to produce embryonic stem cell is to that of its These autologous stem cells have the potential to of cell in the body, and thus be in tissue and organ replacement applications applications be in the treatment of such as end-stage and for which is limited of tissue which has also been cell an alternative source of cells that may be 1 the of with tissue engineering to tissues and organs. to from the for and it has been that die of that could have been with embryonic stem tissues current allogeneic tissue rejection is a because of and immunosuppressive are usually to and The use of tissue and derived from may lead to the of that are with of tissues. 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