Tissue Engineering, Stem Cells, and Cloning

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 cell of the 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 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 that has the as its cell This 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. As a result, with the variety of and potentially with immunosuppressive may be for and tissue of cell has limitations that require further can be widely in replacement the of the is The majority of derived from animal not after implantation In multiple be to produce one for animal The potential for to grow into is to for and However, greater has been in which may be in to the of such as in vitro and which have been developed for this for further are in the multiple of such as and of and cell donor cells and that will readily produce viable sources of cells. Furthermore, have been found in if they to including with an and of the liver, and and These may be to the of the cells, which the of the or while the sequences in where the and the structure of the are not to an embryonic of may the of embryonic at the is usually not in from cells, but from early embryonic to an embryonic state may help to the and the of cells. Tissue Engineering of the including have been toward the development of several cell types and tissues and for clinical application. biomaterials without cells, such as and acellular matrices from small and have been used animal for the of tissue of these acellular collagen matrices derived from bladder have also been seeded with autologous cells for laboratory has been able to with collagen Acellular collagen matrices derived from bladder submucosa by laboratory have been used and In animal of the were and replaced with acellular matrix in an and and infiltration, and the were able to through the These were in a clinical of patients with and bladder submucosa used as an matrix for in patients with and functional were in these patients with to a The use of an matrix to be for patients with conditions and the for autologous thus and donor Tissue engineering of the a collagen of a with a tissue is the on the and matrix is to the in an on the after of on the side and after on the the techniques are not for The collagen matrices are able to only when used in an However, if a is the collagen matrices should be seeded with autologous cells to the of and tissue development collagen matrices seeded with autologous cells can be used for are used as tissues for bladder replacement or However, tissues are to specific whereas bladder tissue is for the of Because of the with the use of have alternative materials and tissues for bladder replacement or The of the use of cell strategies for bladder on the ability to use donor tissue and to provide the conditions for and and cells can be expanded in seeded onto and to and form of cells These principles were toward the of tissue engineered in an animal that a with replacement with a tissue engineered organ in and cells were expanded from an autologous bladder biopsy sample and seeded onto a biodegradable The from this that it is possible to that are and is for a variety of such as and conditions such as and One of the major limitations of reconstructive is the of sufficient autologous autologous derived from the cells, may be in The major components of the are and cells. The of autologous functional and structural tissue be and cells were expanded and seeded on acellular collagen matrices and implanted in a the appropriate of tissue and structural and functional including and that it is possible to autologous functional laboratory is on the of the engineered of the may have with and may not have sufficient tissue for the of engineering functional tissue autologous cells and cells were then grown and expanded in These cells were seeded onto biodegradable which were then used for tissue replacement in the autologous after implantation, and the presence of normal tissue and organ studies that the functional of these tissues were to of normal studies that use these engineered are several including and can normal development or has been because of the of native The of engineering tissue in vivo and cells of were and expanded in These cells were seeded onto biodegradable and the were then implanted into for to and the presence of tissue studies in the functional properties to of normal When these were used for autologous were in the whereas the were to be the principles of both tissue engineering and in an to produce tissue in a large animal fibroblasts from were obtained by and single donor cells were isolated and into the space of donor The were implanted into to allow for further in vivo cells were expanded in and seeded onto biodegradable The which of the cells and the were then implanted into the space of the same from which the cells were to allow for tissue The is a organ with multiple cell types and a functional that it one of the most to in tissue engineering of the have been toward the development of support of biologic and synthetic components and vivo replacement are to be However, be for patients with end-stage if these could be implanted without the for an or immunosuppressive cells were seeded on scaffolds of three The of the three of scaffold were to that into a This a with a for the These scaffolds with the were transplanted into the same from which the and wk after Combining and tissue engineering to produce of the seeded with cells, after implantation, the of a the their and the of the of cells that after primary and with allogeneic cells, cells, or donor of the including and specific and that the implanted cells and of the implants extensive and of the cells into and the the and the that the of into the with the presence of the of specific in the and the presence of protein Because studies have shown that the the donor to be a potential source of in proteins by cells could a cell response specific for when the cells are implanted into the donor used of the of the and donor to potential in the two acid were to the and the and as a result, a of two could be the of for is no to the of these acid on also to be a potential source of in the cells. in have been shown to both rejection in vivo and expansion in vitro which could the use of these in patients with rejection of major human tested for a possible cell response to the in vivo and of cells in vitro. that the cells no of a cell that rejection will not occur in the presence of This may a in the of stem cell These studies that cells derived from can be expanded in culture, and transplanted in vivo with the use of biodegradable scaffolds on which the single cells can into tissue that are to that of the host. These studies were the of the use of for of tissues in or synthetic materials have been used as replacement for However, these materials growth potential and may the at for such as or have been autologous cells and biodegradable scaffolds and have been in and The advantage from the use these is that they degrade in vivo and thus allow the new tissue to form without the presence of of these techniques from the laboratory to the clinical setting have where autologous cells were and seeded onto a biodegradable scaffold The autologous used to a that been after implantation, no of or were in the and have limited capacity and thus a for the patients with can be with mechanical and may lead to if were expanded and onto biodegradable scaffolds to engineered for use in large in When to a the engineered able to loading and of the large in the engineered able to provide a functional that able to undergo when to structural and functional treatment are for patients have such as and because of the limited of autologous tissue in the Tissue engineering in the may be a viable alternative for the treatment of these because cells could be and grown into tissue in with the of from both and have been from expanded in then seeded onto biodegradable scaffolds The were then implanted as replacement tissue in The to undergo and while its structural support and Furthermore, if native tissue is engineered may be derived from cells as well can be used in the treatment of both and The in and with this the of this and the with Several are alternative implant materials that be for human use The ideal for the treatment of and should be and for human this studies were to the of injectable chondrocytes in vivo initially that a of and with could serve as a synthetic substrate for the injectable delivery and of in Alginate and its degradation can be on the concentration of of the The use of autologous for the treatment of in of the for an ideal injectable derived from an biopsy can be readily grown and expanded in can be achieved in vitro and in vivo by chondrocytes on synthetic biodegradable In these the matrix replaced the alginate as the polysaccharide This for the treatment of in a These studies that chondrocytes can be easily and with alginate in the can be easily and the tissue formed is able to without of clinical were by the engineered with were at the The patients a rate as with other injectable in of not in patients treatment The patients were have a biocompatible of engineered autologous tissue a with were also with chondrocytes at three different 1 an rate of at both and chondrocytes for the treatment of of a with of the same after of autologous The potential use of for the treatment of has been were directly into the and bladder of with a in an Tissue to after the as well as of of the into regenerative The that a of the in techniques of derived cells have been used for the treatment of in a The that can be and after and the of further the of the use of cells of as an injectable The use of injectable precursor cells has also been for use in the treatment of to or precursor cells are the cells found in that proliferate to form and eventually and new precursor cells have been shown to an in the of In a autologous precursor cells were into a of and both replacement of as well as of functional were in the tissue This is the of the replacement of both tissue and its by the of precursor cells. As a result, precursor cells may be a for in patients with with and are dependent on replacement to restore and maintain of and its and replacement such as and and may be with and such as and and density Because cells of the are the major source of in implantation of heterologous cells or tissue has been as a for replacement these were limited by the of the tissues and cells to produce of cells in biocompatible and has been an to against a host response as well as to maintain of the cells while the of desired cell were used as a novel for delivery in vivo were in over the of the that cells may be a potential for The engineering of large will require a of and to deliver to One possible of is through the use of delivery of such as growth with the implantation of cells to of engineered tissues. from were and with an and with human cells The of cells were in and the engineered tissues were to 8 wk after The cells were to form with by and with of their whereas engineered of cells a of cells with of over and no cells. These that a of and cells may be for and in engineered The delivery of may help to growth for a variety of cells with the were used for delivery on human cell The of to of cell and in delivery of via cells may be for a variety of Tissue engineering are for of tissue and organ within the human Because tissue engineering the of cell transplantation, materials science, and engineering, have the techniques of cell culture, transplantation, and are for the of this engineered tissues are at different of development, with some used a few in and some in the that engineered tissues may have an expanded clinical in the because they a viable for require tissue major advances in the of stem cell tissue engineering, and techniques have it possible to these to the of regenerative