4 Tissue engineering and regenerative therapies

Adult tissue resident or somatic stem cells
Adult tissue resident or somatic stem cells
Stem cells resident in the di ff erent tissues and organs are responsible for providing replacements for specialised cells that have reached the end of  their functional lifespan either through natural attrition or because of  damage and disease. In certain tissues and organs, notably the bone marrow and gut, stem cells regularly divide and di ff erentiate into specialised cells to replace senescent or damaged cells in the blood and the gastrointestinal mucosa, respectively . Stem cells in other organs, such as the heart or central nervous system, are less able to e ff ect repair or replacement. SSCs have the capacity to di ff erentiate into a limited number of  specialised cell types (multipotent); among the best characterised types are haemato poietic stem cells. In tissue engineering and regenerative medi cine, mesenchymal stem or stromal cell (MSC) populations are widely described but have been more di ffi cult to characterise, particularly in terms of stem cell attrib utes relating to clinical use. Mesenchymal stem and stromal cells 1 Building on earlier observations in the 1960s relating to bone marrow-derived cell populations, in the 1990s the term ‘mesenchymal stem cell’ (MSC) was used in relation to therapy , based on observations that some of  these cells, under the right conditions, could di ff erentiate into cell types relating to muscu 2 loskeletal, adipose and other tissues. In 2005 the International Society for Cellular Therapy (now the International Society for Cell and Gene Therapy; ISCT) proposed that these cells be termed multipotent ‘mesenchymal stromal cells’ with the same abbreviation MSC, and that the term mesenchymal stem demonstrate stem cell activity by clearly stated criteria’. They 4 went on to describe minimum criteria in 2006 : adherence to plastic, expression of  certain surface markers (CD105, CD73 and CD90) but a lack of  expression of  others (CD45, CD34, CD14 or CD11b, CD79 α or CD19 and HLA-DR) and, finally , the ability to di ff erentiate into osteoblasts, adipocytes and chondroblasts in vitro , often described by authors as trilineage di ff erentiation. The importance of  nomenclature relates to the mechanism by which such cells might achieve a clinical e ff ect. The earlier term mesenchymal ‘stem’ cell implies that cells directly con - tribute to re pair and regeneration by di ff erentiation, whereas using the term ‘stromal’ can encompass paracrine and secre - tory behaviour, in which cells are envisaged to work with other cells to influence the outcome of  repair and regenera tion ( Figure 4.3 ) . As a consequence, the term mesenchymal ‘stem’ cell has been highlighted as a cause of  potential confusion, - whereby pa tients might wrongly infer that the cell constitutes 5,6 - a ‘stem cell therapy’. In 2019, the ISCT gave continued sup - port for the term mesenchymal stromal cell but recommended that it be: supplemented with the tissue source of  the cell; intended unless rigorous evidence for stemness exits; associ - 7 ated with robust functional assays demonstrating properties. A further consideration is the manufacture and delivery of such cells. MSCs can be isolated from bone marrow (iliac crest aspiration) or from subcutaneous fat (liposuction/lipoaspira - tion). Cells can be delivered at the point of  care, using bedside systems, or isolated in vitro on the basis of  their adherence to plastic and subsequently further characterised. Therefore, they - can be used shortly after extraction or after expansion of  their numbers by in vitro culture. Furthermore, MSCs can be di ff er - entiated into the desired lineage in vitro by addition of  suitable growth factors and chemicals. The wide variety of  cell type, source and manufacturing process represent important opportunities for treatment.
Cell culture
Osteoblast Chondrocyte Adipocyte
In vitro
trilineage differentiation
Figure 4.3
Proposed characteristics of mesenchymal stromal cells relevant to tissue engineering and regenerative medicine.
Mesenchymal
stromal cell
Interaction with in
/f_l
ammatory
and immune processes
B-cell
Macrophage
Natural killer cell
Dendritic cell
T cell
the understanding of  which will be greatly improved by molecular biology techniques and functional assay . In terms of agreed nomenclature, a report on consensus has described key parameters in the abbreviation DOSES: D /uni00A0 – /uni00A0 donor, O /uni00A0 – /uni00A0 origin tissue, S /uni00A0 – /uni00A0 separation method, E /uni00A0 – /uni00A0 exhibited characteristics, 8 S /uni00A0 – /uni00A0 site of  delivery . The relative ease of  cell acquisition has meant that autologous MSCs have been used in clinical settings and they represent a great opportunity for new treatment development. Before widespread adoption, more translational research is required to understand and refine the therapeutic mechanism of  action and conduct well-designed clinical trials to establish the evidence of  e ff ectiveness. Adult tissue resident or somatic stem cells
Stem cells resident in the di ff erent tissues and organs are responsible for providing replacements for specialised cells that have reached the end of  their functional lifespan either through natural attrition or because of  damage and disease. In certain tissues and organs, notably the bone marrow and gut, stem cells regularly divide and di ff erentiate into specialised cells to replace senescent or damaged cells in the blood and the gastrointestinal mucosa, respectively . Stem cells in other organs, such as the heart or central nervous system, are less able to e ff ect repair or replacement. SSCs have the capacity to di ff erentiate into a limited number of  specialised cell types (multipotent); among the best characterised types are haemato poietic stem cells. In tissue engineering and regenerative medi cine, mesenchymal stem or stromal cell (MSC) populations are widely described but have been more di ffi cult to characterise, particularly in terms of stem cell attrib utes relating to clinical use. Mesenchymal stem and stromal cells 1 Building on earlier observations in the 1960s relating to bone marrow-derived cell populations, in the 1990s the term ‘mesenchymal stem cell’ (MSC) was used in relation to therapy , based on observations that some of  these cells, under the right conditions, could di ff erentiate into cell types relating to muscu 2 loskeletal, adipose and other tissues. In 2005 the International Society for Cellular Therapy (now the International Society for Cell and Gene Therapy; ISCT) proposed that these cells be termed multipotent ‘mesenchymal stromal cells’ with the same abbreviation MSC, and that the term mesenchymal stem demonstrate stem cell activity by clearly stated criteria’. They 4 went on to describe minimum criteria in 2006 : adherence to plastic, expression of  certain surface markers (CD105, CD73 and CD90) but a lack of  expression of  others (CD45, CD34, CD14 or CD11b, CD79 α or CD19 and HLA-DR) and, finally , the ability to di ff erentiate into osteoblasts, adipocytes and chondroblasts in vitro , often described by authors as trilineage di ff erentiation. The importance of  nomenclature relates to the mechanism by which such cells might achieve a clinical e ff ect. The earlier term mesenchymal ‘stem’ cell implies that cells directly con - tribute to re pair and regeneration by di ff erentiation, whereas using the term ‘stromal’ can encompass paracrine and secre - tory behaviour, in which cells are envisaged to work with other cells to influence the outcome of  repair and regenera tion ( Figure 4.3 ) . As a consequence, the term mesenchymal ‘stem’ cell has been highlighted as a cause of  potential confusion, - whereby pa tients might wrongly infer that the cell constitutes 5,6 - a ‘stem cell therapy’. In 2019, the ISCT gave continued sup - port for the term mesenchymal stromal cell but recommended that it be: supplemented with the tissue source of  the cell; intended unless rigorous evidence for stemness exits; associ - 7 ated with robust functional assays demonstrating properties. A further consideration is the manufacture and delivery of such cells. MSCs can be isolated from bone marrow (iliac crest aspiration) or from subcutaneous fat (liposuction/lipoaspira - tion). Cells can be delivered at the point of  care, using bedside systems, or isolated in vitro on the basis of  their adherence to plastic and subsequently further characterised. Therefore, they - can be used shortly after extraction or after expansion of  their numbers by in vitro culture. Furthermore, MSCs can be di ff er - entiated into the desired lineage in vitro by addition of  suitable growth factors and chemicals. The wide variety of  cell type, source and manufacturing process represent important opportunities for treatment.
Cell culture
Osteoblast Chondrocyte Adipocyte
In vitro
trilineage differentiation
Figure 4.3
Proposed characteristics of mesenchymal stromal cells relevant to tissue engineering and regenerative medicine.
Mesenchymal
stromal cell
Interaction with in
/f_l
ammatory
and immune processes
B-cell
Macrophage
Natural killer cell
Dendritic cell
T cell
the understanding of  which will be greatly improved by molecular biology techniques and functional assay . In terms of agreed nomenclature, a report on consensus has described key parameters in the abbreviation DOSES: D /uni00A0 – /uni00A0 donor, O /uni00A0 – /uni00A0 origin tissue, S /uni00A0 – /uni00A0 separation method, E /uni00A0 – /uni00A0 exhibited characteristics, 8 S /uni00A0 – /uni00A0 site of  delivery . The relative ease of  cell acquisition has meant that autologous MSCs have been used in clinical settings and they represent a great opportunity for new treatment development. Before widespread adoption, more translational research is required to understand and refine the therapeutic mechanism of  action and conduct well-designed clinical trials to establish the evidence of  e ff ectiveness. Adult tissue resident or somatic stem cells
Stem cells resident in the di ff erent tissues and organs are responsible for providing replacements for specialised cells that have reached the end of  their functional lifespan either through natural attrition or because of  damage and disease. In certain tissues and organs, notably the bone marrow and gut, stem cells regularly divide and di ff erentiate into specialised cells to replace senescent or damaged cells in the blood and the gastrointestinal mucosa, respectively . Stem cells in other organs, such as the heart or central nervous system, are less able to e ff ect repair or replacement. SSCs have the capacity to di ff erentiate into a limited number of  specialised cell types (multipotent); among the best characterised types are haemato poietic stem cells. In tissue engineering and regenerative medi cine, mesenchymal stem or stromal cell (MSC) populations are widely described but have been more di ffi cult to characterise, particularly in terms of stem cell attrib utes relating to clinical use. Mesenchymal stem and stromal cells 1 Building on earlier observations in the 1960s relating to bone marrow-derived cell populations, in the 1990s the term ‘mesenchymal stem cell’ (MSC) was used in relation to therapy , based on observations that some of  these cells, under the right conditions, could di ff erentiate into cell types relating to muscu 2 loskeletal, adipose and other tissues. In 2005 the International Society for Cellular Therapy (now the International Society for Cell and Gene Therapy; ISCT) proposed that these cells be termed multipotent ‘mesenchymal stromal cells’ with the same abbreviation MSC, and that the term mesenchymal stem demonstrate stem cell activity by clearly stated criteria’. They 4 went on to describe minimum criteria in 2006 : adherence to plastic, expression of  certain surface markers (CD105, CD73 and CD90) but a lack of  expression of  others (CD45, CD34, CD14 or CD11b, CD79 α or CD19 and HLA-DR) and, finally , the ability to di ff erentiate into osteoblasts, adipocytes and chondroblasts in vitro , often described by authors as trilineage di ff erentiation. The importance of  nomenclature relates to the mechanism by which such cells might achieve a clinical e ff ect. The earlier term mesenchymal ‘stem’ cell implies that cells directly con - tribute to re pair and regeneration by di ff erentiation, whereas using the term ‘stromal’ can encompass paracrine and secre - tory behaviour, in which cells are envisaged to work with other cells to influence the outcome of  repair and regenera tion ( Figure 4.3 ) . As a consequence, the term mesenchymal ‘stem’ cell has been highlighted as a cause of  potential confusion, - whereby pa tients might wrongly infer that the cell constitutes 5,6 - a ‘stem cell therapy’. In 2019, the ISCT gave continued sup - port for the term mesenchymal stromal cell but recommended that it be: supplemented with the tissue source of  the cell; intended unless rigorous evidence for stemness exits; associ - 7 ated with robust functional assays demonstrating properties. A further consideration is the manufacture and delivery of such cells. MSCs can be isolated from bone marrow (iliac crest aspiration) or from subcutaneous fat (liposuction/lipoaspira - tion). Cells can be delivered at the point of  care, using bedside systems, or isolated in vitro on the basis of  their adherence to plastic and subsequently further characterised. Therefore, they - can be used shortly after extraction or after expansion of  their numbers by in vitro culture. Furthermore, MSCs can be di ff er - entiated into the desired lineage in vitro by addition of  suitable growth factors and chemicals. The wide variety of  cell type, source and manufacturing process represent important opportunities for treatment.
Cell culture
Osteoblast Chondrocyte Adipocyte
In vitro
trilineage differentiation
Figure 4.3
Proposed characteristics of mesenchymal stromal cells relevant to tissue engineering and regenerative medicine.
Mesenchymal
stromal cell
Interaction with in
/f_l
ammatory
and immune processes
B-cell
Macrophage
Natural killer cell
Dendritic cell
T cell
the understanding of  which will be greatly improved by molecular biology techniques and functional assay . In terms of agreed nomenclature, a report on consensus has described key parameters in the abbreviation DOSES: D /uni00A0 – /uni00A0 donor, O /uni00A0 – /uni00A0 origin tissue, S /uni00A0 – /uni00A0 separation method, E /uni00A0 – /uni00A0 exhibited characteristics, 8 S /uni00A0 – /uni00A0 site of  delivery . The relative ease of  cell acquisition has meant that autologous MSCs have been used in clinical settings and they represent a great opportunity for new treatment development. Before widespread adoption, more translational research is required to understand and refine the therapeutic mechanism of  action and conduct well-designed clinical trials to establish the evidence of  e ff ectiveness.

CELLS
CELLS
Although both fully di ff erentiated cells (somatic cells) and stem cells are being used and developed for tissue engineering and regenerative therapy , most of  the focus is on the use of  stem cells, particularly somatic stem cells (SSCs) and induced pluri - potent stem cells (iPSCs). The major features of  the di ff erent cell types are listed in Table 4.2 . - CELLS
Although both fully di ff erentiated cells (somatic cells) and stem cells are being used and developed for tissue engineering and regenerative therapy , most of  the focus is on the use of  stem cells, particularly somatic stem cells (SSCs) and induced pluri - potent stem cells (iPSCs). The major features of  the di ff erent cell types are listed in Table 4.2 . - CELLS
Although both fully di ff erentiated cells (somatic cells) and stem cells are being used and developed for tissue engineering and regenerative therapy , most of  the focus is on the use of  stem cells, particularly somatic stem cells (SSCs) and induced pluri - potent stem cells (iPSCs). The major features of  the di ff erent cell types are listed in Table 4.2 . -

Embryonic stem cells
Embryonic stem cells
In the embryo, stem cells are able to give rise to all of  the di ff er ent cell types of  the body at the gamete stage (totipotent) and nearly all cell types (pluripotent) when at the blastocyst stage ( Figure 4.1 ). ESCs can be obtained from the inner cell mass of the early human blastocyst (days 4–5), using embryos that have been cr eated through in vitro treatment of  infertility and are surplus to those needed for reimplantation. The technique for isolating and growing human ESCs in culture was developed 9 by James Thomson in 1998. ESCs have proliferative ability and possess key character istics of  self-renewal and pluripotency . However, their use in the clinic has major limitations, one of  which is ethical. The surplus embryos used for derivation of  ESCs would otherwise be discarded but, because they need to be destr oyed to obtain ESCs, the approach has raised major ethical and political debate. The dominant view in many countries, including the UK, is that the potential therapeutic benefits of  ESCs justify their use but there are very strict guidelines for their derivation; to date their clinical use has been limited. Cells from ESCs would be allogeneic and therefore be at risk of  immunological rejection. Embryonic stem cells
In the embryo, stem cells are able to give rise to all of  the di ff er ent cell types of  the body at the gamete stage (totipotent) and nearly all cell types (pluripotent) when at the blastocyst stage ( Figure 4.1 ). ESCs can be obtained from the inner cell mass of the early human blastocyst (days 4–5), using embryos that have been cr eated through in vitro treatment of  infertility and are surplus to those needed for reimplantation. The technique for isolating and growing human ESCs in culture was developed 9 by James Thomson in 1998. ESCs have proliferative ability and possess key character istics of  self-renewal and pluripotency . However, their use in the clinic has major limitations, one of  which is ethical. The surplus embryos used for derivation of  ESCs would otherwise be discarded but, because they need to be destr oyed to obtain ESCs, the approach has raised major ethical and political debate. The dominant view in many countries, including the UK, is that the potential therapeutic benefits of  ESCs justify their use but there are very strict guidelines for their derivation; to date their clinical use has been limited. Cells from ESCs would be allogeneic and therefore be at risk of  immunological rejection. Embryonic stem cells
In the embryo, stem cells are able to give rise to all of  the di ff er ent cell types of  the body at the gamete stage (totipotent) and nearly all cell types (pluripotent) when at the blastocyst stage ( Figure 4.1 ). ESCs can be obtained from the inner cell mass of the early human blastocyst (days 4–5), using embryos that have been cr eated through in vitro treatment of  infertility and are surplus to those needed for reimplantation. The technique for isolating and growing human ESCs in culture was developed 9 by James Thomson in 1998. ESCs have proliferative ability and possess key character istics of  self-renewal and pluripotency . However, their use in the clinic has major limitations, one of  which is ethical. The surplus embryos used for derivation of  ESCs would otherwise be discarded but, because they need to be destr oyed to obtain ESCs, the approach has raised major ethical and political debate. The dominant view in many countries, including the UK, is that the potential therapeutic benefits of  ESCs justify their use but there are very strict guidelines for their derivation; to date their clinical use has been limited. Cells from ESCs would be allogeneic and therefore be at risk of  immunological rejection.

Exemplars  cells as a therapy
Exemplars: cells as a therapy
as The delivery of  cells into damaged tissues has long been used to facilitate enhanced regeneration. The first modern example of  successful cell therapy was bone marrow transplantation for the treatment of  leukaemia, which was successfully performed in 1956. Since then, cells have been used for the regeneration of a range of  di ff erent tissues. The following section details exemplars of  the use of  cells as therapeutic agents, for the regeneration of  skin and cartilage (articular and auricular). Skin The tissue that was one of  the first regenerated using human cells was skin. The pioneering work of  Rheinwald and Green in this area led to the identification of  optimal conditions for the growth and maintenance of keratinocytes harvested from skin in culture. The first tissue engineered skin consisted of a layer of  keratinocytes grown on a collagenous membrane, which could be directly applied to a healing wound. In the original skin tissue engineering process, the keratinocytes were grown onto a membrane and were cultured in the same system as a layer of confluent but (unable to divide) fibroblasts. The fibroblasts are required in this culture because they provide a series of  paracrine factors that allow for the keratinocytes to maintain their phenotype and also mature when lifted to the air–liquid interface. The fibroblasts had to be mitotically inhibited because they tend to proliferate at a greater rate than the keratinocytes and can consequently overtake the complex culture. As a consequence of  the need for multiple steps and cell types, in addition to the air-lift required for stratification James George Rheinwald , b. 1948, American research scientist. Howard Green , 1925–2015, George Higginson Professor of  Cell Biology , Harvard Medical School, Boston, MA, USA, pioneer in the science of  skin regeneration. of  the epidermal keratinocyte layer to occur, the process is relatively slow (requiring up to a month). This means that the burn wound to be resurfaced using this process needs to be closed and protected prior to application. This process has been r efined over the years and has resulted in the develop - ment of  a range of  technologies, some of  which are still on the market. Despite a reasonable level of  integration, the skin itself lacks many of  the features of  normal human skin, for example colora tion, and is not in widespread use in the clinic. Articular cartilage regeneration Microfracture or microdrilling ( Figure 4.5 ) into subchondral bone at the base of  a chondral defect creates a communication between the intra-articular and subchondral spaces, allowing blood, containing bone marrow stromal cells, to arrive at the defect. Used clinically for more than 30 year s, the formation of the clot creates an environment that allows for the reformation of  cartilage within the defect, which subsequently provides a level of  mechanical function. Although widely used in the clinic with success, the cartilage formed is typically fibrocartilage, which is mechanically inferior to the native articular cartilage and lacks longer term durability .
Skin biopsy – keratinocytes
Figure 4.4
Schematic diagram showing the principles of induced pluripotent stem cell (iPSC) therapy. Mononuclear cells from peripheral blood
or keratinocytes from a skin biopsy ar
e cultured
in vitro
and then reprogrammed to become iPSCs by addition of reprogramming factors. The
iPSCs are then expanded and selected differ
entiation factors added to promote differentiation of iPSCs into the desired specialised cell type
for use as therapy.
Reprogramming
factors
Induced pluripotent stem cells
Differentiation
factors
Cell therapy
Differentiated cells
Examples include:
chondrocytes
Disease
neurones
modelling
myocytes
pancreatic islet cells
To overcome these limitations, research has focused on ways to create an environment in the chondral defect that is more conducive to the formation of articular or hyaline carti lage. Autologous chondrocyte implantation (ACI) is one such method ( Figure 4.5 ). In the first step a small number of are harvested from healthy cartilage within a patient’s joint, for example the knee. The cells are then expanded, to increase the cell number, in a laborator y (with good manufacturing practice [GMP] quality standards). In a second procedural step, cells are reintroduced into the defect, classically with a surgically positioned ‘roof ’ or patch over the defect, prior to injection of the cells below . In 2017, following the evaluation of evidence, the UK’s National Institute for Health and Care Excellence (NICE) recommended ACI for use in the National Health Service (NHS). The recommendation was for defined patients who have symptomatic articular cartilage defects of  the knee, with a cost-e ff ectiveness estimate thought likely to be less than £20 /uni00A0 000 per quality-adjusted life year (QALY) gained.
Autologous
chondrocyte
Figure 4.5
Examples of
(a)
endogenous cell targeting (microfracture)
and
(b)
cells being used as a therapy (autologous chondrocyte
implantation). (Adapted with permission from Makris E, Gomoll A,
Malizos K
et al
. Repair and tissue engineering techniques for articular
cartilage.
Nat Rev Rheumatol
2015;
11
, 21–34.)
Exemplars: cells as a therapy
as The delivery of  cells into damaged tissues has long been used to facilitate enhanced regeneration. The first modern example of  successful cell therapy was bone marrow transplantation for the treatment of  leukaemia, which was successfully performed in 1956. Since then, cells have been used for the regeneration of a range of  di ff erent tissues. The following section details exemplars of  the use of  cells as therapeutic agents, for the regeneration of  skin and cartilage (articular and auricular). Skin The tissue that was one of  the first regenerated using human cells was skin. The pioneering work of  Rheinwald and Green in this area led to the identification of  optimal conditions for the growth and maintenance of keratinocytes harvested from skin in culture. The first tissue engineered skin consisted of a layer of  keratinocytes grown on a collagenous membrane, which could be directly applied to a healing wound. In the original skin tissue engineering process, the keratinocytes were grown onto a membrane and were cultured in the same system as a layer of confluent but (unable to divide) fibroblasts. The fibroblasts are required in this culture because they provide a series of  paracrine factors that allow for the keratinocytes to maintain their phenotype and also mature when lifted to the air–liquid interface. The fibroblasts had to be mitotically inhibited because they tend to proliferate at a greater rate than the keratinocytes and can consequently overtake the complex culture. As a consequence of  the need for multiple steps and cell types, in addition to the air-lift required for stratification James George Rheinwald , b. 1948, American research scientist. Howard Green , 1925–2015, George Higginson Professor of  Cell Biology , Harvard Medical School, Boston, MA, USA, pioneer in the science of  skin regeneration. of  the epidermal keratinocyte layer to occur, the process is relatively slow (requiring up to a month). This means that the burn wound to be resurfaced using this process needs to be closed and protected prior to application. This process has been r efined over the years and has resulted in the develop - ment of  a range of  technologies, some of  which are still on the market. Despite a reasonable level of  integration, the skin itself lacks many of  the features of  normal human skin, for example colora tion, and is not in widespread use in the clinic. Articular cartilage regeneration Microfracture or microdrilling ( Figure 4.5 ) into subchondral bone at the base of  a chondral defect creates a communication between the intra-articular and subchondral spaces, allowing blood, containing bone marrow stromal cells, to arrive at the defect. Used clinically for more than 30 year s, the formation of the clot creates an environment that allows for the reformation of  cartilage within the defect, which subsequently provides a level of  mechanical function. Although widely used in the clinic with success, the cartilage formed is typically fibrocartilage, which is mechanically inferior to the native articular cartilage and lacks longer term durability .
Skin biopsy – keratinocytes
Figure 4.4
Schematic diagram showing the principles of induced pluripotent stem cell (iPSC) therapy. Mononuclear cells from peripheral blood
or keratinocytes from a skin biopsy ar
e cultured
in vitro
and then reprogrammed to become iPSCs by addition of reprogramming factors. The
iPSCs are then expanded and selected differ
entiation factors added to promote differentiation of iPSCs into the desired specialised cell type
for use as therapy.
Reprogramming
factors
Induced pluripotent stem cells
Differentiation
factors
Cell therapy
Differentiated cells
Examples include:
chondrocytes
Disease
neurones
modelling
myocytes
pancreatic islet cells
To overcome these limitations, research has focused on ways to create an environment in the chondral defect that is more conducive to the formation of articular or hyaline carti lage. Autologous chondrocyte implantation (ACI) is one such method ( Figure 4.5 ). In the first step a small number of are harvested from healthy cartilage within a patient’s joint, for example the knee. The cells are then expanded, to increase the cell number, in a laborator y (with good manufacturing practice [GMP] quality standards). In a second procedural step, cells are reintroduced into the defect, classically with a surgically positioned ‘roof ’ or patch over the defect, prior to injection of the cells below . In 2017, following the evaluation of evidence, the UK’s National Institute for Health and Care Excellence (NICE) recommended ACI for use in the National Health Service (NHS). The recommendation was for defined patients who have symptomatic articular cartilage defects of  the knee, with a cost-e ff ectiveness estimate thought likely to be less than £20 /uni00A0 000 per quality-adjusted life year (QALY) gained.
Autologous
chondrocyte
Figure 4.5
Examples of
(a)
endogenous cell targeting (microfracture)
and
(b)
cells being used as a therapy (autologous chondrocyte
implantation). (Adapted with permission from Makris E, Gomoll A,
Malizos K
et al
. Repair and tissue engineering techniques for articular
cartilage.
Nat Rev Rheumatol
2015;
11
, 21–34.)
Exemplars: cells as a therapy
as The delivery of  cells into damaged tissues has long been used to facilitate enhanced regeneration. The first modern example of  successful cell therapy was bone marrow transplantation for the treatment of  leukaemia, which was successfully performed in 1956. Since then, cells have been used for the regeneration of a range of  di ff erent tissues. The following section details exemplars of  the use of  cells as therapeutic agents, for the regeneration of  skin and cartilage (articular and auricular). Skin The tissue that was one of  the first regenerated using human cells was skin. The pioneering work of  Rheinwald and Green in this area led to the identification of  optimal conditions for the growth and maintenance of keratinocytes harvested from skin in culture. The first tissue engineered skin consisted of a layer of  keratinocytes grown on a collagenous membrane, which could be directly applied to a healing wound. In the original skin tissue engineering process, the keratinocytes were grown onto a membrane and were cultured in the same system as a layer of confluent but (unable to divide) fibroblasts. The fibroblasts are required in this culture because they provide a series of  paracrine factors that allow for the keratinocytes to maintain their phenotype and also mature when lifted to the air–liquid interface. The fibroblasts had to be mitotically inhibited because they tend to proliferate at a greater rate than the keratinocytes and can consequently overtake the complex culture. As a consequence of  the need for multiple steps and cell types, in addition to the air-lift required for stratification James George Rheinwald , b. 1948, American research scientist. Howard Green , 1925–2015, George Higginson Professor of  Cell Biology , Harvard Medical School, Boston, MA, USA, pioneer in the science of  skin regeneration. of  the epidermal keratinocyte layer to occur, the process is relatively slow (requiring up to a month). This means that the burn wound to be resurfaced using this process needs to be closed and protected prior to application. This process has been r efined over the years and has resulted in the develop - ment of  a range of  technologies, some of  which are still on the market. Despite a reasonable level of  integration, the skin itself lacks many of  the features of  normal human skin, for example colora tion, and is not in widespread use in the clinic. Articular cartilage regeneration Microfracture or microdrilling ( Figure 4.5 ) into subchondral bone at the base of  a chondral defect creates a communication between the intra-articular and subchondral spaces, allowing blood, containing bone marrow stromal cells, to arrive at the defect. Used clinically for more than 30 year s, the formation of the clot creates an environment that allows for the reformation of  cartilage within the defect, which subsequently provides a level of  mechanical function. Although widely used in the clinic with success, the cartilage formed is typically fibrocartilage, which is mechanically inferior to the native articular cartilage and lacks longer term durability .
Skin biopsy – keratinocytes
Figure 4.4
Schematic diagram showing the principles of induced pluripotent stem cell (iPSC) therapy. Mononuclear cells from peripheral blood
or keratinocytes from a skin biopsy ar
e cultured
in vitro
and then reprogrammed to become iPSCs by addition of reprogramming factors. The
iPSCs are then expanded and selected differ
entiation factors added to promote differentiation of iPSCs into the desired specialised cell type
for use as therapy.
Reprogramming
factors
Induced pluripotent stem cells
Differentiation
factors
Cell therapy
Differentiated cells
Examples include:
chondrocytes
Disease
neurones
modelling
myocytes
pancreatic islet cells
To overcome these limitations, research has focused on ways to create an environment in the chondral defect that is more conducive to the formation of articular or hyaline carti lage. Autologous chondrocyte implantation (ACI) is one such method ( Figure 4.5 ). In the first step a small number of are harvested from healthy cartilage within a patient’s joint, for example the knee. The cells are then expanded, to increase the cell number, in a laborator y (with good manufacturing practice [GMP] quality standards). In a second procedural step, cells are reintroduced into the defect, classically with a surgically positioned ‘roof ’ or patch over the defect, prior to injection of the cells below . In 2017, following the evaluation of evidence, the UK’s National Institute for Health and Care Excellence (NICE) recommended ACI for use in the National Health Service (NHS). The recommendation was for defined patients who have symptomatic articular cartilage defects of  the knee, with a cost-e ff ectiveness estimate thought likely to be less than £20 /uni00A0 000 per quality-adjusted life year (QALY) gained.
Autologous
chondrocyte
Figure 4.5
Examples of
(a)
endogenous cell targeting (microfracture)
and
(b)
cells being used as a therapy (autologous chondrocyte
implantation). (Adapted with permission from Makris E, Gomoll A,
Malizos K
et al
. Repair and tissue engineering techniques for articular
cartilage.
Nat Rev Rheumatol
2015;
11
, 21–34.)

Exemplars  materials in development
Exemplars: materials in development
Synthetic and engineered polymers Synthetic polymers are those made from chemical processing of natural components or from complex synthesis from precursors such as fossil fuels. There are a great many synthetic polymers that are used in the body , including polymethyl  methacrylate - cells (PMMA), which is used to make intraocular lenses and as a cement in joint replacement; Dacron™ (Terylene™), used in vascular grafts; and a range of  resorbable materials, including poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and copolymers thereof  (PLGA). These resorbable polymers have been used in the body for some time as sutures and in other degradable structures. Moreover, they have become widely used in regenerative medicine for the produc - tion of  sca ff olds, on which cells can be seeded and grown prior to, during or after implantation into the body . Synthetic mate - rials can be processed in a range of  di ff erent ways in order to provide a structure optimised to the intended purpose. P orous monoliths can be produced by foaming processes, whic h can - include but are not limited to infiltration and then expansion of  supercritical carbon dioxide in order to create a foamed structure. The pore sizes of  such structures may be tailored by modifying process conditions or material compositions ( Figure 4.6 ). Such polymers can also be formed as spheres by using emul - sion processing or they can be processed into a mat of  fibres using a process known as electrospinning, whereby charged threads of  polymer are drawn using an electric field and sub - sequently deposited onto a surface ( Figur e 4.7 ). Manufactured in this way , the materials tend to have a very high surface area and can exhibit structural features across the same length scales as the fibrous components of  the extracellular matrix. As a consequence, electrospun patches have been designed for the re pair of  anatomical structures such as the rotator cu ff and have even been used to explore how modifications in matrix geometry may result in pathology . Bioceramics A range of  ceramics have been used for the delivery of  com - pounds that can trigger the regeneration of  mineralised tissues.
(b)
Figure 4.6
Electron micrograph of a poly(lactic acid) scaffold foamed
using supercritical carbon dioxide
(a)
with pore structure assessed
using micro-computed tomography
(b)
. (Adapted with permission
from Collins NJ, Leeke GA, Bridson RH
et al
. The in
/f_l
uence of silica
on pore diameter and distribution
in PLA scaffolds produced using
supercritical CO
.
J Mater Sci: Mater Med
2008;
19
: 1497–502.)
2
® The most widely used such material is Bioglass , a glassy material (a ceramic with no crystal structure) containing calcium, silicate and phosphate ions (Na O–CaO–P O 2 2 5 When placed into an aqueous environment, such as within body fluids, the surface of  the ceramic material can break down, releasing the component ions into the surrounding liquid. This can result in the deposition of  a bone-like mineral across the surface of  the material itself, which can encourage the mate rial to bond to surrounding hard and soft tissues. In addition, the eluted ions can trigger specific biological responses that include the recruitment and di ff er entiation of mesenchymal ® cell populations. Bioglass has been used in a range of medical products and has recently been widely applied in remineralis ® ing toothpaste formulations (as Novamin ). The incorporation of  other ions, such as strontium and lithium, into the glassy matrix can drive specific biological processes, enhancing the ® process of  bone formation. Bioglass can be manufactured as a foamed structure and as a monolith. It is most frequently β mer matrices to drive the process of  bone integration. Hydrogels Hydrogels are an emerging and increasingly researched class of  materials in regenerative medicine. They consist of hydrophilic polymers that are typically dispersed in an aqueous component to form a hydrocolloid. Interactions between the individual polymer chains can then be formed by modifying the temperature, adding ions or adding a chemical cross-linking agent. The resulting network retains water and forms a solid but highly hydrated structure (typically c . 99wt% water). The high water content of  these materials means that nutrients, oxygen and metabolic by-products can di ff use through them. As a consequence, they have been widely used for the encapsu - lation of  cells, typically allowing the maintenance of  high levels of  viability . Alginate, a seaweed-derived polysaccharide blend, for example, has been used to protect pancreatic islets fr om immunological attack. In recent years, this approach has been refined to allow for pancreatic islets to be shipped between –SiO ). medical centres ( Figure 4.8 ). Other materials that form gels 2 include chitosan, gellan, collagen and hyaluronic acid. In addition to these biologically derived materials, hydrogels may also be formed from synthetic polymers, including poly(eth - ylene glycol), and these can be chemically modified to provide - specific biological stimuli to entrapped cells. Additive layer manufacturing and hydrogels As described above, hydrogels have been used for cell encap - sulation. The potential to modify both the chemical and - mechanical properties exhibited by these materials makes them perfect candidates for the growth of  tissues outside the body , prior to eventual implantation into a defect site inside the bod y . As a consequence, hydrogel-based structures have provided sca ff olding for many di ff erent engineered tissues, ranging from bone through to brain. However, a major issue
Figure 4.7
Electrospun poly(caprolactone)
/f_i
bres intended for use in
tendon reconstruction (courtesy of Dr Anita Ghag).
(a)
Pump
Vibrating
technology
100
/uni00A0/uni03BC
m
Figure 4.8
Schematic of
(a)
pancreatic
-cell encapsulation and
Ellis MJ, Grover LM. Encapsulation and
/f_l
uidization maintains the viability and glucose sensitivity of beta-cells.
3(8): 1750–7.)
(b)
Bioreactor
column
Feeding
vessel
Peristaltic
pump
(b)
bioreactor cultivation. (Adapted with permission from Nikravesh N, Cox SC,
ACS Biomater Sci Eng
2017;
with hydrogels is that they are normally extremely weak during the gelation process, meaning that they will slump and lose their shape when cast. In order to get around this major issue, printed structures may be suspended in a secondary (often shear thinning) medium that provides both a level of support during the printing process and time for the hydrogel to develop su ffi cient mechanical integrity to support its own weight. Once this point has been reached, the structure can be removed from the supporting medium and washed prior to onward culturing. A wide range of  substances have been used to provide support, ® including Pluronic and agarose; however, the most widely used method currently is known as free-form reversible embedding of  suspended hydrogels (FRESH), which uses gelatin. The FRESH method utilises a macerated gelatin-based support matrix. The particulate nature of  the gelatin means that the printed hydrogel (which may or may not contain cells) can be deposited within the bed and will be supported while the mechanical properties exhibited by the as-deposited hydrogel become fully developed. The supporting matrix can then be melted (at 37°C) to leave the complex hydrogel structure. Other suspended printing methods have been developed that do not require the use of  an animal-derived support medium (and that are therefore compatible with translation to the clinic) or the elevation of  temperature to remove the supporting phase. One such method, suspended layer additive manufacturing (SLAM), uses a supportive matrix formed from hydrogel partic - ulate systems that shear thin but exhibit rapid elastic recover y; this allows for the production of  high-resolution prints that can be removed from the supporting bed by the application of  a simple wash with water. This method enab les the production of  structures exhibiting complex morphology , such as the carotid artery ( Figure 4.9 ) , and can even be used to produce structures formed or modified from multiple material types, meaning that structures can be tailored with high resolution. With clinical relevance, SLAM has been used to man ufacture osteochondral plugs containing both bony and cartilaginous regions. At present, these technologies are most likely to have an impact on disease modelling and drug candidate evaluation, but in the longer term they may emerge as key technologies facilitating tissue regeneration.
Fluid gel print bed
Cell containing hydrogel
Cross-linker (causes the
construct to solidify)
Figure 4.9
Schematic showing the formation of complex hydrogel structures using a suspended manufacturing process where a cell containing
hydrogel is deposited into a supportive bed. This process allows for the production of hydrogels in complex geometries such as the carotid
arteries shown here. (Adapted with permission from Senior JJ, Cooke ME, Grover LM, Smith AM. Fabrication of complex hydrogel structures
using suspended layer additive manufacturing (SLAM).
Adv Funct Mater
2019;
29
: 1904845.)
Exemplars: materials in development
Synthetic and engineered polymers Synthetic polymers are those made from chemical processing of natural components or from complex synthesis from precursors such as fossil fuels. There are a great many synthetic polymers that are used in the body , including polymethyl  methacrylate - cells (PMMA), which is used to make intraocular lenses and as a cement in joint replacement; Dacron™ (Terylene™), used in vascular grafts; and a range of  resorbable materials, including poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and copolymers thereof  (PLGA). These resorbable polymers have been used in the body for some time as sutures and in other degradable structures. Moreover, they have become widely used in regenerative medicine for the produc - tion of  sca ff olds, on which cells can be seeded and grown prior to, during or after implantation into the body . Synthetic mate - rials can be processed in a range of  di ff erent ways in order to provide a structure optimised to the intended purpose. P orous monoliths can be produced by foaming processes, whic h can - include but are not limited to infiltration and then expansion of  supercritical carbon dioxide in order to create a foamed structure. The pore sizes of  such structures may be tailored by modifying process conditions or material compositions ( Figure 4.6 ). Such polymers can also be formed as spheres by using emul - sion processing or they can be processed into a mat of  fibres using a process known as electrospinning, whereby charged threads of  polymer are drawn using an electric field and sub - sequently deposited onto a surface ( Figur e 4.7 ). Manufactured in this way , the materials tend to have a very high surface area and can exhibit structural features across the same length scales as the fibrous components of  the extracellular matrix. As a consequence, electrospun patches have been designed for the re pair of  anatomical structures such as the rotator cu ff and have even been used to explore how modifications in matrix geometry may result in pathology . Bioceramics A range of  ceramics have been used for the delivery of  com - pounds that can trigger the regeneration of  mineralised tissues.
(b)
Figure 4.6
Electron micrograph of a poly(lactic acid) scaffold foamed
using supercritical carbon dioxide
(a)
with pore structure assessed
using micro-computed tomography
(b)
. (Adapted with permission
from Collins NJ, Leeke GA, Bridson RH
et al
. The in
/f_l
uence of silica
on pore diameter and distribution
in PLA scaffolds produced using
supercritical CO
.
J Mater Sci: Mater Med
2008;
19
: 1497–502.)
2
® The most widely used such material is Bioglass , a glassy material (a ceramic with no crystal structure) containing calcium, silicate and phosphate ions (Na O–CaO–P O 2 2 5 When placed into an aqueous environment, such as within body fluids, the surface of  the ceramic material can break down, releasing the component ions into the surrounding liquid. This can result in the deposition of  a bone-like mineral across the surface of  the material itself, which can encourage the mate rial to bond to surrounding hard and soft tissues. In addition, the eluted ions can trigger specific biological responses that include the recruitment and di ff er entiation of mesenchymal ® cell populations. Bioglass has been used in a range of medical products and has recently been widely applied in remineralis ® ing toothpaste formulations (as Novamin ). The incorporation of  other ions, such as strontium and lithium, into the glassy matrix can drive specific biological processes, enhancing the ® process of  bone formation. Bioglass can be manufactured as a foamed structure and as a monolith. It is most frequently β mer matrices to drive the process of  bone integration. Hydrogels Hydrogels are an emerging and increasingly researched class of  materials in regenerative medicine. They consist of hydrophilic polymers that are typically dispersed in an aqueous component to form a hydrocolloid. Interactions between the individual polymer chains can then be formed by modifying the temperature, adding ions or adding a chemical cross-linking agent. The resulting network retains water and forms a solid but highly hydrated structure (typically c . 99wt% water). The high water content of  these materials means that nutrients, oxygen and metabolic by-products can di ff use through them. As a consequence, they have been widely used for the encapsu - lation of  cells, typically allowing the maintenance of  high levels of  viability . Alginate, a seaweed-derived polysaccharide blend, for example, has been used to protect pancreatic islets fr om immunological attack. In recent years, this approach has been refined to allow for pancreatic islets to be shipped between –SiO ). medical centres ( Figure 4.8 ). Other materials that form gels 2 include chitosan, gellan, collagen and hyaluronic acid. In addition to these biologically derived materials, hydrogels may also be formed from synthetic polymers, including poly(eth - ylene glycol), and these can be chemically modified to provide - specific biological stimuli to entrapped cells. Additive layer manufacturing and hydrogels As described above, hydrogels have been used for cell encap - sulation. The potential to modify both the chemical and - mechanical properties exhibited by these materials makes them perfect candidates for the growth of  tissues outside the body , prior to eventual implantation into a defect site inside the bod y . As a consequence, hydrogel-based structures have provided sca ff olding for many di ff erent engineered tissues, ranging from bone through to brain. However, a major issue
Figure 4.7
Electrospun poly(caprolactone)
/f_i
bres intended for use in
tendon reconstruction (courtesy of Dr Anita Ghag).
(a)
Pump
Vibrating
technology
100
/uni00A0/uni03BC
m
Figure 4.8
Schematic of
(a)
pancreatic
-cell encapsulation and
Ellis MJ, Grover LM. Encapsulation and
/f_l
uidization maintains the viability and glucose sensitivity of beta-cells.
3(8): 1750–7.)
(b)
Bioreactor
column
Feeding
vessel
Peristaltic
pump
(b)
bioreactor cultivation. (Adapted with permission from Nikravesh N, Cox SC,
ACS Biomater Sci Eng
2017;
with hydrogels is that they are normally extremely weak during the gelation process, meaning that they will slump and lose their shape when cast. In order to get around this major issue, printed structures may be suspended in a secondary (often shear thinning) medium that provides both a level of support during the printing process and time for the hydrogel to develop su ffi cient mechanical integrity to support its own weight. Once this point has been reached, the structure can be removed from the supporting medium and washed prior to onward culturing. A wide range of  substances have been used to provide support, ® including Pluronic and agarose; however, the most widely used method currently is known as free-form reversible embedding of  suspended hydrogels (FRESH), which uses gelatin. The FRESH method utilises a macerated gelatin-based support matrix. The particulate nature of  the gelatin means that the printed hydrogel (which may or may not contain cells) can be deposited within the bed and will be supported while the mechanical properties exhibited by the as-deposited hydrogel become fully developed. The supporting matrix can then be melted (at 37°C) to leave the complex hydrogel structure. Other suspended printing methods have been developed that do not require the use of  an animal-derived support medium (and that are therefore compatible with translation to the clinic) or the elevation of  temperature to remove the supporting phase. One such method, suspended layer additive manufacturing (SLAM), uses a supportive matrix formed from hydrogel partic - ulate systems that shear thin but exhibit rapid elastic recover y; this allows for the production of  high-resolution prints that can be removed from the supporting bed by the application of  a simple wash with water. This method enab les the production of  structures exhibiting complex morphology , such as the carotid artery ( Figure 4.9 ) , and can even be used to produce structures formed or modified from multiple material types, meaning that structures can be tailored with high resolution. With clinical relevance, SLAM has been used to man ufacture osteochondral plugs containing both bony and cartilaginous regions. At present, these technologies are most likely to have an impact on disease modelling and drug candidate evaluation, but in the longer term they may emerge as key technologies facilitating tissue regeneration.
Fluid gel print bed
Cell containing hydrogel
Cross-linker (causes the
construct to solidify)
Figure 4.9
Schematic showing the formation of complex hydrogel structures using a suspended manufacturing process where a cell containing
hydrogel is deposited into a supportive bed. This process allows for the production of hydrogels in complex geometries such as the carotid
arteries shown here. (Adapted with permission from Senior JJ, Cooke ME, Grover LM, Smith AM. Fabrication of complex hydrogel structures
using suspended layer additive manufacturing (SLAM).
Adv Funct Mater
2019;
29
: 1904845.)
Exemplars: materials in development
Synthetic and engineered polymers Synthetic polymers are those made from chemical processing of natural components or from complex synthesis from precursors such as fossil fuels. There are a great many synthetic polymers that are used in the body , including polymethyl  methacrylate - cells (PMMA), which is used to make intraocular lenses and as a cement in joint replacement; Dacron™ (Terylene™), used in vascular grafts; and a range of  resorbable materials, including poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and copolymers thereof  (PLGA). These resorbable polymers have been used in the body for some time as sutures and in other degradable structures. Moreover, they have become widely used in regenerative medicine for the produc - tion of  sca ff olds, on which cells can be seeded and grown prior to, during or after implantation into the body . Synthetic mate - rials can be processed in a range of  di ff erent ways in order to provide a structure optimised to the intended purpose. P orous monoliths can be produced by foaming processes, whic h can - include but are not limited to infiltration and then expansion of  supercritical carbon dioxide in order to create a foamed structure. The pore sizes of  such structures may be tailored by modifying process conditions or material compositions ( Figure 4.6 ). Such polymers can also be formed as spheres by using emul - sion processing or they can be processed into a mat of  fibres using a process known as electrospinning, whereby charged threads of  polymer are drawn using an electric field and sub - sequently deposited onto a surface ( Figur e 4.7 ). Manufactured in this way , the materials tend to have a very high surface area and can exhibit structural features across the same length scales as the fibrous components of  the extracellular matrix. As a consequence, electrospun patches have been designed for the re pair of  anatomical structures such as the rotator cu ff and have even been used to explore how modifications in matrix geometry may result in pathology . Bioceramics A range of  ceramics have been used for the delivery of  com - pounds that can trigger the regeneration of  mineralised tissues.
(b)
Figure 4.6
Electron micrograph of a poly(lactic acid) scaffold foamed
using supercritical carbon dioxide
(a)
with pore structure assessed
using micro-computed tomography
(b)
. (Adapted with permission
from Collins NJ, Leeke GA, Bridson RH
et al
. The in
/f_l
uence of silica
on pore diameter and distribution
in PLA scaffolds produced using
supercritical CO
.
J Mater Sci: Mater Med
2008;
19
: 1497–502.)
2
® The most widely used such material is Bioglass , a glassy material (a ceramic with no crystal structure) containing calcium, silicate and phosphate ions (Na O–CaO–P O 2 2 5 When placed into an aqueous environment, such as within body fluids, the surface of  the ceramic material can break down, releasing the component ions into the surrounding liquid. This can result in the deposition of  a bone-like mineral across the surface of  the material itself, which can encourage the mate rial to bond to surrounding hard and soft tissues. In addition, the eluted ions can trigger specific biological responses that include the recruitment and di ff er entiation of mesenchymal ® cell populations. Bioglass has been used in a range of medical products and has recently been widely applied in remineralis ® ing toothpaste formulations (as Novamin ). The incorporation of  other ions, such as strontium and lithium, into the glassy matrix can drive specific biological processes, enhancing the ® process of  bone formation. Bioglass can be manufactured as a foamed structure and as a monolith. It is most frequently β mer matrices to drive the process of  bone integration. Hydrogels Hydrogels are an emerging and increasingly researched class of  materials in regenerative medicine. They consist of hydrophilic polymers that are typically dispersed in an aqueous component to form a hydrocolloid. Interactions between the individual polymer chains can then be formed by modifying the temperature, adding ions or adding a chemical cross-linking agent. The resulting network retains water and forms a solid but highly hydrated structure (typically c . 99wt% water). The high water content of  these materials means that nutrients, oxygen and metabolic by-products can di ff use through them. As a consequence, they have been widely used for the encapsu - lation of  cells, typically allowing the maintenance of  high levels of  viability . Alginate, a seaweed-derived polysaccharide blend, for example, has been used to protect pancreatic islets fr om immunological attack. In recent years, this approach has been refined to allow for pancreatic islets to be shipped between –SiO ). medical centres ( Figure 4.8 ). Other materials that form gels 2 include chitosan, gellan, collagen and hyaluronic acid. In addition to these biologically derived materials, hydrogels may also be formed from synthetic polymers, including poly(eth - ylene glycol), and these can be chemically modified to provide - specific biological stimuli to entrapped cells. Additive layer manufacturing and hydrogels As described above, hydrogels have been used for cell encap - sulation. The potential to modify both the chemical and - mechanical properties exhibited by these materials makes them perfect candidates for the growth of  tissues outside the body , prior to eventual implantation into a defect site inside the bod y . As a consequence, hydrogel-based structures have provided sca ff olding for many di ff erent engineered tissues, ranging from bone through to brain. However, a major issue
Figure 4.7
Electrospun poly(caprolactone)
/f_i
bres intended for use in
tendon reconstruction (courtesy of Dr Anita Ghag).
(a)
Pump
Vibrating
technology
100
/uni00A0/uni03BC
m
Figure 4.8
Schematic of
(a)
pancreatic
-cell encapsulation and
Ellis MJ, Grover LM. Encapsulation and
/f_l
uidization maintains the viability and glucose sensitivity of beta-cells.
3(8): 1750–7.)
(b)
Bioreactor
column
Feeding
vessel
Peristaltic
pump
(b)
bioreactor cultivation. (Adapted with permission from Nikravesh N, Cox SC,
ACS Biomater Sci Eng
2017;
with hydrogels is that they are normally extremely weak during the gelation process, meaning that they will slump and lose their shape when cast. In order to get around this major issue, printed structures may be suspended in a secondary (often shear thinning) medium that provides both a level of support during the printing process and time for the hydrogel to develop su ffi cient mechanical integrity to support its own weight. Once this point has been reached, the structure can be removed from the supporting medium and washed prior to onward culturing. A wide range of  substances have been used to provide support, ® including Pluronic and agarose; however, the most widely used method currently is known as free-form reversible embedding of  suspended hydrogels (FRESH), which uses gelatin. The FRESH method utilises a macerated gelatin-based support matrix. The particulate nature of  the gelatin means that the printed hydrogel (which may or may not contain cells) can be deposited within the bed and will be supported while the mechanical properties exhibited by the as-deposited hydrogel become fully developed. The supporting matrix can then be melted (at 37°C) to leave the complex hydrogel structure. Other suspended printing methods have been developed that do not require the use of  an animal-derived support medium (and that are therefore compatible with translation to the clinic) or the elevation of  temperature to remove the supporting phase. One such method, suspended layer additive manufacturing (SLAM), uses a supportive matrix formed from hydrogel partic - ulate systems that shear thin but exhibit rapid elastic recover y; this allows for the production of  high-resolution prints that can be removed from the supporting bed by the application of  a simple wash with water. This method enab les the production of  structures exhibiting complex morphology , such as the carotid artery ( Figure 4.9 ) , and can even be used to produce structures formed or modified from multiple material types, meaning that structures can be tailored with high resolution. With clinical relevance, SLAM has been used to man ufacture osteochondral plugs containing both bony and cartilaginous regions. At present, these technologies are most likely to have an impact on disease modelling and drug candidate evaluation, but in the longer term they may emerge as key technologies facilitating tissue regeneration.
Fluid gel print bed
Cell containing hydrogel
Cross-linker (causes the
construct to solidify)
Figure 4.9
Schematic showing the formation of complex hydrogel structures using a suspended manufacturing process where a cell containing
hydrogel is deposited into a supportive bed. This process allows for the production of hydrogels in complex geometries such as the carotid
arteries shown here. (Adapted with permission from Senior JJ, Cooke ME, Grover LM, Smith AM. Fabrication of complex hydrogel structures
using suspended layer additive manufacturing (SLAM).
Adv Funct Mater
2019;
29
: 1904845.)

Exemplars  molecules in action
Exemplars: molecules in action
Healing skin without scarring All tissues in the body , when damaged, are subject to a conserved process by which cells are recruited to the wound to remove damaged tissue fragments and then deposit and subsequently remodel the tissue (see Chapter 3 ). In the case of  the skin, the initial damage results in bleeding into the wound followed by haemostasis and, when the platelets within the clot degranulate, the secretion of  proinflammatory factors. The tumour necrosis factor alpha (TNF α ), interleukin (IL) 1 and IL-6, platelet- derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) recruit monocytes and endothelial cells into the wound bed to begin the wound-healing process. These cells release further cytokines, such as transforming growth factor beta 1 (TGF β 1), that mediate the formation of  new extracellular matrix (normally initially collagen III in skin). Over time, the collagen III is remodelled and replaced with collagen I, which is normally deposited in a basket weave pattern. This process very e ffi ciently closes wounds and, when not too severe, healing can be with minimal scar formation. In the case of  severe damage or infection, the inflammatory response can ‘overshoot’, resulting in the overproduction of cytokines such as TGF β 1, which drive the recruitment of  excess myofibroblasts to the wound site. This leads to the deposition and subsequent overcontraction of  collagen matrix, which can result in the classic appearance of  a scar. A number of  interventions have targeted this process with the aim of preventing the formation of a scar on the skin. Of note was the identification and isolation of  TGF β 3, which plays a role in regulating extracellular matrix formation. This molecule was shown to regulate wound healing by blocking the TGF β 1 and -2 receptors on the cell surface and by modulating the penetration of  cells in a newly forming tissue. TGF subsequently developed as a therapeutic agent for use within the skin, with the aim of  preventing scar formation. As TGF is a large, protein-based drug it was challenging to manufacture and purify and so was expensive in comparison with small- molecule drugs. Although this molecule had significant poten tial as an antiscarring agent in phase I and II trials, ultimately it was not adopted. This is an exemplar of  a translational approach, working from a proposed mechanism through to stepwise clinical trials to test e ff ectiveness. Molecular delivery to prevent ocular fibrosis Another example of  the function of  a tissue being significantly impaired by fibrosis is the eye. When the cornea is damaged, as long as the wound is kept clean, re-epithelialisation of  the surface can occur relatively quickly with full restoration of the corneal surface within 7 days or so. However, if  there is an infection on the surface of the eye there can be a potent inflammatory response that results in the rapid and disordered deposition of  collagenous tissue across the ocular surface. patient can become blind. Recent work to create a therapeutic - agent to prevent ocular scarring has focused on the localised delivery of  a TGF β 1 antagonist called decorin. Decorin is a proteoglycan with a high a ffi nity for collagen I, which it decorates the surface of  in normal extracellular matrix. When free in solution, decorin also has an a ffi nity to seven di ff erent cytokine molecules, one of  which is TGF β 1, and has been shown to modulate fibrosis. A decorin-containing eye drop is able to deliver a sustained dose of  decorin to the surface of  the cornea following damage and subsequent infection. The drop has been shown to be e ff ective in preclinical models of  ocular fibrosis, facilitating the restoration of  a transparent ocular surface with a significant reduction in markers that are usually indicative of scar formation ( Figure 4.10 ). Challenges to the delivery of molecules Despite the opportunities a ff orded by molecules in tissue regeneration, major challenges remain that relate to getting the therapeutic agent to the right part of  the body at the right time. Small molecules tend to di ff use rapidly through excipient (delivery) materials and so are rapidly released into the tissue environment, meaning that a strategy for sustained delivery is a necessity if  repeat administration of  the drug molecule is to be avoided. A number of  implantable materials exist that allow for the slow localised release of  molecules at the desired site of  application. The majority of  these are dense, degrad - able polymers that can be implanted locally and allowed to degrade, thus enabling sustained release of  the therapeutic agent. The issues are potentially even more significant for large biomolecules such as proteins, which tend to be v ery sensitive to local environmental changes, with associated reductions in bioactivity . They can also have extremely high activities at very low concentrations and can be extremely expensive. In addi - tion, excipients for these therapeutic products must be chosen very carefully so that potency is maintained and storage time maximised. Exemplars: molecules in action
Healing skin without scarring All tissues in the body , when damaged, are subject to a conserved process by which cells are recruited to the wound to remove damaged tissue fragments and then deposit and subsequently remodel the tissue (see Chapter 3 ). In the case of  the skin, the initial damage results in bleeding into the wound followed by haemostasis and, when the platelets within the clot degranulate, the secretion of  proinflammatory factors. The tumour necrosis factor alpha (TNF α ), interleukin (IL) 1 and IL-6, platelet- derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) recruit monocytes and endothelial cells into the wound bed to begin the wound-healing process. These cells release further cytokines, such as transforming growth factor beta 1 (TGF β 1), that mediate the formation of  new extracellular matrix (normally initially collagen III in skin). Over time, the collagen III is remodelled and replaced with collagen I, which is normally deposited in a basket weave pattern. This process very e ffi ciently closes wounds and, when not too severe, healing can be with minimal scar formation. In the case of  severe damage or infection, the inflammatory response can ‘overshoot’, resulting in the overproduction of cytokines such as TGF β 1, which drive the recruitment of  excess myofibroblasts to the wound site. This leads to the deposition and subsequent overcontraction of  collagen matrix, which can result in the classic appearance of  a scar. A number of  interventions have targeted this process with the aim of preventing the formation of a scar on the skin. Of note was the identification and isolation of  TGF β 3, which plays a role in regulating extracellular matrix formation. This molecule was shown to regulate wound healing by blocking the TGF β 1 and -2 receptors on the cell surface and by modulating the penetration of  cells in a newly forming tissue. TGF subsequently developed as a therapeutic agent for use within the skin, with the aim of  preventing scar formation. As TGF is a large, protein-based drug it was challenging to manufacture and purify and so was expensive in comparison with small- molecule drugs. Although this molecule had significant poten tial as an antiscarring agent in phase I and II trials, ultimately it was not adopted. This is an exemplar of  a translational approach, working from a proposed mechanism through to stepwise clinical trials to test e ff ectiveness. Molecular delivery to prevent ocular fibrosis Another example of  the function of  a tissue being significantly impaired by fibrosis is the eye. When the cornea is damaged, as long as the wound is kept clean, re-epithelialisation of  the surface can occur relatively quickly with full restoration of the corneal surface within 7 days or so. However, if  there is an infection on the surface of the eye there can be a potent inflammatory response that results in the rapid and disordered deposition of  collagenous tissue across the ocular surface. patient can become blind. Recent work to create a therapeutic - agent to prevent ocular scarring has focused on the localised delivery of  a TGF β 1 antagonist called decorin. Decorin is a proteoglycan with a high a ffi nity for collagen I, which it decorates the surface of  in normal extracellular matrix. When free in solution, decorin also has an a ffi nity to seven di ff erent cytokine molecules, one of  which is TGF β 1, and has been shown to modulate fibrosis. A decorin-containing eye drop is able to deliver a sustained dose of  decorin to the surface of  the cornea following damage and subsequent infection. The drop has been shown to be e ff ective in preclinical models of  ocular fibrosis, facilitating the restoration of  a transparent ocular surface with a significant reduction in markers that are usually indicative of scar formation ( Figure 4.10 ). Challenges to the delivery of molecules Despite the opportunities a ff orded by molecules in tissue regeneration, major challenges remain that relate to getting the therapeutic agent to the right part of  the body at the right time. Small molecules tend to di ff use rapidly through excipient (delivery) materials and so are rapidly released into the tissue environment, meaning that a strategy for sustained delivery is a necessity if  repeat administration of  the drug molecule is to be avoided. A number of  implantable materials exist that allow for the slow localised release of  molecules at the desired site of  application. The majority of  these are dense, degrad - able polymers that can be implanted locally and allowed to degrade, thus enabling sustained release of  the therapeutic agent. The issues are potentially even more significant for large biomolecules such as proteins, which tend to be v ery sensitive to local environmental changes, with associated reductions in bioactivity . They can also have extremely high activities at very low concentrations and can be extremely expensive. In addi - tion, excipients for these therapeutic products must be chosen very carefully so that potency is maintained and storage time maximised. Exemplars: molecules in action
Healing skin without scarring All tissues in the body , when damaged, are subject to a conserved process by which cells are recruited to the wound to remove damaged tissue fragments and then deposit and subsequently remodel the tissue (see Chapter 3 ). In the case of  the skin, the initial damage results in bleeding into the wound followed by haemostasis and, when the platelets within the clot degranulate, the secretion of  proinflammatory factors. The tumour necrosis factor alpha (TNF α ), interleukin (IL) 1 and IL-6, platelet- derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) recruit monocytes and endothelial cells into the wound bed to begin the wound-healing process. These cells release further cytokines, such as transforming growth factor beta 1 (TGF β 1), that mediate the formation of  new extracellular matrix (normally initially collagen III in skin). Over time, the collagen III is remodelled and replaced with collagen I, which is normally deposited in a basket weave pattern. This process very e ffi ciently closes wounds and, when not too severe, healing can be with minimal scar formation. In the case of  severe damage or infection, the inflammatory response can ‘overshoot’, resulting in the overproduction of cytokines such as TGF β 1, which drive the recruitment of  excess myofibroblasts to the wound site. This leads to the deposition and subsequent overcontraction of  collagen matrix, which can result in the classic appearance of  a scar. A number of  interventions have targeted this process with the aim of preventing the formation of a scar on the skin. Of note was the identification and isolation of  TGF β 3, which plays a role in regulating extracellular matrix formation. This molecule was shown to regulate wound healing by blocking the TGF β 1 and -2 receptors on the cell surface and by modulating the penetration of  cells in a newly forming tissue. TGF subsequently developed as a therapeutic agent for use within the skin, with the aim of  preventing scar formation. As TGF is a large, protein-based drug it was challenging to manufacture and purify and so was expensive in comparison with small- molecule drugs. Although this molecule had significant poten tial as an antiscarring agent in phase I and II trials, ultimately it was not adopted. This is an exemplar of  a translational approach, working from a proposed mechanism through to stepwise clinical trials to test e ff ectiveness. Molecular delivery to prevent ocular fibrosis Another example of  the function of  a tissue being significantly impaired by fibrosis is the eye. When the cornea is damaged, as long as the wound is kept clean, re-epithelialisation of  the surface can occur relatively quickly with full restoration of the corneal surface within 7 days or so. However, if  there is an infection on the surface of the eye there can be a potent inflammatory response that results in the rapid and disordered deposition of  collagenous tissue across the ocular surface. patient can become blind. Recent work to create a therapeutic - agent to prevent ocular scarring has focused on the localised delivery of  a TGF β 1 antagonist called decorin. Decorin is a proteoglycan with a high a ffi nity for collagen I, which it decorates the surface of  in normal extracellular matrix. When free in solution, decorin also has an a ffi nity to seven di ff erent cytokine molecules, one of  which is TGF β 1, and has been shown to modulate fibrosis. A decorin-containing eye drop is able to deliver a sustained dose of  decorin to the surface of  the cornea following damage and subsequent infection. The drop has been shown to be e ff ective in preclinical models of  ocular fibrosis, facilitating the restoration of  a transparent ocular surface with a significant reduction in markers that are usually indicative of scar formation ( Figure 4.10 ). Challenges to the delivery of molecules Despite the opportunities a ff orded by molecules in tissue regeneration, major challenges remain that relate to getting the therapeutic agent to the right part of  the body at the right time. Small molecules tend to di ff use rapidly through excipient (delivery) materials and so are rapidly released into the tissue environment, meaning that a strategy for sustained delivery is a necessity if  repeat administration of  the drug molecule is to be avoided. A number of  implantable materials exist that allow for the slow localised release of  molecules at the desired site of  application. The majority of  these are dense, degrad - able polymers that can be implanted locally and allowed to degrade, thus enabling sustained release of  the therapeutic agent. The issues are potentially even more significant for large biomolecules such as proteins, which tend to be v ery sensitive to local environmental changes, with associated reductions in bioactivity . They can also have extremely high activities at very low concentrations and can be extremely expensive. In addi - tion, excipients for these therapeutic products must be chosen very carefully so that potency is maintained and storage time maximised.

FURTHER READING
FURTHER READING
16 : Fisher S. Handbook of  regenerative medicine and tissue engineering . New Y ork: Hayle Medical, 2015. Wagner WR, Sakiyama-Elbert SE, Zhang G, Yaszemski MJ. Biomate - rials science: an introduction to materials in medicine , 4th edn. Oxford: Academic Press, 2020. FURTHER READING
16 : Fisher S. Handbook of  regenerative medicine and tissue engineering . New Y ork: Hayle Medical, 2015. Wagner WR, Sakiyama-Elbert SE, Zhang G, Yaszemski MJ. Biomate - rials science: an introduction to materials in medicine , 4th edn. Oxford: Academic Press, 2020. FURTHER READING
16 : Fisher S. Handbook of  regenerative medicine and tissue engineering . New Y ork: Hayle Medical, 2015. Wagner WR, Sakiyama-Elbert SE, Zhang G, Yaszemski MJ. Biomate - rials science: an introduction to materials in medicine , 4th edn. Oxford: Academic Press, 2020.

In vitro differentiation of stem cells to speciali
In vitro differentiation of stem cells to specialised tissue cells
There is an enormous research e ff ort aimed at better under - standing the factors responsible for cell fate decisions and establishing e ff ective and reproducible protocols that can be used to di ff erentiate stem cells in vitro into the desired type of  specialised cell. Typically , such protocols use culture in chemically defined media containing cocktails of  small mole - cules that stimulate or inhibit key signalling pathways, along with cytokines, growth factors and chemicals. It is becoming increasingly clear that exposure to certain biomaterials and the physical attributes of  a sca ff old, including its surface character - istics, also promote stem cell di ff erentiation along a particular lineage. Mechanical stress also influences cell fate decisions. After stem cells have been subjected to in vitro di ff erentiation, it is essential that the purity of  the di ff erentiated cells and the absence of  undi ff erentiated stem cells are confirmed to reduce the risk of tumour transmission. The cells must also be fully - characterised and their function confirmed before they are used for therapeutic purposes. In vitro differentiation of stem cells to specialised tissue cells
There is an enormous research e ff ort aimed at better under - standing the factors responsible for cell fate decisions and establishing e ff ective and reproducible protocols that can be used to di ff erentiate stem cells in vitro into the desired type of  specialised cell. Typically , such protocols use culture in chemically defined media containing cocktails of  small mole - cules that stimulate or inhibit key signalling pathways, along with cytokines, growth factors and chemicals. It is becoming increasingly clear that exposure to certain biomaterials and the physical attributes of  a sca ff old, including its surface character - istics, also promote stem cell di ff erentiation along a particular lineage. Mechanical stress also influences cell fate decisions. After stem cells have been subjected to in vitro di ff erentiation, it is essential that the purity of  the di ff erentiated cells and the absence of  undi ff erentiated stem cells are confirmed to reduce the risk of tumour transmission. The cells must also be fully - characterised and their function confirmed before they are used for therapeutic purposes.

In vitro differentiation of stem cells to specialised tissue cells
In vitro differentiation of stem cells to specialised tissue cells
There is an enormous research e ff ort aimed at better under - standing the factors responsible for cell fate decisions and establishing e ff ective and reproducible protocols that can be used to di ff erentiate stem cells in vitro into the desired type of  specialised cell. Typically , such protocols use culture in chemically defined media containing cocktails of  small mole - cules that stimulate or inhibit key signalling pathways, along with cytokines, growth factors and chemicals. It is becoming increasingly clear that exposure to certain biomaterials and the physical attributes of  a sca ff old, including its surface character - istics, also promote stem cell di ff erentiation along a particular lineage. Mechanical stress also influences cell fate decisions. After stem cells have been subjected to in vitro di ff erentiation, it is essential that the purity of  the di ff erentiated cells and the absence of  undi ff erentiated stem cells are confirmed to reduce the risk of tumour transmission. The cells must also be fully - characterised and their function confirmed before they are used for therapeutic purposes.

Induced pluripotent stem cells
Induced pluripotent stem cells
10 The discovery in 2006 by Shinya Yamanaka, building on the 11 earlier work of  John Gurdon, that certain types of  specialised adult cells could be reprogrammed using genetic manipulation to become embryonic-like iPSCs was a major breakthrough. Using retroviral or lentiviral transfection to introduce a combi nation of  transcription factors (OCT3/4, SOX2 and either Kruppel-like factor and C-MYC [together designated the OSKM reprogramming factors] or NANOG and LIN28), it was shown that specialised somatic cells can be reprogrammed to become stem cells . Moreover, iPSCs proliferate in vitro e ffi ciently as ESCs and are pluripotent, thereby circumventing concerns about the use of  human embryos. Importantly , the development of  iPSCs also means that, at least in principle, an intended recipient of  stem cell therapy can themselves provide James Thomson , b. 1958, Professor and Director of  Regenerative Biology , Morgridge Institute for Research, University of  Wisconsin-Madison, Madison, WI, USA. Shinya Yamanaka , b. 1962, Japanese stem cell researcher, winner of  the Nobel Prize in Physiology or Medicine in 2012. Sir John Bertrand Gurdon , b. 1933, British developmental biologist, winner of  the Nobel Prize in Physiology or Medicine in 2012 with Shinya Yamanaka. that can then be directed to di ff erentiate into the desired specialised cell type for therapy; because such cells would be autologous they would not provoke an immunological rejection response ( Figure 4.4 ). Alternatively , iPSCs could be obtained from a number of  volunteer donors selected on the basis of  their HLA type and stored to create a national or international tissue bank of iPSCs. Lines of iPSCs could then be c hosen from the bank to provide a fully or partially matched cell transplant for recipients, eliminating or reducing the need for immunosuppression to prevent immunological rejection. One of  the problems of  reprogramming somatic cells to become iPSCs using retroviruses is that genomic integration of  the virus may lead to activation of  oncogenic genes, caus - ing tumorigenesis. To reduce this risk, non-retr oviral vectors have been used (such as adenovirus and Sendai virus vectors, which do not insert their own genes into the host cell genome) or plasmids, episomal vectors and synthetic RNA. There has - also been much recent progress in identifying combinations of  small molecules, growth factors and chemicals that mimic the e ff ect of viral transfection with transcription factors and obviate the need for viral vectors altogether. The production process from sourcing cells (e.g. skin fibroblasts or peripheral blood mononuclear cells) to obtaining an adequate number of validated iPSCs may take several weeks. - Induced pluripotent stem cells
10 The discovery in 2006 by Shinya Yamanaka, building on the 11 earlier work of  John Gurdon, that certain types of  specialised adult cells could be reprogrammed using genetic manipulation to become embryonic-like iPSCs was a major breakthrough. Using retroviral or lentiviral transfection to introduce a combi nation of  transcription factors (OCT3/4, SOX2 and either Kruppel-like factor and C-MYC [together designated the OSKM reprogramming factors] or NANOG and LIN28), it was shown that specialised somatic cells can be reprogrammed to become stem cells . Moreover, iPSCs proliferate in vitro e ffi ciently as ESCs and are pluripotent, thereby circumventing concerns about the use of  human embryos. Importantly , the development of  iPSCs also means that, at least in principle, an intended recipient of  stem cell therapy can themselves provide James Thomson , b. 1958, Professor and Director of  Regenerative Biology , Morgridge Institute for Research, University of  Wisconsin-Madison, Madison, WI, USA. Shinya Yamanaka , b. 1962, Japanese stem cell researcher, winner of  the Nobel Prize in Physiology or Medicine in 2012. Sir John Bertrand Gurdon , b. 1933, British developmental biologist, winner of  the Nobel Prize in Physiology or Medicine in 2012 with Shinya Yamanaka. that can then be directed to di ff erentiate into the desired specialised cell type for therapy; because such cells would be autologous they would not provoke an immunological rejection response ( Figure 4.4 ). Alternatively , iPSCs could be obtained from a number of  volunteer donors selected on the basis of  their HLA type and stored to create a national or international tissue bank of iPSCs. Lines of iPSCs could then be c hosen from the bank to provide a fully or partially matched cell transplant for recipients, eliminating or reducing the need for immunosuppression to prevent immunological rejection. One of  the problems of  reprogramming somatic cells to become iPSCs using retroviruses is that genomic integration of  the virus may lead to activation of  oncogenic genes, caus - ing tumorigenesis. To reduce this risk, non-retr oviral vectors have been used (such as adenovirus and Sendai virus vectors, which do not insert their own genes into the host cell genome) or plasmids, episomal vectors and synthetic RNA. There has - also been much recent progress in identifying combinations of  small molecules, growth factors and chemicals that mimic the e ff ect of viral transfection with transcription factors and obviate the need for viral vectors altogether. The production process from sourcing cells (e.g. skin fibroblasts or peripheral blood mononuclear cells) to obtaining an adequate number of validated iPSCs may take several weeks. - Induced pluripotent stem cells
10 The discovery in 2006 by Shinya Yamanaka, building on the 11 earlier work of  John Gurdon, that certain types of  specialised adult cells could be reprogrammed using genetic manipulation to become embryonic-like iPSCs was a major breakthrough. Using retroviral or lentiviral transfection to introduce a combi nation of  transcription factors (OCT3/4, SOX2 and either Kruppel-like factor and C-MYC [together designated the OSKM reprogramming factors] or NANOG and LIN28), it was shown that specialised somatic cells can be reprogrammed to become stem cells . Moreover, iPSCs proliferate in vitro e ffi ciently as ESCs and are pluripotent, thereby circumventing concerns about the use of  human embryos. Importantly , the development of  iPSCs also means that, at least in principle, an intended recipient of  stem cell therapy can themselves provide James Thomson , b. 1958, Professor and Director of  Regenerative Biology , Morgridge Institute for Research, University of  Wisconsin-Madison, Madison, WI, USA. Shinya Yamanaka , b. 1962, Japanese stem cell researcher, winner of  the Nobel Prize in Physiology or Medicine in 2012. Sir John Bertrand Gurdon , b. 1933, British developmental biologist, winner of  the Nobel Prize in Physiology or Medicine in 2012 with Shinya Yamanaka. that can then be directed to di ff erentiate into the desired specialised cell type for therapy; because such cells would be autologous they would not provoke an immunological rejection response ( Figure 4.4 ). Alternatively , iPSCs could be obtained from a number of  volunteer donors selected on the basis of  their HLA type and stored to create a national or international tissue bank of iPSCs. Lines of iPSCs could then be c hosen from the bank to provide a fully or partially matched cell transplant for recipients, eliminating or reducing the need for immunosuppression to prevent immunological rejection. One of  the problems of  reprogramming somatic cells to become iPSCs using retroviruses is that genomic integration of  the virus may lead to activation of  oncogenic genes, caus - ing tumorigenesis. To reduce this risk, non-retr oviral vectors have been used (such as adenovirus and Sendai virus vectors, which do not insert their own genes into the host cell genome) or plasmids, episomal vectors and synthetic RNA. There has - also been much recent progress in identifying combinations of  small molecules, growth factors and chemicals that mimic the e ff ect of viral transfection with transcription factors and obviate the need for viral vectors altogether. The production process from sourcing cells (e.g. skin fibroblasts or peripheral blood mononuclear cells) to obtaining an adequate number of validated iPSCs may take several weeks. -

Introduction
INTRODUCTION
Tissue engineering and regenerative medicine are relatively new but rapidly expanding multidisciplinary fields relevant to surgery that have the potential to transform the treatment of  a wide range of  human diseases. The ability of  tissues to undergo spontaneous repair and regeneration is highly variable and, in most cases, limited. This has driven the devel opment of  approaches that harness the biology at the site of tissue damage to mediate regeneration through the localised delivery of cells, materials and molecules. The continuing impro vement in our understanding of how tissues are formed and how they heal underpins the continuous development of novel approaches. As these technologies improve, translate and build up clinical evidence of  e ff ectiveness, the prospect of actual tissue regeneration, not just repair, will establish clinical utility . In this chapter, we explore the development of  the tissue engineering paradigm ( Figure 4.1 ) and the constituent processes that have been used to enhance tissue healing by considering cells, materials and molecules and the interplay between them, alongside key exemplars. We further highlight the opportunities, challenges and likely future directions for the field.

KEY AREAS OF UNDERPINNING SCIENCE
KEY AREAS OF UNDERPINNING SCIENCE
Advances in tissue engineering and more broadly regenera tive medicine are underpinned by developments in both the physical and biological sciences, building on the classical tissue engineering paradigm ( Figure 4.1 ). An improved understand ing of  developmental biology and the cues that direct stem cell fa te have been key to advancement of  the field. A better understanding of  the stem cell niche has enabled scientists to propose changes to molecular and mechanical properties that could bring about modifi  ed cell behaviour . This has been realised by advances in materials science, which have been crit ical in the development of  structures (sca ff olds), onto and into used to localise cells and molecules to a specific site within the body . Notwithstanding the potential o ff ered by these therapies, it should be emphasised that the whole field is still at a relatively early stage of development. Although there are examples where tissue engineering and regenerative therapies have already been introduced into clinical practice, for example the repair of  damaged cartilage, most potential regenerative ther - apies have not yet entered routine surgical practice as there are considerable barriers to be overcome before this translational step can be achieved. We have divided the chapter into sections r elating to cells, materials and molecules, while recognising the interplay and composite therapeutic solutions that ultimately arise.
Pluripotent
cells
O
O
Polymers
n
CH
3
Ceramics
Hydrogel
Figure 4.1
The tissue engineering par-
adigm in the context of regenerative
therapies.
KEY AREAS OF UNDERPINNING SCIENCE
Advances in tissue engineering and more broadly regenera tive medicine are underpinned by developments in both the physical and biological sciences, building on the classical tissue engineering paradigm ( Figure 4.1 ). An improved understand ing of  developmental biology and the cues that direct stem cell fa te have been key to advancement of  the field. A better understanding of  the stem cell niche has enabled scientists to propose changes to molecular and mechanical properties that could bring about modifi  ed cell behaviour . This has been realised by advances in materials science, which have been crit ical in the development of  structures (sca ff olds), onto and into used to localise cells and molecules to a specific site within the body . Notwithstanding the potential o ff ered by these therapies, it should be emphasised that the whole field is still at a relatively early stage of development. Although there are examples where tissue engineering and regenerative therapies have already been introduced into clinical practice, for example the repair of  damaged cartilage, most potential regenerative ther - apies have not yet entered routine surgical practice as there are considerable barriers to be overcome before this translational step can be achieved. We have divided the chapter into sections r elating to cells, materials and molecules, while recognising the interplay and composite therapeutic solutions that ultimately arise.
Pluripotent
cells
O
O
Polymers
n
CH
3
Ceramics
Hydrogel
Figure 4.1
The tissue engineering par-
adigm in the context of regenerative
therapies.
KEY AREAS OF UNDERPINNING SCIENCE
Advances in tissue engineering and more broadly regenera tive medicine are underpinned by developments in both the physical and biological sciences, building on the classical tissue engineering paradigm ( Figure 4.1 ). An improved understand ing of  developmental biology and the cues that direct stem cell fa te have been key to advancement of  the field. A better understanding of  the stem cell niche has enabled scientists to propose changes to molecular and mechanical properties that could bring about modifi  ed cell behaviour . This has been realised by advances in materials science, which have been crit ical in the development of  structures (sca ff olds), onto and into used to localise cells and molecules to a specific site within the body . Notwithstanding the potential o ff ered by these therapies, it should be emphasised that the whole field is still at a relatively early stage of development. Although there are examples where tissue engineering and regenerative therapies have already been introduced into clinical practice, for example the repair of  damaged cartilage, most potential regenerative ther - apies have not yet entered routine surgical practice as there are considerable barriers to be overcome before this translational step can be achieved. We have divided the chapter into sections r elating to cells, materials and molecules, while recognising the interplay and composite therapeutic solutions that ultimately arise.
Pluripotent
cells
O
O
Polymers
n
CH
3
Ceramics
Hydrogel
Figure 4.1
The tissue engineering par-
adigm in the context of regenerative
therapies.

Learning objectives
Learning objectives
To understand: The potential opportunities afforded by tissue engineering • and regenerative therapy The nature of stem cells, including somatic and adult stem • cells, embryonic stem cells and induced pluripotent stem cells Learning objectives
To understand: The potential opportunities afforded by tissue engineering • and regenerative therapy The nature of stem cells, including somatic and adult stem • cells, embryonic stem cells and induced pluripotent stem cells Learning objectives
To understand: The potential opportunities afforded by tissue engineering • and regenerative therapy The nature of stem cells, including somatic and adult stem • cells, embryonic stem cells and induced pluripotent stem cells

MATERIALS
MATERIALS
A large number of  materials have been used in tissue engineer ing and regenerative medicine, either as delivery vehicles or as sca ff olds; it is beyond the scope of this chapter to describe them extensively (see Further reading ). In simple terms, materials can be either natural or synthetic and further characterised by their mechanical properties, which will often need to take account of  porosity . A further layer of  complexity relates to specific molecules within and upon the material and the intended targeting or seeding with cells. In this section, we will focus on synthetic polymers, hydrogels and osteoinductive ® ceramics such as Bioglass . These materials have been selected because of  their broad relevance in tissue engineering and regenerative medicine. MATERIALS
A large number of  materials have been used in tissue engineer ing and regenerative medicine, either as delivery vehicles or as sca ff olds; it is beyond the scope of this chapter to describe them extensively (see Further reading ). In simple terms, materials can be either natural or synthetic and further characterised by their mechanical properties, which will often need to take account of  porosity . A further layer of  complexity relates to specific molecules within and upon the material and the intended targeting or seeding with cells. In this section, we will focus on synthetic polymers, hydrogels and osteoinductive ® ceramics such as Bioglass . These materials have been selected because of  their broad relevance in tissue engineering and regenerative medicine. MATERIALS
A large number of  materials have been used in tissue engineer ing and regenerative medicine, either as delivery vehicles or as sca ff olds; it is beyond the scope of this chapter to describe them extensively (see Further reading ). In simple terms, materials can be either natural or synthetic and further characterised by their mechanical properties, which will often need to take account of  porosity . A further layer of  complexity relates to specific molecules within and upon the material and the intended targeting or seeding with cells. In this section, we will focus on synthetic polymers, hydrogels and osteoinductive ® ceramics such as Bioglass . These materials have been selected because of  their broad relevance in tissue engineering and regenerative medicine.

MOLECULES
MOLECULES
Modification of the tissue environment during healing can be achieved by the delivery of  molecules that are selected and able to specifically modify biological responses at the site of  injury . In terms of  tissue regeneration, these molecules can modulate the inflammatory environment, either by enhancement or by angiogenesis. This approach has been used to optimise tissue regeneration across several applications, with specific exam ples, for the skin and the eye, described below . MOLECULES
Modification of the tissue environment during healing can be achieved by the delivery of  molecules that are selected and able to specifically modify biological responses at the site of  injury . In terms of  tissue regeneration, these molecules can modulate the inflammatory environment, either by enhancement or by angiogenesis. This approach has been used to optimise tissue regeneration across several applications, with specific exam ples, for the skin and the eye, described below . MOLECULES
Modification of the tissue environment during healing can be achieved by the delivery of  molecules that are selected and able to specifically modify biological responses at the site of  injury . In terms of  tissue regeneration, these molecules can modulate the inflammatory environment, either by enhancement or by angiogenesis. This approach has been used to optimise tissue regeneration across several applications, with specific exam ples, for the skin and the eye, described below .

OPPORTUNITIES
OPPORTUNITIES
The potential impact of  tissue engineering and regenerative therapies is so far-reaching that practising surgeons should be aware of  the resulting opportunities to improve patient management. Several conditions that could benefit from this approach are of particular relevance to surgeons because they are closely involved in assessment and treatment ( Table 4.1 Selected examples include the repair or replacement of injured or diseased cartilage, skin, pancreatic islets, bladder, intestine, heart tissue, arteries, larynx and bronchus. A longer term goal Burrill Bernard Crohn , 1884–1983, gastroenterologist, Mount Sinai Hospital, New Y ork, NY , USA, described regional ileitis in 1932. - in tissue engineering is the replacement of  diseased whole organs such as the liver and kidney , although the technical challenges here are enormous. ). Surgeons are integral to many of  the multidisciplinary research teams currently undertaking translational research in this field and will play a vital role in the future delivery and
The role and range of materials and scaffolds for tissue
•
engineering
The role and range of molecules and their delivery
•
The main challenges, safety issues and future directions
•
TABLE 4.1
Examples of tissues created by tissue
engineering and conditions they may be used to treat.
Tissue
Conditions treated
Skin
Burns and skin defects after excision or
trauma
Eye
Retinal and corneal disease
Cardiac muscle
Heart failure
Heart valves
Congenital and acquired valvular heart
disease
Cartilage and bone
Degenerative and traumatic bone and
joint disorders
Trachea and bronchus
Congenital and acquired stenosis and
resection for malignancy
Bladder
Congenital bladder malformation and
cystectomy
Anal/bladder sphincter
Incontinence
Pancreatic islets
Insulin-dependent diabetes
Large blood vessels
Atheromatous, aneurysmal and
traumatic arterial disease
Oesophagus
Benign stenosis and resection for
malignancy
Small intestine
Intestinal failure after surgical resection
for Crohn’s disease, cancer or
ischaemia
apeutic application, tissue engineering also has the potential to provide in vitro tissues that can be used to model human disease and to test therapeutic drugs for e ffi cacy and toxicity . However, it is important to emphasise that, while the potential benefit of  cell therapy and tissue engineering is undeniable, there are many technical, regulatory and safety issues to be addressed for it to have wide clinical impact. Summary box 4.1 Tissue engineering and regenerative therapies /uni25CF /uni25CF /uni25CF
These have potential to provide:
Treatment for a wide range of diseases
Clinical applications – underpinned by translational research,
delivery strategies and clinical evidence
Models to test therapeutic ef
/f_i
cacy and toxicity
OPPORTUNITIES
The potential impact of  tissue engineering and regenerative therapies is so far-reaching that practising surgeons should be aware of  the resulting opportunities to improve patient management. Several conditions that could benefit from this approach are of particular relevance to surgeons because they are closely involved in assessment and treatment ( Table 4.1 Selected examples include the repair or replacement of injured or diseased cartilage, skin, pancreatic islets, bladder, intestine, heart tissue, arteries, larynx and bronchus. A longer term goal Burrill Bernard Crohn , 1884–1983, gastroenterologist, Mount Sinai Hospital, New Y ork, NY , USA, described regional ileitis in 1932. - in tissue engineering is the replacement of  diseased whole organs such as the liver and kidney , although the technical challenges here are enormous. ). Surgeons are integral to many of  the multidisciplinary research teams currently undertaking translational research in this field and will play a vital role in the future delivery and
The role and range of materials and scaffolds for tissue
•
engineering
The role and range of molecules and their delivery
•
The main challenges, safety issues and future directions
•
TABLE 4.1
Examples of tissues created by tissue
engineering and conditions they may be used to treat.
Tissue
Conditions treated
Skin
Burns and skin defects after excision or
trauma
Eye
Retinal and corneal disease
Cardiac muscle
Heart failure
Heart valves
Congenital and acquired valvular heart
disease
Cartilage and bone
Degenerative and traumatic bone and
joint disorders
Trachea and bronchus
Congenital and acquired stenosis and
resection for malignancy
Bladder
Congenital bladder malformation and
cystectomy
Anal/bladder sphincter
Incontinence
Pancreatic islets
Insulin-dependent diabetes
Large blood vessels
Atheromatous, aneurysmal and
traumatic arterial disease
Oesophagus
Benign stenosis and resection for
malignancy
Small intestine
Intestinal failure after surgical resection
for Crohn’s disease, cancer or
ischaemia
apeutic application, tissue engineering also has the potential to provide in vitro tissues that can be used to model human disease and to test therapeutic drugs for e ffi cacy and toxicity . However, it is important to emphasise that, while the potential benefit of  cell therapy and tissue engineering is undeniable, there are many technical, regulatory and safety issues to be addressed for it to have wide clinical impact. Summary box 4.1 Tissue engineering and regenerative therapies /uni25CF /uni25CF /uni25CF
These have potential to provide:
Treatment for a wide range of diseases
Clinical applications – underpinned by translational research,
delivery strategies and clinical evidence
Models to test therapeutic ef
/f_i
cacy and toxicity
OPPORTUNITIES
The potential impact of  tissue engineering and regenerative therapies is so far-reaching that practising surgeons should be aware of  the resulting opportunities to improve patient management. Several conditions that could benefit from this approach are of particular relevance to surgeons because they are closely involved in assessment and treatment ( Table 4.1 Selected examples include the repair or replacement of injured or diseased cartilage, skin, pancreatic islets, bladder, intestine, heart tissue, arteries, larynx and bronchus. A longer term goal Burrill Bernard Crohn , 1884–1983, gastroenterologist, Mount Sinai Hospital, New Y ork, NY , USA, described regional ileitis in 1932. - in tissue engineering is the replacement of  diseased whole organs such as the liver and kidney , although the technical challenges here are enormous. ). Surgeons are integral to many of  the multidisciplinary research teams currently undertaking translational research in this field and will play a vital role in the future delivery and
The role and range of materials and scaffolds for tissue
•
engineering
The role and range of molecules and their delivery
•
The main challenges, safety issues and future directions
•
TABLE 4.1
Examples of tissues created by tissue
engineering and conditions they may be used to treat.
Tissue
Conditions treated
Skin
Burns and skin defects after excision or
trauma
Eye
Retinal and corneal disease
Cardiac muscle
Heart failure
Heart valves
Congenital and acquired valvular heart
disease
Cartilage and bone
Degenerative and traumatic bone and
joint disorders
Trachea and bronchus
Congenital and acquired stenosis and
resection for malignancy
Bladder
Congenital bladder malformation and
cystectomy
Anal/bladder sphincter
Incontinence
Pancreatic islets
Insulin-dependent diabetes
Large blood vessels
Atheromatous, aneurysmal and
traumatic arterial disease
Oesophagus
Benign stenosis and resection for
malignancy
Small intestine
Intestinal failure after surgical resection
for Crohn’s disease, cancer or
ischaemia
apeutic application, tissue engineering also has the potential to provide in vitro tissues that can be used to model human disease and to test therapeutic drugs for e ffi cacy and toxicity . However, it is important to emphasise that, while the potential benefit of  cell therapy and tissue engineering is undeniable, there are many technical, regulatory and safety issues to be addressed for it to have wide clinical impact. Summary box 4.1 Tissue engineering and regenerative therapies /uni25CF /uni25CF /uni25CF
These have potential to provide:
Treatment for a wide range of diseases
Clinical applications – underpinned by translational research,
delivery strategies and clinical evidence
Models to test therapeutic ef
/f_i
cacy and toxicity

REFERENCES
REFERENCES
1 Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV . Osteogenesis in transplants of  bone marrow cells. J Embryol Exp Morphol 1966; 381–90. 2 Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9 (5): 641–50. 3 Horwitz EM, Le Blanc K, Dominici M et al. ; International Society for Cellular Therapy . Clarification of  the nomenclature for MSC: CRISPR-Cas9 , clustered regularly interspaced short palindromic repeats and associated protein 9. Resulted in the Nobel Prize in Chemistry being awarded to Emmanuelle Charpentier and Jennifer A. Doudna in 2020. Cytotherapy 2005; 7 (5): 393–5. 4 Dominici M, Le Blanc K, Mueller I et al . Minimal criteria for defin - ing multipotent mesenchymal stromal cells. The International Soci - ety for Cellular Therapy position statement. Cytotherapy 2006; 8 (4): - 315–17. 5 Caplan AI. Mesenchymal stem cells: time to change the name! Stem - Cells Transl Med 2017; 6 : 1445–51. 6 Sipp D, Robey PG, Turner L. Clear up this stem-cell mess. Nature 2018; 561 : 455–7. 7 Viswanathan S, Shi Y , Galipeau J et al . Mesenchymal stem versus ® stromal cells: International Society for Cell & Gene Therapy (ISCT ) Mesenchymal Stromal Cell committee position statement on nomen - clature. Cytotherapy 2019; 21 (10): 1019–24. 8 Murray IR, Chahla J, Safran M et al . International expert consen - sus on a cell therapy communication tool: DOSES. J Bone Joint Surg - 2019; 101 (10): 904–11. 9 Thomson JA, Itskovitz-Eldor J, Shapiro SS et al . Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): - 1145-7. Erratum in: Science 1998; 282 (5395): 1827. - 10 Takahashi K, Yamanaka S. Induction of  pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126 (4): 663-76. 11 Gurdon JB. The developmental capacity of  nuclei taken from intesti - nal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 1962; 10 : 622-40. REFERENCES
1 Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV . Osteogenesis in transplants of  bone marrow cells. J Embryol Exp Morphol 1966; 381–90. 2 Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9 (5): 641–50. 3 Horwitz EM, Le Blanc K, Dominici M et al. ; International Society for Cellular Therapy . Clarification of  the nomenclature for MSC: CRISPR-Cas9 , clustered regularly interspaced short palindromic repeats and associated protein 9. Resulted in the Nobel Prize in Chemistry being awarded to Emmanuelle Charpentier and Jennifer A. Doudna in 2020. Cytotherapy 2005; 7 (5): 393–5. 4 Dominici M, Le Blanc K, Mueller I et al . Minimal criteria for defin - ing multipotent mesenchymal stromal cells. The International Soci - ety for Cellular Therapy position statement. Cytotherapy 2006; 8 (4): - 315–17. 5 Caplan AI. Mesenchymal stem cells: time to change the name! Stem - Cells Transl Med 2017; 6 : 1445–51. 6 Sipp D, Robey PG, Turner L. Clear up this stem-cell mess. Nature 2018; 561 : 455–7. 7 Viswanathan S, Shi Y , Galipeau J et al . Mesenchymal stem versus ® stromal cells: International Society for Cell & Gene Therapy (ISCT ) Mesenchymal Stromal Cell committee position statement on nomen - clature. Cytotherapy 2019; 21 (10): 1019–24. 8 Murray IR, Chahla J, Safran M et al . International expert consen - sus on a cell therapy communication tool: DOSES. J Bone Joint Surg - 2019; 101 (10): 904–11. 9 Thomson JA, Itskovitz-Eldor J, Shapiro SS et al . Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): - 1145-7. Erratum in: Science 1998; 282 (5395): 1827. - 10 Takahashi K, Yamanaka S. Induction of  pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126 (4): 663-76. 11 Gurdon JB. The developmental capacity of  nuclei taken from intesti - nal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 1962; 10 : 622-40. REFERENCES
1 Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV . Osteogenesis in transplants of  bone marrow cells. J Embryol Exp Morphol 1966; 381–90. 2 Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9 (5): 641–50. 3 Horwitz EM, Le Blanc K, Dominici M et al. ; International Society for Cellular Therapy . Clarification of  the nomenclature for MSC: CRISPR-Cas9 , clustered regularly interspaced short palindromic repeats and associated protein 9. Resulted in the Nobel Prize in Chemistry being awarded to Emmanuelle Charpentier and Jennifer A. Doudna in 2020. Cytotherapy 2005; 7 (5): 393–5. 4 Dominici M, Le Blanc K, Mueller I et al . Minimal criteria for defin - ing multipotent mesenchymal stromal cells. The International Soci - ety for Cellular Therapy position statement. Cytotherapy 2006; 8 (4): - 315–17. 5 Caplan AI. Mesenchymal stem cells: time to change the name! Stem - Cells Transl Med 2017; 6 : 1445–51. 6 Sipp D, Robey PG, Turner L. Clear up this stem-cell mess. Nature 2018; 561 : 455–7. 7 Viswanathan S, Shi Y , Galipeau J et al . Mesenchymal stem versus ® stromal cells: International Society for Cell & Gene Therapy (ISCT ) Mesenchymal Stromal Cell committee position statement on nomen - clature. Cytotherapy 2019; 21 (10): 1019–24. 8 Murray IR, Chahla J, Safran M et al . International expert consen - sus on a cell therapy communication tool: DOSES. J Bone Joint Surg - 2019; 101 (10): 904–11. 9 Thomson JA, Itskovitz-Eldor J, Shapiro SS et al . Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): - 1145-7. Erratum in: Science 1998; 282 (5395): 1827. - 10 Takahashi K, Yamanaka S. Induction of  pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126 (4): 663-76. 11 Gurdon JB. The developmental capacity of  nuclei taken from intesti - nal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 1962; 10 : 622-40.

SAFETY CONCERNS
SAFETY CONCERNS
β 3 was The major safety concerns of  cell-based therapy and tissue β 3 engineering are listed in Table 4.3 . One of  the most serious concerns is that of  tumour formation and malignant transfor - mation. The risk of  tumour formation varies according to the - cell type used, the genetic modification strategy used to trans - form the stem cells, the site of transplantation and w hether the cells are autologous or allogeneic. The direct risk of  tumour formation by the transplanted cells relates specifically to ESCs and iPSCs and there appears to be little risk with SSCs. The ability of  stem cells to form tera tomas is one of  the hallmarks /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
TABLE 4.3
Risks of cell-based therapy.
Tumour formation
Genetic and epigenetic abnormalities
Transmission of infection
Poor viability and loss of function
Differentiation to undesired cell types
Rejection (allogeneic cells)
Side effects of immunosuppression (allogeneic cells)
α - α - α - α - Fibronectin Fibronectin Fibronectin Fibronectin Fibronectin Laminin Laminin Laminin Laminin Laminin /uni2032 of  pluripotency , and the risk of  this happening following stem cell therapy may be reduced by ensuring that only cells that have been fully di ff erentiated in vitro , and not those that are still pluripotent, are used for therapy . The risk of  malignancy may also be reduced by the choice of in vitro strategy used to di ff erentiate stem cells prior to use: the use of  viral vectors that do not integrate into the genome or of  non-viral approaches to di ff erentiation reduces the risk of  malignant transformation. There is also interest in developing techniques for directly reprogramming somatic cells to adopt the function of  a the threshold (%) Percentage of pixels above t tac n I α - tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr P = 0.051 the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr - the threshold (%) Percentage of  pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr α α di ff erent cell type without having to make them first revert back to the pluripotent state – so-called transdi ff erentiation. Another major concern is that of  transmitting infection. It is essential that if  allogeneic stem cells are used they are screened to exclude infection and that cells and engineered tissues ar e prepared accor ding to GMP guidelines to avoid bac - terial infection during in vitro culture prior to use. Moreover, if allogeneic cells are used for tissue engineering and regenerative therapy , they may be susceptible to graft rejection and immuno - suppressive therapy may be necessary .
Group 1 Group 2 Group 3
Intact Infection
G/P
(pretreatment)
Day 16 Day 16 Day 16
Day 2
SMA
SMA
SMA
SMA
DAPI
DAPI
DAPI
DAPI
(b)
DAPI DAPI
DAPI
DAPI
(c)
DAPI
DAPI DAPI
DAPI
Figure 4.10
Stained sections through a mouse cornea before (left) and after (second column) injury and subsequent treatment with standard
of care (gentamicin [G] and prednisolone [P]; group 1), standard of care plus a novel
/f_l
uid gel carrier (FG) (group 2) and standard of care plus
the carrier and decorin (Dec) (group 3). Sections are stained for markers of scarring:
(c)
laminin. Importantly, the use of the slow-release decorin resulted in rapid restoration of the corneal structure with a signi
/f_i
cant reduction in
scar markers. DAPI, 4
,6-diamidino-2-phenylindole (
/f_l
uorescent stain that binds strongly to adenine-/thymine-rich regions in DNA). (Adapted
with permission from Hill LJ, Moakes RJA, Vareechon C
et al
. Sustained release of decorin to the surface of the eye enables scarless corneal
regeneration.
npj Regen Med
2018; 3: 23.)
G/P/FG G/P/DecFG
120
100
80
60
40
20
0
SMA
DAPI
120
100
80
60
40
20
0
DAPI
120
100
80
60
40
20
0
DAPI
(a)
-smooth muscle actin (
-SMA),
(b)
/f_i
bronectin and
Tissue engineering and regenerative strategies hold out great hope for e ff ectively repairing or replacing tissues in a wide number of human diseases. The field is moving rapidly , under pinned by new developments in the relevant science in stem cells, materials and molecules. New emerging areas of tech nology include therapeutic signalling by wa y of  extracellular vesicles (EVs) and gene editing of cells using CRISPR-Cas9, and gene therapies. All will require the use of  a translational approach, wher eby the hypothesised mechanism is developed and translated to the clinic, building up robust clinical evidence of  e ffi cacy , by way of  well-designed and well-conducted clinical trials before widespread adoption. It is likely that patient stratification will further refine ther apy options. The ability to phenotype, genotype and profile patients at a molecular level will allow more detailed charac terisation of  patient subgroups and staging of  disease. In addi tion to clinical studies and evidence , the rapid pace of  therapy development will need to be accompanied by the development of new regulatory frameworks, f or example in point-of-care manufacturing. SAFETY CONCERNS
β 3 was The major safety concerns of  cell-based therapy and tissue β 3 engineering are listed in Table 4.3 . One of  the most serious concerns is that of  tumour formation and malignant transfor - mation. The risk of  tumour formation varies according to the - cell type used, the genetic modification strategy used to trans - form the stem cells, the site of transplantation and w hether the cells are autologous or allogeneic. The direct risk of  tumour formation by the transplanted cells relates specifically to ESCs and iPSCs and there appears to be little risk with SSCs. The ability of  stem cells to form tera tomas is one of  the hallmarks /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
TABLE 4.3
Risks of cell-based therapy.
Tumour formation
Genetic and epigenetic abnormalities
Transmission of infection
Poor viability and loss of function
Differentiation to undesired cell types
Rejection (allogeneic cells)
Side effects of immunosuppression (allogeneic cells)
α - α - α - α - Fibronectin Fibronectin Fibronectin Fibronectin Fibronectin Laminin Laminin Laminin Laminin Laminin /uni2032 of  pluripotency , and the risk of  this happening following stem cell therapy may be reduced by ensuring that only cells that have been fully di ff erentiated in vitro , and not those that are still pluripotent, are used for therapy . The risk of  malignancy may also be reduced by the choice of in vitro strategy used to di ff erentiate stem cells prior to use: the use of  viral vectors that do not integrate into the genome or of  non-viral approaches to di ff erentiation reduces the risk of  malignant transformation. There is also interest in developing techniques for directly reprogramming somatic cells to adopt the function of  a the threshold (%) Percentage of pixels above t tac n I α - tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr P = 0.051 the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr - the threshold (%) Percentage of  pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr α α di ff erent cell type without having to make them first revert back to the pluripotent state – so-called transdi ff erentiation. Another major concern is that of  transmitting infection. It is essential that if  allogeneic stem cells are used they are screened to exclude infection and that cells and engineered tissues ar e prepared accor ding to GMP guidelines to avoid bac - terial infection during in vitro culture prior to use. Moreover, if allogeneic cells are used for tissue engineering and regenerative therapy , they may be susceptible to graft rejection and immuno - suppressive therapy may be necessary .
Group 1 Group 2 Group 3
Intact Infection
G/P
(pretreatment)
Day 16 Day 16 Day 16
Day 2
SMA
SMA
SMA
SMA
DAPI
DAPI
DAPI
DAPI
(b)
DAPI DAPI
DAPI
DAPI
(c)
DAPI
DAPI DAPI
DAPI
Figure 4.10
Stained sections through a mouse cornea before (left) and after (second column) injury and subsequent treatment with standard
of care (gentamicin [G] and prednisolone [P]; group 1), standard of care plus a novel
/f_l
uid gel carrier (FG) (group 2) and standard of care plus
the carrier and decorin (Dec) (group 3). Sections are stained for markers of scarring:
(c)
laminin. Importantly, the use of the slow-release decorin resulted in rapid restoration of the corneal structure with a signi
/f_i
cant reduction in
scar markers. DAPI, 4
,6-diamidino-2-phenylindole (
/f_l
uorescent stain that binds strongly to adenine-/thymine-rich regions in DNA). (Adapted
with permission from Hill LJ, Moakes RJA, Vareechon C
et al
. Sustained release of decorin to the surface of the eye enables scarless corneal
regeneration.
npj Regen Med
2018; 3: 23.)
G/P/FG G/P/DecFG
120
100
80
60
40
20
0
SMA
DAPI
120
100
80
60
40
20
0
DAPI
120
100
80
60
40
20
0
DAPI
(a)
-smooth muscle actin (
-SMA),
(b)
/f_i
bronectin and
Tissue engineering and regenerative strategies hold out great hope for e ff ectively repairing or replacing tissues in a wide number of human diseases. The field is moving rapidly , under pinned by new developments in the relevant science in stem cells, materials and molecules. New emerging areas of tech nology include therapeutic signalling by wa y of  extracellular vesicles (EVs) and gene editing of cells using CRISPR-Cas9, and gene therapies. All will require the use of  a translational approach, wher eby the hypothesised mechanism is developed and translated to the clinic, building up robust clinical evidence of  e ffi cacy , by way of  well-designed and well-conducted clinical trials before widespread adoption. It is likely that patient stratification will further refine ther apy options. The ability to phenotype, genotype and profile patients at a molecular level will allow more detailed charac terisation of  patient subgroups and staging of  disease. In addi tion to clinical studies and evidence , the rapid pace of  therapy development will need to be accompanied by the development of new regulatory frameworks, f or example in point-of-care manufacturing. SAFETY CONCERNS
β 3 was The major safety concerns of  cell-based therapy and tissue β 3 engineering are listed in Table 4.3 . One of  the most serious concerns is that of  tumour formation and malignant transfor - mation. The risk of  tumour formation varies according to the - cell type used, the genetic modification strategy used to trans - form the stem cells, the site of transplantation and w hether the cells are autologous or allogeneic. The direct risk of  tumour formation by the transplanted cells relates specifically to ESCs and iPSCs and there appears to be little risk with SSCs. The ability of  stem cells to form tera tomas is one of  the hallmarks /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
TABLE 4.3
Risks of cell-based therapy.
Tumour formation
Genetic and epigenetic abnormalities
Transmission of infection
Poor viability and loss of function
Differentiation to undesired cell types
Rejection (allogeneic cells)
Side effects of immunosuppression (allogeneic cells)
α - α - α - α - Fibronectin Fibronectin Fibronectin Fibronectin Fibronectin Laminin Laminin Laminin Laminin Laminin /uni2032 of  pluripotency , and the risk of  this happening following stem cell therapy may be reduced by ensuring that only cells that have been fully di ff erentiated in vitro , and not those that are still pluripotent, are used for therapy . The risk of  malignancy may also be reduced by the choice of in vitro strategy used to di ff erentiate stem cells prior to use: the use of  viral vectors that do not integrate into the genome or of  non-viral approaches to di ff erentiation reduces the risk of  malignant transformation. There is also interest in developing techniques for directly reprogramming somatic cells to adopt the function of  a the threshold (%) Percentage of pixels above t tac n I α - tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr P = 0.051 the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr - the threshold (%) Percentage of  pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr α α di ff erent cell type without having to make them first revert back to the pluripotent state – so-called transdi ff erentiation. Another major concern is that of  transmitting infection. It is essential that if  allogeneic stem cells are used they are screened to exclude infection and that cells and engineered tissues ar e prepared accor ding to GMP guidelines to avoid bac - terial infection during in vitro culture prior to use. Moreover, if allogeneic cells are used for tissue engineering and regenerative therapy , they may be susceptible to graft rejection and immuno - suppressive therapy may be necessary .
Group 1 Group 2 Group 3
Intact Infection
G/P
(pretreatment)
Day 16 Day 16 Day 16
Day 2
SMA
SMA
SMA
SMA
DAPI
DAPI
DAPI
DAPI
(b)
DAPI DAPI
DAPI
DAPI
(c)
DAPI
DAPI DAPI
DAPI
Figure 4.10
Stained sections through a mouse cornea before (left) and after (second column) injury and subsequent treatment with standard
of care (gentamicin [G] and prednisolone [P]; group 1), standard of care plus a novel
/f_l
uid gel carrier (FG) (group 2) and standard of care plus
the carrier and decorin (Dec) (group 3). Sections are stained for markers of scarring:
(c)
laminin. Importantly, the use of the slow-release decorin resulted in rapid restoration of the corneal structure with a signi
/f_i
cant reduction in
scar markers. DAPI, 4
,6-diamidino-2-phenylindole (
/f_l
uorescent stain that binds strongly to adenine-/thymine-rich regions in DNA). (Adapted
with permission from Hill LJ, Moakes RJA, Vareechon C
et al
. Sustained release of decorin to the surface of the eye enables scarless corneal
regeneration.
npj Regen Med
2018; 3: 23.)
G/P/FG G/P/DecFG
120
100
80
60
40
20
0
SMA
DAPI
120
100
80
60
40
20
0
DAPI
120
100
80
60
40
20
0
DAPI
(a)
-smooth muscle actin (
-SMA),
(b)
/f_i
bronectin and
Tissue engineering and regenerative strategies hold out great hope for e ff ectively repairing or replacing tissues in a wide number of human diseases. The field is moving rapidly , under pinned by new developments in the relevant science in stem cells, materials and molecules. New emerging areas of tech nology include therapeutic signalling by wa y of  extracellular vesicles (EVs) and gene editing of cells using CRISPR-Cas9, and gene therapies. All will require the use of  a translational approach, wher eby the hypothesised mechanism is developed and translated to the clinic, building up robust clinical evidence of  e ffi cacy , by way of  well-designed and well-conducted clinical trials before widespread adoption. It is likely that patient stratification will further refine ther apy options. The ability to phenotype, genotype and profile patients at a molecular level will allow more detailed charac terisation of  patient subgroups and staging of  disease. In addi tion to clinical studies and evidence , the rapid pace of  therapy development will need to be accompanied by the development of new regulatory frameworks, f or example in point-of-care manufacturing.

Somatic cells
Somatic cells
Fully di ff erentiated specialised cells (somatic cells) obtained from normal tissues have been used for tissue engineering and regenerative therapy with some degree of  success. For example, skin has been engineered using cultured epithelial cells grown in vitro and used to treat patients with burn injuries. Chondrocytes have been isolated, expanded in vitro - and implanted into areas of  deficient cartilage in a procedure called autologous chondrocyte implantation. Bladder wall has a
Adult stem
and str omal
Somatic
(differentiated)
cells
cells
Contr olled
delivery
Cells
Materials Molecules
Anti-in
/f_l
ammatory
Osteogenic
also been engineered using a combination of smooth muscle cells and uroepithelial cells expanded in vitro and grown on a sca ff old before reimplantation. Such tissues can be grown using cells obtained from the intended recipient by tissue biopsy (autologous cells) or using cells obtained from unrelated donors (allogeneic cells). The major advantage of the former source is that, after implantation, they are not rejected by the recipient’s immune system; hence there is no requirement for immunosuppression (see Chapter 88 ). For other indications, the use of  fully di ff erentiated special ised cells is not practical in most situations because such cells are not readily available in su ffi cient numbers and they have only limited proliferative ability in vitro , which means that their numbers cannot be readily expanded to su ffi cient levels. To overcome these limitations, the major focus in the field of  cell therapy has been on the use of  stem cells.
Cell type
Somatic cells
SSCs
Ease of availability
Limited
Good
Expansion
in vitro
Limited
Good
Potency
No
Limited
Ethical concerns
No
No
Risk of malignancy
None
Low
Autologous
Yes
Yes
Anticipated future use
Limited
High
hESCs, human embryonic stem cells; iPSCs, induced pluripotent stem cells; SSCs, somatic stem cells.
ethical issues associated with hESCs.
Zygote
Totipotent
cell
Blastocyst
ESCs or iPSCs
Trophoblast
Pluripotent
cell
Primitive
endoderm
Epiblast
Multipotent
cell
Nullipotent
cell
Figure 4.2
Hierarchy of cells according to potency, ranging from stem cells to specialised differentiated cells. ESC, embryonic stem cell; iPSC,
induced pluripotent stem cell. (Adapted with permission from Tewary M, Shakiba N, Zandstra PW. Stem cell bioengineering: building from stem
cell biology.
Nat Rev Genet 2018
;
19
: 595–614.)
hESCs
Fetal cells
iPSCs
Moderate
Moderate
Good
Excellent
Good
Excellent
Excellent
Limited
Excellent
a
Yes
Yes
Yes
Moderate
Moderate
Moderate
No
No
Yes
Limited
Limited
High
a
Note that iPSCs avoid some of the
Somatic cells
Fully di ff erentiated specialised cells (somatic cells) obtained from normal tissues have been used for tissue engineering and regenerative therapy with some degree of  success. For example, skin has been engineered using cultured epithelial cells grown in vitro and used to treat patients with burn injuries. Chondrocytes have been isolated, expanded in vitro - and implanted into areas of  deficient cartilage in a procedure called autologous chondrocyte implantation. Bladder wall has a
Adult stem
and str omal
Somatic
(differentiated)
cells
cells
Contr olled
delivery
Cells
Materials Molecules
Anti-in
/f_l
ammatory
Osteogenic
also been engineered using a combination of smooth muscle cells and uroepithelial cells expanded in vitro and grown on a sca ff old before reimplantation. Such tissues can be grown using cells obtained from the intended recipient by tissue biopsy (autologous cells) or using cells obtained from unrelated donors (allogeneic cells). The major advantage of the former source is that, after implantation, they are not rejected by the recipient’s immune system; hence there is no requirement for immunosuppression (see Chapter 88 ). For other indications, the use of  fully di ff erentiated special ised cells is not practical in most situations because such cells are not readily available in su ffi cient numbers and they have only limited proliferative ability in vitro , which means that their numbers cannot be readily expanded to su ffi cient levels. To overcome these limitations, the major focus in the field of  cell therapy has been on the use of  stem cells.
Cell type
Somatic cells
SSCs
Ease of availability
Limited
Good
Expansion
in vitro
Limited
Good
Potency
No
Limited
Ethical concerns
No
No
Risk of malignancy
None
Low
Autologous
Yes
Yes
Anticipated future use
Limited
High
hESCs, human embryonic stem cells; iPSCs, induced pluripotent stem cells; SSCs, somatic stem cells.
ethical issues associated with hESCs.
Zygote
Totipotent
cell
Blastocyst
ESCs or iPSCs
Trophoblast
Pluripotent
cell
Primitive
endoderm
Epiblast
Multipotent
cell
Nullipotent
cell
Figure 4.2
Hierarchy of cells according to potency, ranging from stem cells to specialised differentiated cells. ESC, embryonic stem cell; iPSC,
induced pluripotent stem cell. (Adapted with permission from Tewary M, Shakiba N, Zandstra PW. Stem cell bioengineering: building from stem
cell biology.
Nat Rev Genet 2018
;
19
: 595–614.)
hESCs
Fetal cells
iPSCs
Moderate
Moderate
Good
Excellent
Good
Excellent
Excellent
Limited
Excellent
a
Yes
Yes
Yes
Moderate
Moderate
Moderate
No
No
Yes
Limited
Limited
High
a
Note that iPSCs avoid some of the
Somatic cells
Fully di ff erentiated specialised cells (somatic cells) obtained from normal tissues have been used for tissue engineering and regenerative therapy with some degree of  success. For example, skin has been engineered using cultured epithelial cells grown in vitro and used to treat patients with burn injuries. Chondrocytes have been isolated, expanded in vitro - and implanted into areas of  deficient cartilage in a procedure called autologous chondrocyte implantation. Bladder wall has a
Adult stem
and str omal
Somatic
(differentiated)
cells
cells
Contr olled
delivery
Cells
Materials Molecules
Anti-in
/f_l
ammatory
Osteogenic
also been engineered using a combination of smooth muscle cells and uroepithelial cells expanded in vitro and grown on a sca ff old before reimplantation. Such tissues can be grown using cells obtained from the intended recipient by tissue biopsy (autologous cells) or using cells obtained from unrelated donors (allogeneic cells). The major advantage of the former source is that, after implantation, they are not rejected by the recipient’s immune system; hence there is no requirement for immunosuppression (see Chapter 88 ). For other indications, the use of  fully di ff erentiated special ised cells is not practical in most situations because such cells are not readily available in su ffi cient numbers and they have only limited proliferative ability in vitro , which means that their numbers cannot be readily expanded to su ffi cient levels. To overcome these limitations, the major focus in the field of  cell therapy has been on the use of  stem cells.
Cell type
Somatic cells
SSCs
Ease of availability
Limited
Good
Expansion
in vitro
Limited
Good
Potency
No
Limited
Ethical concerns
No
No
Risk of malignancy
None
Low
Autologous
Yes
Yes
Anticipated future use
Limited
High
hESCs, human embryonic stem cells; iPSCs, induced pluripotent stem cells; SSCs, somatic stem cells.
ethical issues associated with hESCs.
Zygote
Totipotent
cell
Blastocyst
ESCs or iPSCs
Trophoblast
Pluripotent
cell
Primitive
endoderm
Epiblast
Multipotent
cell
Nullipotent
cell
Figure 4.2
Hierarchy of cells according to potency, ranging from stem cells to specialised differentiated cells. ESC, embryonic stem cell; iPSC,
induced pluripotent stem cell. (Adapted with permission from Tewary M, Shakiba N, Zandstra PW. Stem cell bioengineering: building from stem
cell biology.
Nat Rev Genet 2018
;
19
: 595–614.)
hESCs
Fetal cells
iPSCs
Moderate
Moderate
Good
Excellent
Good
Excellent
Excellent
Limited
Excellent
a
Yes
Yes
Yes
Moderate
Moderate
Moderate
No
No
Yes
Limited
Limited
High
a
Note that iPSCs avoid some of the

Stem cells
Stem cells
Stem cells are undi ff erentiated or non-specialised cells that are able, through cell division, to renew themselves indefinitely (self-renewal). Crucially , they are also able, when provided with appropriate stimuli, to di ff erentiate into one or more of  the di ff erent types of  specialised cell found in tissues and organs (potency). Because of  their unique ability to undergo self-renewal when cultured in vitro and to be directed to di ff er - entiate into specialised cell types, they have enormous potential - for use as cell-based therapies or, by way of  their di ff erent char - acteristics, to otherwise contribute to regenerative medicine. Stem cells can be classified in di ff erent ways, for example by using their characteristic level of  potency such as pluripotent and multipotent ( Figure 4.2 ) or with reference to the tissue from which they are derived, f or example the early embryo
Lineage potential
Capable of giving rise to
all cell types of body and
extraembryonic tissues
Capable of giving rise to
all cell types of body
Stem cell
Reprogramming
potency
Capable of giving rise to all
cell types of a particular
tissue or organ
Not capable of giving rise
to other cell types
whether they are derived by reprogramming adult specialised cells to a pluripotent state (iPSCs). Stem cells
Stem cells are undi ff erentiated or non-specialised cells that are able, through cell division, to renew themselves indefinitely (self-renewal). Crucially , they are also able, when provided with appropriate stimuli, to di ff erentiate into one or more of  the di ff erent types of  specialised cell found in tissues and organs (potency). Because of  their unique ability to undergo self-renewal when cultured in vitro and to be directed to di ff er - entiate into specialised cell types, they have enormous potential - for use as cell-based therapies or, by way of  their di ff erent char - acteristics, to otherwise contribute to regenerative medicine. Stem cells can be classified in di ff erent ways, for example by using their characteristic level of  potency such as pluripotent and multipotent ( Figure 4.2 ) or with reference to the tissue from which they are derived, f or example the early embryo
Lineage potential
Capable of giving rise to
all cell types of body and
extraembryonic tissues
Capable of giving rise to
all cell types of body
Stem cell
Reprogramming
potency
Capable of giving rise to all
cell types of a particular
tissue or organ
Not capable of giving rise
to other cell types
whether they are derived by reprogramming adult specialised cells to a pluripotent state (iPSCs). Stem cells
Stem cells are undi ff erentiated or non-specialised cells that are able, through cell division, to renew themselves indefinitely (self-renewal). Crucially , they are also able, when provided with appropriate stimuli, to di ff erentiate into one or more of  the di ff erent types of  specialised cell found in tissues and organs (potency). Because of  their unique ability to undergo self-renewal when cultured in vitro and to be directed to di ff er - entiate into specialised cell types, they have enormous potential - for use as cell-based therapies or, by way of  their di ff erent char - acteristics, to otherwise contribute to regenerative medicine. Stem cells can be classified in di ff erent ways, for example by using their characteristic level of  potency such as pluripotent and multipotent ( Figure 4.2 ) or with reference to the tissue from which they are derived, f or example the early embryo
Lineage potential
Capable of giving rise to
all cell types of body and
extraembryonic tissues
Capable of giving rise to
all cell types of body
Stem cell
Reprogramming
potency
Capable of giving rise to all
cell types of a particular
tissue or organ
Not capable of giving rise
to other cell types
whether they are derived by reprogramming adult specialised cells to a pluripotent state (iPSCs).