- Email: [email protected]
Introduction to tissue engineering
18 Introduction to tissue engineering L E E D. K. B U T T E R Y University of Nottingham, UK ANNE E. BISHOP Imperial College London, UK
18.1
Introduction
Tissue engineering can best be defined by its goal: the design and construction in the laboratory of living, functional components that can be used for the regeneration of malfunctioning tissues. Although considered as being a relatively new field, the first documented report of tissue engineering emerged in 1933, when tumour cells were wrapped in a polymer membrane and implanted into a pig. Tissue engineering is an interdisciplinary field that brings together the principles of the life sciences and medicine with those of engineering and has three basic components: cells, scaffolds and signals. Its development over the past decade has been the result of a variety of factors: increased knowledge and availability of stem cells, genomics, proteomics, the advent of new biomaterials as potential templates for tissue growth, improvements in bioreactor design and increased understanding of healing processes have all contributed. However, although tissue engineering research is evolving rapidly, there has been a hiatus in the commercial development and, hence, clinical application of engineered products. The key challenges to industrial development include problems in devising cost-efficient, scalable processes, guaranteeing product viability and satisfying regulators (Chapter 23). However, progress continues and the number of people currently benefiting from tissue engineering is set to expand exponentially in the coming years. In this chapter, we describe how tissues can be engineered and some of their current applications.
18.2
The challenge
The challenge for tissue engineering is to optimise the isolation, proliferation and differentiation of cells, and to design scaffolds or delivery systems that are conducive to supporting and co-ordinating growth of three-dimensional tissues in the laboratory. One idealistic strategy would be to harvest stem cells from a patient, expand them in cell culture (Chapter 20), and seed them 193
194
Biomaterials, artificial organs and tissue engineering
on a scaffold. Stem cells can become many types of specific mature cells, via a process call differentiation, when given the specific biological stimuli. The scaffold should then act as a template and stimulus for proliferation (multiplication) and differentiation of the stem cells into the specific cells that will generate specific new tissue. The tissue can either be grown on a scaffold that will completely disappear (resorb) as the new tissue grows, so that only the new tissue will be implanted, or a ‘biocomposite’ of the scaffold and new tissue can be implanted (Chapter 19). After implantation, the tissueengineered construct must then be able to survive, restore normal function, e.g. biochemistry and both mechanical and structural integrity, and integrate with the surrounding tissues. Using cells from the same patient eliminates the problem of immunorejection that can occur with transplants from donors.
18.3
Cell sources
Probably the single most important element in the success of tissue engineering is the ability to generate appropriate numbers of cells (too many cells can be just as detrimental as too few) and the capacity for those cells to differentiate from, and maintain, the correct phenotype and perform specific biological functions. For example, cells must produce an extracellular matrix (a proteinbased matrix such as collagen in bone) in the correct organisation, secrete cytokines and other signalling molecules, and interact with neighbouring cells/tissues. Immediately, this raises a number of potential problems, not least of which is obtaining appropriate cell numbers to promote repair. Tissue engineers have looked at virtually all tissues in the body. In some cases, it has been possible to repair/replace tissue using, as a starting material, the relevant cells from the same patient or from a close relative, such as knee repair using autologous chondrocytes. Non-specific cell types have also been used, including dermal fibroblasts for heart valve engineering. See Chapter 6 for descriptions of cell types and definitions.
Primary cells Primary cells are mature cells of a specific tissue type that are harvested from explant material removed by surgical procedure. An example is primary human osteoblasts that are harvested from the femoral heads removed during total hip replacement operations. Primary cells are the most desirable with regard to immunological compatibility but, in general, they are differentiated, post-mitotic cells. This means that they are no longer able to divide and their proliferation potential is low. This might be compounded by the tendency of some cell types to de-differentiate during ex vivo cultivation and express an inappropriate phenotype, e.g. articular chondrocytes in culture often produce fibrocartilage as opposed to hyaline cartilage. This has stimulated studies to
Introduction to tissue engineering
195
find and develop alternative cell sources for tissue engineering strategies, and stem cells might represent a solution to the limitations of primary cells obtained from explanted tissues.
Stem cells Stem cells are commonly defined as undifferentiated cells that can proliferate and have the capacity to both self-renew and differentiate to one or more types of specialised cells. However, there has been some reconsideration of this definition recently following the observation of de-differentiation and trans-differentiation of certain mature cell types. In view of this, it has been suggested that it should be made broader and applicable to a biological function that can be induced in a range of cell types, including differentiated cells, rather than a single entity. Stem cells can be isolated from embryos, fetuses or from adult tissue, but the range of cell types to which they can differentiate varies. Embryonic stem cells are the most pluripotent, i.e. they have the potential to become most different types of cells under the right conditions. For tissue engineering, stem cells potentially can provide a virtually inexhaustible cell source. Current research is focussed on promoting stem cell differentiation to required lineages, purification of consequent cells, confirmation that there is no residual carcinogenic potential in the cell population and implantation in a form that will replace, or augment the function of, diseased or injured tissues. An initial step is the selection of the most appropriate stem cell to form the required tissue.
‘Adult’ stem cells Everyone carries around their own repository of stem cells that exist in various tissue niches, including bone marrow, brain, liver and skin, as well as in the circulation. Originally, these cells were considered to have only oligolineage (monopotent) potential but it is now known that they can show a considerable degree of plasticity. In theory, therefore, these cells could be removed from a patient, incorporated into a tissue construct and put back into the same individual when repair becomes necessary, bypassing the need for immunosuppression. Clearly, adult-derived progenitor cells need to be investigated and their clinical usefulness established. However, as mentioned above, for some stem cell types, problems with accessibility (e.g. it is not easy or desirable to tap stem cells in the brain!), low frequency (e.g. in bone marrow there is roughly 1 stem cell per 100 000 cells and this might also be affected by age and disease), restricted differentiation potential and poor growth may limit their applicability to tissue engineering.
196
Biomaterials, artificial organs and tissue engineering
Embryonic stem cells Embryonic stem cells, whilst raising ethical concerns in some quarters, remain the most plastic cell source available to tissue engineers. Murine embryonic stem (ES) cells were first described more than two decades ago, when they were isolated from the inner cell mass of the developing blastocyst (the early development phase of an embryo) and grown in the laboratory. ES cells have since been shown to be totipotent, differentiating to all lineages, including the germline and trophoblast. In vitro, murine ES cells were shown to proliferate indefinitely in the undifferentiated state and retain the capacity to differentiate to all mature somatic phenotypes when they received the appropriate signals. The initial isolation of ES cell lines heralded a major breakthrough for developmental biology as it provided a simple model system for studying the processes of early embryonic development and cellular differentiation. However, it also opened the way for tissue engineering applications; if ES cells could be derived from human blastocysts, their capacity for multilineage differentiation might be exploited, e.g. for cell-based therapies in which virtually any tissue or cell type could be produced on demand in the laboratory. Human ES cells were eventually derived in 1998, providing a tremendous boost for tissue engineering. Human ES cells show several important differences from murine ES cells in vitro. Human ES cells tend to grow more slowly, usually forming flat, rather than spherical, colonies and are dissociated more easily into single cells than their murine counterparts. Unlike murine, human ES cells are also unresponsive to leukaemia inhibitory factor (LIF). Therefore, to keep them undifferentiated, they require culture on murine embryonic fibroblast feeder (MEF) feeder layers in the presence of basic fibroblast growth factor or on specific substrates such as matrigel or laminin in MEF-conditioned medium. Differentiation of ES cells is often initiated via the formation of distinct cellular aggregates or embryoid bodies (EBs), which are made up of cells of the three basic germ layers; ectoderm, endoderm and mesoderm. Most ES cells form EBs spontaneously and they have proved useful in studying early events in mammalian development. Within just a few days, EBs can grow to comprise many thousands of cells and are a rich source of progenitor cells of all germ layers. Progenitor cells (sometimes called transit-amplifying cells) are ‘committed’ to forming a particular cell type. They retain a finite capacity to proliferate and help define the ‘bulk’, structure and function of tissues.
18.4
Culture conditions
As indicated above, by manipulating the culture conditions under which stem cells are maintained, it is possible to control or restrict the available differentiation pathways and selectively generate cultures enriched with a
Introduction to tissue engineering
197
particular phenotype. Such manipulations include stimulation of cells with particular cytokines, growth factors, amino acids, other proteins and active ions and co-culture with the target cell/tissue type. Often, cell sorting techniques such as fluorescence-activated cell sorting (FACS) are used to purify further a particular cell type. Utilising these approaches, virtually every cell type in the body has been derived from stem cells, mainly in vitro but stable stem cell-derived grafts have been established in vivo. Stem cells are also amenable to genetic manipulation, in particular ES cells, and have been instrumental in the creation of transgenic and gene knockout animals permitting more detailed investigation of the genome and the specific functions of a particular gene. This genetic tractability also offers the potential to introduce genes to promote lineage-restricted differentiation and provides a basis for gene therapy to introduce therapeutic genes and, potentially, to modulate the immune response allowing implantation of ‘non-self’ cells/tissues. Chapter 20 describes the cell culture techniques that can be employed in the development of tissue-engineered constructs.
18.5
Three-dimensional interactions
The normal function of most cells and tissues is, in addition to soluble factors, dependent on spatial interaction with neighbouring cells and with a substratum or extracellular matrix (ECM). Cell–cell and cell–ECM interactions are co-ordinated by members of several families of membrane spanning proteins, called adhesion molecules. These are fundamental to cell adhesion, helping to define 3-D cellular organization and also to participate directly in cell signalling, controlling cell recruitment, growth, differentiation, immune recognition and modulation of inflammation. Consequently, recapitulating the function of the ECM and 3-D cell interactions is an important aspect of generating viable constructs for in vivo tissue replacement. A number of natural and synthetic materials have been used to produce 3D scaffolds to function as an artificial ECM (Chapter 19). Scaffolds for tissue repair ideally should be non-toxic, have good biocompatibility, be biodegradable and be capable of interacting specifically with the cell type(s) of interest. Work with such materials has shown how scaffolds can also be made to be bioactive through adsorption with biomolecules and that such modifications can enable specific recruitment and adhesion of specific cell types.
18.6
Cell reprogramming
In the light of the success of cloning Dolly the sheep, at the Roslin Institute, and the various other animals that have followed, much interest has been
198
Biomaterials, artificial organs and tissue engineering
generated in understanding the mechanisms of nuclear cloning/reprogramming and potentially harnessing them for tissue repair strategies. In nuclear cloning, an enucleated oocyte is fused with the nucleus of a somatic cell. This stimulates ‘rewinding’ of the genetic programme of the somatic nucleus to generate a totipotent cell. This cell can be used to generate an ES cell line that could be used to generate specific cell/tissue types that would be genetically identical to the donor somatic cell (therapeutic cloning). If the egg is implanted into a surrogate mother, it can potentially form an intact embryo (reproductive cloning). The factors and mechanisms that induce this remarkable transformation are not known but it seems likely that a component, e.g. cytokine, hormone etc. of the enucleated oocyte stimulates this reprogramming process.
18.7
The way forward
In this chapter, we have discussed some of the most recent developments in the use of stem cells for tissue repair and regeneration. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use in the clinic is commonplace. As mentioned above, there is much debate about whether adult stem cells can be used instead of ES cells. The opinion of these authors and many others working in this field is that it is too early to disregard one or other of these cell sources (see Table 18.1, which lists a few ‘pros’ and ‘cons’ associated Table 18.1 Comparisons of ES cell and ‘adult’ stem cells and application to tissue repair ES cells
Bone marrow stromal stem cells
In vitro proliferation
Indefinite
Unclear, probably finite ~ 50 population doublings
Stability
Karyotype may change with prolonged culture
Niche may change with age or disease affecting cell numbers and differentiation
Accessibility
Several established cell lines. Consistency of lines likely but not confirmed
Some cell lines, but generally requires aspiration of tissue. May be variations associated with sample site/technique and accessibility
3-D interactions
Typically grow as aggregates and differentiate as EBs
Generally monolayer cultures
Repair in vivo
Yes, a few animal studies. Existing lines can not be used in humans
Yes, shown in many animal studies, autologous and allogeneic transplants in humans
Introduction to tissue engineering
199
with ES cells and ‘adult’ stem cells). There is certainly a need for rationalisation but this can only be exercised once we have carefully compared and contrasted these various cells under the appropriate experimental conditions. Some characteristics that might help resolve the issue of cell source can already be applied to the debate. Accessibility of cells is obviously important. In terms of adult stem cells, it is already clear that some cells, like neural stem cells, pose significant difficulties in harvesting (at least in living donors). Even cells that are more accessible, such as marrow stem cells, are harvested using an invasive aspiration procedure. There are also issues concerning the incidence/ abundance of adult stem cells and their numbers and potency that might decline with increasing age or be affected by disease. With regard to ES cells, concerns have been raised by certain religions, and the proposed practice of therapeutic cloning tends to be misrepresented in the media. The creation of ES cells can be offset somewhat by the fact that there are potentially ‘large’ numbers of embryos created by in vitro fertilisation programmes that are surplus to requirements (normally destroyed) and could potentially be used for derivation of ES cells. In the UK the government has, with considerably foresight, paved the way for extensive but carefully monitored stem cell research through its legislation and the creation by the Medical Research Council (MRC) of a stem cell bank. As a final word of warning, for both adult and embryonic stem cells, their stability, potential to transmit harmful pathogens or genetic mutations, risk of forming unwanted tissues or even teratocarcinomas have yet to be fully evaluated.
18.8
Summary
In this chapter, some of the key challenges facing tissue engineers are addressed. Cell sources are discussed, including autologous primary and embryonic and adult stem cells, and the means by which the cells can be propagated and encouraged to differentiate towards specific lineages. The requirements for scaffolds to produce 3-D tissue constructs are outlined, and the contentious issue of stem cell cloning is presented in the context of its applications in tissue engineering. Finally, an appraisal of the future of tissue engineering is given, concentrating on the potential applications of stem cells.
18.9
Key definitions
Blastocyst:
early development phase of an embryo.
Cytokines: regulatory proteins that are released by cells of the immune system and act as intercellular mediators in the generation of an immune response.
200
Biomaterials, artificial organs and tissue engineering
Ectoderm: outermost of the three primary germ layers of an embryo, from which the epidermis, nervous tissue and sensory organs develop. Endoderm: innermost of the three primary germ layers of an embryo, developing into the gastrointestinal tract, lungs and associated structures. Karyotype: characterisation of the chromosomal complement of an individual or a species, including number, form and size of the chromosomes. Mesoderm: middle embryonic germ layer, from which connective tissue, muscle, bone, and the urogenital and circulatory systems develop. Oocyte:
cell from which an ovum develops (a female gametocyte).
Phenotype:
characteristics of a specific cell type.
Plasticity (of cells):
the potential for cell differentiation.
Pluripotent: a pluripotent stem cell has the potential to differentiate into several cell types. Somatic cell:
any type of cell that is not involved in reproduction.
Stromal cell: a cell with a structural function, found in bone marrow, that supports haemopoietic (blood-generating) cells. Teratocarcinoma: a malignant tumour consisting of different types of tissue, caused by the development of independent germ cells. Totipotent: cell type. Trophoblast:
a totipotent stem cell has the potential to differentiate to any a cell of the outermost layer of the blastocyst (trophoderm).
18.10 Reading list Atala A. and Lanza R., Methods of Tissue Engineering, Philadelphia, Academic Press, 2001. Hollinger J.O., Bone Tissue Engineering, Boca Raton, Florida, CRC Press, 2004. Lanza R., Langer R. and Vacanti J. (eds), Principles of Tissue Engineering, Philadelphia, Academic Press, 2000. Marshak D.R., Gardner R.L. and Gottlieb D. (eds), Stem Cell Biology, New York, Cold Spring Harbor Laboratory Press, 2001. Palsson B. (ed), Tissue Engineering, London, Prentice Hall, 2004. The following websites provide useful information on the principles and ethics of stem cells: http://www.roslin.ac.uk http://www.doh.gov.uk/cegc/index.htm http://www.royalsoc.ac.uk/policy/ http://www.nih.gov/news/stemcell/primer.htm http://www.imperial.ac.uk/medicine/is/tissue
18.1
Introduction
Tissue engineering can best be defined by its goal: the design and construction in the laboratory of living, functional components that can be used for the regeneration of malfunctioning tissues. Although considered as being a relatively new field, the first documented report of tissue engineering emerged in 1933, when tumour cells were wrapped in a polymer membrane and implanted into a pig. Tissue engineering is an interdisciplinary field that brings together the principles of the life sciences and medicine with those of engineering and has three basic components: cells, scaffolds and signals. Its development over the past decade has been the result of a variety of factors: increased knowledge and availability of stem cells, genomics, proteomics, the advent of new biomaterials as potential templates for tissue growth, improvements in bioreactor design and increased understanding of healing processes have all contributed. However, although tissue engineering research is evolving rapidly, there has been a hiatus in the commercial development and, hence, clinical application of engineered products. The key challenges to industrial development include problems in devising cost-efficient, scalable processes, guaranteeing product viability and satisfying regulators (Chapter 23). However, progress continues and the number of people currently benefiting from tissue engineering is set to expand exponentially in the coming years. In this chapter, we describe how tissues can be engineered and some of their current applications.
18.2
The challenge
The challenge for tissue engineering is to optimise the isolation, proliferation and differentiation of cells, and to design scaffolds or delivery systems that are conducive to supporting and co-ordinating growth of three-dimensional tissues in the laboratory. One idealistic strategy would be to harvest stem cells from a patient, expand them in cell culture (Chapter 20), and seed them 193
194
Biomaterials, artificial organs and tissue engineering
on a scaffold. Stem cells can become many types of specific mature cells, via a process call differentiation, when given the specific biological stimuli. The scaffold should then act as a template and stimulus for proliferation (multiplication) and differentiation of the stem cells into the specific cells that will generate specific new tissue. The tissue can either be grown on a scaffold that will completely disappear (resorb) as the new tissue grows, so that only the new tissue will be implanted, or a ‘biocomposite’ of the scaffold and new tissue can be implanted (Chapter 19). After implantation, the tissueengineered construct must then be able to survive, restore normal function, e.g. biochemistry and both mechanical and structural integrity, and integrate with the surrounding tissues. Using cells from the same patient eliminates the problem of immunorejection that can occur with transplants from donors.
18.3
Cell sources
Probably the single most important element in the success of tissue engineering is the ability to generate appropriate numbers of cells (too many cells can be just as detrimental as too few) and the capacity for those cells to differentiate from, and maintain, the correct phenotype and perform specific biological functions. For example, cells must produce an extracellular matrix (a proteinbased matrix such as collagen in bone) in the correct organisation, secrete cytokines and other signalling molecules, and interact with neighbouring cells/tissues. Immediately, this raises a number of potential problems, not least of which is obtaining appropriate cell numbers to promote repair. Tissue engineers have looked at virtually all tissues in the body. In some cases, it has been possible to repair/replace tissue using, as a starting material, the relevant cells from the same patient or from a close relative, such as knee repair using autologous chondrocytes. Non-specific cell types have also been used, including dermal fibroblasts for heart valve engineering. See Chapter 6 for descriptions of cell types and definitions.
Primary cells Primary cells are mature cells of a specific tissue type that are harvested from explant material removed by surgical procedure. An example is primary human osteoblasts that are harvested from the femoral heads removed during total hip replacement operations. Primary cells are the most desirable with regard to immunological compatibility but, in general, they are differentiated, post-mitotic cells. This means that they are no longer able to divide and their proliferation potential is low. This might be compounded by the tendency of some cell types to de-differentiate during ex vivo cultivation and express an inappropriate phenotype, e.g. articular chondrocytes in culture often produce fibrocartilage as opposed to hyaline cartilage. This has stimulated studies to
Introduction to tissue engineering
195
find and develop alternative cell sources for tissue engineering strategies, and stem cells might represent a solution to the limitations of primary cells obtained from explanted tissues.
Stem cells Stem cells are commonly defined as undifferentiated cells that can proliferate and have the capacity to both self-renew and differentiate to one or more types of specialised cells. However, there has been some reconsideration of this definition recently following the observation of de-differentiation and trans-differentiation of certain mature cell types. In view of this, it has been suggested that it should be made broader and applicable to a biological function that can be induced in a range of cell types, including differentiated cells, rather than a single entity. Stem cells can be isolated from embryos, fetuses or from adult tissue, but the range of cell types to which they can differentiate varies. Embryonic stem cells are the most pluripotent, i.e. they have the potential to become most different types of cells under the right conditions. For tissue engineering, stem cells potentially can provide a virtually inexhaustible cell source. Current research is focussed on promoting stem cell differentiation to required lineages, purification of consequent cells, confirmation that there is no residual carcinogenic potential in the cell population and implantation in a form that will replace, or augment the function of, diseased or injured tissues. An initial step is the selection of the most appropriate stem cell to form the required tissue.
‘Adult’ stem cells Everyone carries around their own repository of stem cells that exist in various tissue niches, including bone marrow, brain, liver and skin, as well as in the circulation. Originally, these cells were considered to have only oligolineage (monopotent) potential but it is now known that they can show a considerable degree of plasticity. In theory, therefore, these cells could be removed from a patient, incorporated into a tissue construct and put back into the same individual when repair becomes necessary, bypassing the need for immunosuppression. Clearly, adult-derived progenitor cells need to be investigated and their clinical usefulness established. However, as mentioned above, for some stem cell types, problems with accessibility (e.g. it is not easy or desirable to tap stem cells in the brain!), low frequency (e.g. in bone marrow there is roughly 1 stem cell per 100 000 cells and this might also be affected by age and disease), restricted differentiation potential and poor growth may limit their applicability to tissue engineering.
196
Biomaterials, artificial organs and tissue engineering
Embryonic stem cells Embryonic stem cells, whilst raising ethical concerns in some quarters, remain the most plastic cell source available to tissue engineers. Murine embryonic stem (ES) cells were first described more than two decades ago, when they were isolated from the inner cell mass of the developing blastocyst (the early development phase of an embryo) and grown in the laboratory. ES cells have since been shown to be totipotent, differentiating to all lineages, including the germline and trophoblast. In vitro, murine ES cells were shown to proliferate indefinitely in the undifferentiated state and retain the capacity to differentiate to all mature somatic phenotypes when they received the appropriate signals. The initial isolation of ES cell lines heralded a major breakthrough for developmental biology as it provided a simple model system for studying the processes of early embryonic development and cellular differentiation. However, it also opened the way for tissue engineering applications; if ES cells could be derived from human blastocysts, their capacity for multilineage differentiation might be exploited, e.g. for cell-based therapies in which virtually any tissue or cell type could be produced on demand in the laboratory. Human ES cells were eventually derived in 1998, providing a tremendous boost for tissue engineering. Human ES cells show several important differences from murine ES cells in vitro. Human ES cells tend to grow more slowly, usually forming flat, rather than spherical, colonies and are dissociated more easily into single cells than their murine counterparts. Unlike murine, human ES cells are also unresponsive to leukaemia inhibitory factor (LIF). Therefore, to keep them undifferentiated, they require culture on murine embryonic fibroblast feeder (MEF) feeder layers in the presence of basic fibroblast growth factor or on specific substrates such as matrigel or laminin in MEF-conditioned medium. Differentiation of ES cells is often initiated via the formation of distinct cellular aggregates or embryoid bodies (EBs), which are made up of cells of the three basic germ layers; ectoderm, endoderm and mesoderm. Most ES cells form EBs spontaneously and they have proved useful in studying early events in mammalian development. Within just a few days, EBs can grow to comprise many thousands of cells and are a rich source of progenitor cells of all germ layers. Progenitor cells (sometimes called transit-amplifying cells) are ‘committed’ to forming a particular cell type. They retain a finite capacity to proliferate and help define the ‘bulk’, structure and function of tissues.
18.4
Culture conditions
As indicated above, by manipulating the culture conditions under which stem cells are maintained, it is possible to control or restrict the available differentiation pathways and selectively generate cultures enriched with a
Introduction to tissue engineering
197
particular phenotype. Such manipulations include stimulation of cells with particular cytokines, growth factors, amino acids, other proteins and active ions and co-culture with the target cell/tissue type. Often, cell sorting techniques such as fluorescence-activated cell sorting (FACS) are used to purify further a particular cell type. Utilising these approaches, virtually every cell type in the body has been derived from stem cells, mainly in vitro but stable stem cell-derived grafts have been established in vivo. Stem cells are also amenable to genetic manipulation, in particular ES cells, and have been instrumental in the creation of transgenic and gene knockout animals permitting more detailed investigation of the genome and the specific functions of a particular gene. This genetic tractability also offers the potential to introduce genes to promote lineage-restricted differentiation and provides a basis for gene therapy to introduce therapeutic genes and, potentially, to modulate the immune response allowing implantation of ‘non-self’ cells/tissues. Chapter 20 describes the cell culture techniques that can be employed in the development of tissue-engineered constructs.
18.5
Three-dimensional interactions
The normal function of most cells and tissues is, in addition to soluble factors, dependent on spatial interaction with neighbouring cells and with a substratum or extracellular matrix (ECM). Cell–cell and cell–ECM interactions are co-ordinated by members of several families of membrane spanning proteins, called adhesion molecules. These are fundamental to cell adhesion, helping to define 3-D cellular organization and also to participate directly in cell signalling, controlling cell recruitment, growth, differentiation, immune recognition and modulation of inflammation. Consequently, recapitulating the function of the ECM and 3-D cell interactions is an important aspect of generating viable constructs for in vivo tissue replacement. A number of natural and synthetic materials have been used to produce 3D scaffolds to function as an artificial ECM (Chapter 19). Scaffolds for tissue repair ideally should be non-toxic, have good biocompatibility, be biodegradable and be capable of interacting specifically with the cell type(s) of interest. Work with such materials has shown how scaffolds can also be made to be bioactive through adsorption with biomolecules and that such modifications can enable specific recruitment and adhesion of specific cell types.
18.6
Cell reprogramming
In the light of the success of cloning Dolly the sheep, at the Roslin Institute, and the various other animals that have followed, much interest has been
198
Biomaterials, artificial organs and tissue engineering
generated in understanding the mechanisms of nuclear cloning/reprogramming and potentially harnessing them for tissue repair strategies. In nuclear cloning, an enucleated oocyte is fused with the nucleus of a somatic cell. This stimulates ‘rewinding’ of the genetic programme of the somatic nucleus to generate a totipotent cell. This cell can be used to generate an ES cell line that could be used to generate specific cell/tissue types that would be genetically identical to the donor somatic cell (therapeutic cloning). If the egg is implanted into a surrogate mother, it can potentially form an intact embryo (reproductive cloning). The factors and mechanisms that induce this remarkable transformation are not known but it seems likely that a component, e.g. cytokine, hormone etc. of the enucleated oocyte stimulates this reprogramming process.
18.7
The way forward
In this chapter, we have discussed some of the most recent developments in the use of stem cells for tissue repair and regeneration. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use in the clinic is commonplace. As mentioned above, there is much debate about whether adult stem cells can be used instead of ES cells. The opinion of these authors and many others working in this field is that it is too early to disregard one or other of these cell sources (see Table 18.1, which lists a few ‘pros’ and ‘cons’ associated Table 18.1 Comparisons of ES cell and ‘adult’ stem cells and application to tissue repair ES cells
Bone marrow stromal stem cells
In vitro proliferation
Indefinite
Unclear, probably finite ~ 50 population doublings
Stability
Karyotype may change with prolonged culture
Niche may change with age or disease affecting cell numbers and differentiation
Accessibility
Several established cell lines. Consistency of lines likely but not confirmed
Some cell lines, but generally requires aspiration of tissue. May be variations associated with sample site/technique and accessibility
3-D interactions
Typically grow as aggregates and differentiate as EBs
Generally monolayer cultures
Repair in vivo
Yes, a few animal studies. Existing lines can not be used in humans
Yes, shown in many animal studies, autologous and allogeneic transplants in humans
Introduction to tissue engineering
199
with ES cells and ‘adult’ stem cells). There is certainly a need for rationalisation but this can only be exercised once we have carefully compared and contrasted these various cells under the appropriate experimental conditions. Some characteristics that might help resolve the issue of cell source can already be applied to the debate. Accessibility of cells is obviously important. In terms of adult stem cells, it is already clear that some cells, like neural stem cells, pose significant difficulties in harvesting (at least in living donors). Even cells that are more accessible, such as marrow stem cells, are harvested using an invasive aspiration procedure. There are also issues concerning the incidence/ abundance of adult stem cells and their numbers and potency that might decline with increasing age or be affected by disease. With regard to ES cells, concerns have been raised by certain religions, and the proposed practice of therapeutic cloning tends to be misrepresented in the media. The creation of ES cells can be offset somewhat by the fact that there are potentially ‘large’ numbers of embryos created by in vitro fertilisation programmes that are surplus to requirements (normally destroyed) and could potentially be used for derivation of ES cells. In the UK the government has, with considerably foresight, paved the way for extensive but carefully monitored stem cell research through its legislation and the creation by the Medical Research Council (MRC) of a stem cell bank. As a final word of warning, for both adult and embryonic stem cells, their stability, potential to transmit harmful pathogens or genetic mutations, risk of forming unwanted tissues or even teratocarcinomas have yet to be fully evaluated.
18.8
Summary
In this chapter, some of the key challenges facing tissue engineers are addressed. Cell sources are discussed, including autologous primary and embryonic and adult stem cells, and the means by which the cells can be propagated and encouraged to differentiate towards specific lineages. The requirements for scaffolds to produce 3-D tissue constructs are outlined, and the contentious issue of stem cell cloning is presented in the context of its applications in tissue engineering. Finally, an appraisal of the future of tissue engineering is given, concentrating on the potential applications of stem cells.
18.9
Key definitions
Blastocyst:
early development phase of an embryo.
Cytokines: regulatory proteins that are released by cells of the immune system and act as intercellular mediators in the generation of an immune response.
200
Biomaterials, artificial organs and tissue engineering
Ectoderm: outermost of the three primary germ layers of an embryo, from which the epidermis, nervous tissue and sensory organs develop. Endoderm: innermost of the three primary germ layers of an embryo, developing into the gastrointestinal tract, lungs and associated structures. Karyotype: characterisation of the chromosomal complement of an individual or a species, including number, form and size of the chromosomes. Mesoderm: middle embryonic germ layer, from which connective tissue, muscle, bone, and the urogenital and circulatory systems develop. Oocyte:
cell from which an ovum develops (a female gametocyte).
Phenotype:
characteristics of a specific cell type.
Plasticity (of cells):
the potential for cell differentiation.
Pluripotent: a pluripotent stem cell has the potential to differentiate into several cell types. Somatic cell:
any type of cell that is not involved in reproduction.
Stromal cell: a cell with a structural function, found in bone marrow, that supports haemopoietic (blood-generating) cells. Teratocarcinoma: a malignant tumour consisting of different types of tissue, caused by the development of independent germ cells. Totipotent: cell type. Trophoblast:
a totipotent stem cell has the potential to differentiate to any a cell of the outermost layer of the blastocyst (trophoderm).
18.10 Reading list Atala A. and Lanza R., Methods of Tissue Engineering, Philadelphia, Academic Press, 2001. Hollinger J.O., Bone Tissue Engineering, Boca Raton, Florida, CRC Press, 2004. Lanza R., Langer R. and Vacanti J. (eds), Principles of Tissue Engineering, Philadelphia, Academic Press, 2000. Marshak D.R., Gardner R.L. and Gottlieb D. (eds), Stem Cell Biology, New York, Cold Spring Harbor Laboratory Press, 2001. Palsson B. (ed), Tissue Engineering, London, Prentice Hall, 2004. The following websites provide useful information on the principles and ethics of stem cells: http://www.roslin.ac.uk http://www.doh.gov.uk/cegc/index.htm http://www.royalsoc.ac.uk/policy/ http://www.nih.gov/news/stemcell/primer.htm http://www.imperial.ac.uk/medicine/is/tissue