Jurnal Anthony Regenerasi Kulit

J. R. Soc.
Interface (2007) 4, 413–437

doi:10.1098/rsif.2006.0179
Published online 5 December 2006
REVIEW
Tissue engineering of replacement skin: the

crossroads of biomaterials, wound healing,
embryonic development, stem cells
and regeneration
Anthony D. Metcalfe and Mark W. J. Ferguson*
UK Centre for Tissue Engineering, Faculty of Life Sciences, University of Manchester,
3.239 Stopford Building, Oxford Road, Manchester M13 9PT, UK
Advanced therapies combating acute and chronic skin wounds are likely to be brought about
using our knowledge of regenerative medicine coupled with appropriately tissue-engineered
skin substitutes. At the present time, there are no models of an artificial skin that completely
replicate normal uninjured skin. Natural biopolymers such as collagen and fibronectin have
been investigated as potential sources of biomaterial to which cells can attach. The first
generation of degradable polymers used in tissue engineering were adapted from other
surgical uses and have drawbacks in terms of mechanical and degradation properties. This
has led to the development of synthetic degradable gels primarily as a way to deliver cells
and/or molecules in situ, the so-called smart matrix technology. Tissue or organ repair is
usually accompanied by fibrotic reactions that result in the production of a scar. Certain
mammalian tissues, however, have a capacity for complete regeneration without scarring;
good examples include embryonic or foetal skin and the ear of the MRL/MpJ mouse.
Investigations of these model systems reveal that in order to achieve such complete
regeneration, the inflammatory response is altered such that the extent of fibrosis and
scarring is diminished. From studies on the limited examples of mammalian regeneration, it
may also be possible to exploit such models to further clarify the regenerative process. The
challenge is to identify the factors and cytokines expressed during regeneration and
incorporate them to create a smart matrix for use in a skin equivalent. Recent advances in the
use of DNA microarray and proteomic technology are likely to aid the identification of such
molecules. This, coupled with recent advances in non-viral gene delivery and stem cell
technologies, may also contribute to novel approaches that would generate a skin
replacement whose materials technology was based not only upon intelligent design, but
also upon the molecules involved in the process of regeneration.
Keywords: skin; regeneration; stem cells; tissue engineering; scarring
1. OVERVIEW acute and chronic skin wounds. At the present time, there
are no models of bioengineered skin that completely
Tissue engineering is emerging as an interdisciplinary
replicate the anatomy, physiology, biological stability or
field in biomedical engineering that aims to regenerate
aesthetic nature of uninjured skin. Skin substitutes
new biological material for replacing diseased or damaged
should have some essential characteristics which include:
tissues or organs. To achieve this, not only is a source of
being easy to handle and apply to the wound site; provide
cells required, but also an artificial extracellular matrix
vital barrier function with appropriate water flux; be
(ECM) upon which the cells can be supported. In humans,
readily adherent; have appropriate physical and mechan-
skin represents approximately one-tenth of the body
ical properties; undergo controlled degradation; be
mass, and damage such as trauma, disease, burn or
sterile, non-toxic, non-antigenic; and evoke minimal
surgery to a part of this major organ has dramatic
inflammatory reactivity. Additionally, they should
consequences. Engineering skin substitutes represent a
incorporate into the host with minimal scarring and
prospective source of advanced therapy in combating
pain and facilitate angiogenesis, while still being cost
effective. The ultimate goal of the tissue engineer is to
*Author for correspondence ([email protected]). satisfy most if not all of these criteria when producing
Received 8 August 2006

Accepted 8 September 2006 413 This journal is q 2006 The Royal Society
414 Review. Tissue engineering of replacement skin A. D. Metcalfe and M. W. J. Ferguson
epidermis
dermis
hair follicle
hypodermis
adipose tissue
nerves
blood vessels
sweat gland
Figure 1. A schematic of the structure of skin.
novel, smart skin replacement therapies. Such matrices science, regeneration biology and current advances in
should attempt to model the properties of ECM in that, as proteomics and genomics could be incorporated into a
far as it is feasible, they have the ability to release a multidisciplinary translational research approach for
multitude of growth factors, cytokines and bioactive developing novel smart matrices.
peptide fragments in a temporally and spatially specific
event-driven manner. This timed and focal release of
cytokines, enzymes and pharmacological agents should 2. SKIN AND PRESENT CONCEPTS OF TISSUE
promote optimal tissue repair and regeneration of full- ENGINEERING
thickness wounds. Tissue engineering generally requires
The skin, the largest organ of the body in vertebrates, is
an artificial ECM that can be assimilated into the body composed of the epidermis and dermis with a complex
when the new tissue is regenerated. Materials for such a nerve and blood supply (figure 1). A third layer, the
matrix could be naturally occurring substances such as hypodermis, is composed mainly of fat and a layer of
collagen or could be prepared from biodegradable loose connective tissue. These three layers play an
polymers. Resorption, along with adequate cell adhesion important role in protecting the body from any
onto the matrix surface, gives the biological materials an mechanical damage such as wounding. The epidermis
attractive potential in tissue regeneration. is thin and totally cellular, but has sufficient thickness
A major consideration when developing a tissue to provide vital barrier function. Mammalian epidermis
engineering strategy for promoting repair and regen- and its appendages (hair, nail, sweat and sebaceous
eration is to identify suitable sources of cells and glands) maintain homeostasis by constant recycling of
mechanisms by which they can function and interact the basal cell layer. The epidermis is also exposed to
properly. These cells would also have to be abundant ultraviolet radiation, and the resulting damage is one of
enough to be able to carry out the regeneration process the factors contributing to the constant sloughing of
completely. Developmental biology conundrums such cells from the stratum corneum, which are replaced by
as regional specification of the embryo posed a great migrating cells from the basal layers (Alonso & Fuchs
many questions 20 years ago but by now are almost 2003). The dermis situated directly below the epidermis
solved. Unfortunately, progress towards a definitive constitutes the bulk of the skin and is composed of
understanding of morphogenetic movements, the absol- collagen with some elastin and glycosaminoglycans
ute timing of developmental events and the regen- (GAGs). The major cell type present in the dermis is
eration of missing parts has been slower (Slack 2003). fibroblast, which is capable of producing remodelling
Recently, however, some of the major advances in enzymes such as proteases and collagenases, which play
molecular biology have been applied to the under- an important role in the wound healing process. The
standing of wound healing, development and regenera- hypodermis is the layer located beneath the dermis and
tive processes (Harty et al. 2003). Tissue engineering as contains a considerable amount of adipose tissue that is
a discipline is becoming more aware of this knowledge well vascularized and contributes to both the mechan-
base and there are now moves towards designing ical and the thermoregulatory properties of the skin.
artificial tissues and organs using both cells and The challenge facing the tissue engineer is to
specifically designed materials. This review article combine novel materials with living cells to produce a
outlines how our present understanding of materials skin equivalent which is both functional and durable,
J. R. Soc. Interface (2007)

Review. Tissue engineering of replacement skin A. D. Metcalfe and M. W. J. Ferguson 415
and allows the integration and manipulation of the 4. NATURAL BIOMATERIAL SOURCES FOR
cell biology of host cells and the multitude of signals TISSUE ENGINEERING
that control their behaviour. Conventionally, tissue- Cells adhere and interact with their environment using
engineered skin exists as cells grown in vitro and integrins, focal adhesions and their ability to stimulate
subsequently seeded onto a scaffold or some porous downstream signalling pathways. Such mechanotrans-
material which is then placed in vivo at the site of duction signals conveyed to cells via their adhesion to
injury. At present, there are a number of different the matrix also clearly regulate the development of
engineered skin substitutes available for clinical use, various tissues, and this is becoming a focus point in
all of which fail to fulfil the criteria for fully functional designing intelligent matrices. There are obviously
skin (reviewed extensively in Supp & Boyce 2005). many different naturally derived biomaterials that
could be considered for use in a tissue engineering
context. For the purposes of this review, we have
3. BIOMATERIALS: TAKING CUES FROM concentrated on just two examples of frequently used
NATURE TO CREATE ARTIFICIAL natural scaffolds: collagen and fibronectin.
EXTRACELLULAR MATRIX
Engineering of skin substitutes implies deliberate 4.1. Collagen
design and fabrication according to specific functional
One of the most abundant naturally occurring proteins
objectives (Boyce & Warden 2002). So far, that
responsible for maintaining structural integrity is
design specification in skin has relied upon the
collagen. Collagens present in the skin are mainly
creation of both artificial dermal and epidermal synthesized by fibroblasts and myofibroblasts. In
components which when combined produce a replace- tissues, such as skin, tendons and bone, that undergo
ment skin, which can be grafted in place (reviewed in shear and tensile stresses, collagen is arranged in fibrils.
Supp & Boyce 2005). Materials used as artificial ECM Type I collagen is present in the dermis, fasciae and
to date include those derived from naturally occurring tendons and is a major component of scar tissue. Of the
materials and those manufactured synthetically. 20 different types of collagen, collagens I, II, III, V and
Examples of natural materials include polypeptides, XI assemble into fibrils. Other collagens form networks,
hydroxyapatites, hyaluronan, glycosaminoglycans e.g. collagen IV, the major component of basement
(GAGs), fibronectin, collagen, chitosan and alginates. membrane. Collagens can also form transmembrane
Such materials have the advantage that they have proteins, beaded structures or associate with fibril
low toxicity and a low chronic inflammatory response. surfaces. Collagen has been used for some time in the
Examples of synthetic materials include polyglycolide, design of skin substitutes (Supp & Boyce 2005) and
polylactide and polylactide coglycolide, which are recently has been used to create a model of endothe-
used for sutures and meshes (Vats 2003); other lialized, reconstructed dermis that promotes the
examples include polytetrafluoroethylene and poly- spontaneous formation of a human capillary-like
ethylene terephthalate. network (Hudon et al. 2003; Tremblay et al. 2005).
Matrices used routinely in therapeutic applications Butler & Orgill (2005) describe a tissue engineering
are made from polymers that are often resorbed or technique that combines disaggregated autologous
degraded in the body. Two key challenges exist here. keratinocytes and a highly porous, acellular collagen–
First, the primary generation of degradable polymers glycosaminoglycan matrix that has been shown in a
widely used in tissue engineering were adapted from porcine model to regenerate dermis and epidermis
other surgical uses and have deficiencies in terms of in vivo. Such skin substitute technology may have
mechanical and degradation properties (Griffith 2002). useful applications, if it can be proven to work in a
To overcome this, new classes of degradable materials clinical setting.
are being developed, considered later in this review.
The second major challenge is how to fabricate these
polymers into scaffolds that have defined shapes and 4.2. Fibronectin
a complex porous internal architecture that can A major multifunctional component of the ECM is
direct tissue growth (Griffith & Schwartz 2006). New fibronectin. In designing a fully functional skin sub-
technologies are emerging for accurate manufacture, stitute, there are a multitude of considerations, not
creating materials of defined pore size using novel least of which are the mechanical forces and
technologies using three-dimensional printing (Mironov interactions brought about by wound contraction.
et al. 2003; Seitz et al. 2005) and electrospinning This involves a complex interplay between the fibro-
(Luu et al. 2003; Li et al. 2005). Equally importantly, blast cytoskeleton and integrins with their ECM
these new polymers and their degradation products ligands. In dermal fibroblasts, most of these
must also be non-toxic and non-immunogenic upon interactions are mediated by the b1-type integrins
implantation and degradation. A major disadvantage (Sethi et al. 2002). Integrins such as a5b1 and avb3 are
of synthetic materials is the lack of cell-recognition the major receptors that direct fibronectin matrix
signals. Manufacturing processes are now being assembly (Ruoslahti 1991; Wu et al. 1996). These and
developed which will incorporate into biomaterials, other integrins also interact with the actin cytoskele-
cell-adhesion peptides, which are known to be involved ton, another essential factor in matrix assembly (Hynes
in cellular interactions. 1990). One of the major problems with currently

available skin substitutes is their failure to vascularize. 6. CULTURING SKIN COMPONENTS

Mechanical stretching is known to affect fibronectin IN THE LABORATORY
function (Rosso et al. 2005). Vogel & Baneyx (2003)
Severely damaged full-thickness skin is incapable of
have shown that excessive tension generated by cells in
spontaneous regeneration in mammals. To create a fully
contact with biomaterials render fibronectin fibrils non-
functional skin substitute, complete recapitulation of
angiogenic, which may provide an explanation towards
ontogenesis must occur and this is not yet possible in vitro
why biomaterials fail to vascularize. Fibrin, associated
(Boyce & Warden 2002). However, the phenotype
with fibronectin, has been shown to support keratino-
expressed by human skin cells in culture closely resembles
cyte and fibroblast growth both in vitro and in vivo, and
wound healing physiology (Clark 1996), which includes
may enhance cellular motility in the wound (Currie
cytogenesis, morphogenesis and histogenesis but not
et al. 2001). When used as a delivery system for
organogenesis. The challenge is not only that cells can
cultured keratinocytes and fibroblasts, fibrin glue may
provide similar advantages to those proven with be guided to follow the wound healing process, but also,
conventional skin grafts (Currie et al. 2001). Fibrin more importantly, for them to drive the processes of
matrix has been shown to be a suitable delivery vehicle developmental regeneration. Then it may be possible to
for exogenous growth factors that may in the future be provide full function to an artificial skin substitute
used to accelerate wound healing (Gwak et al. 2005). produced in this way. There are some examples in the
literature where individual components of the skin have
been cultured and transplanted successfully but not as
5. NEW AGE BIOPOLYMERS part of a fully functional skin replacement. Connective
Protein components of the ECM, such as collagen, fibrin, tissue cells are thought to repopulate grafts from
laminin or elastin as well as mixtures of these com- the wound bed, but many skin substitutes contain
ponents in Matrigel (Raeber et al. 2005), are widely used dermal fibroblasts to facilitate such repair processes
as cell-culture substrates due to their inherent resem- (Hansbrough et al. 1992; Parenteau et al. 1992; Boyce
blance to the natural ECM and complex signalling et al. 1993). Melanocytes have also been used to
capabilities. Most biopolymers form complex three- repopulate burn scars and for the treatment of vitiligo
dimensional fibrillar matrices in a self-assembly and/or (Lerner et al. 1987) as well as being added to various
enzyme-catalysed process. The matrix architecture can polymer scaffolds (Boyce et al. 1993; Swope et al. 1997).
be altered by varying solid content or gelation con- However, full restoration of skin sensation has not been
ditions, or by adding molecules that induce fibre demonstrated with either split thickness skin grafts or
aggregation or interfere with the cross-linking engineered skin, and sweat and sebaceous glands have
mechanisms (Kuntz & Saltzman 1997; Hayen et al. only been transplanted experimentally (Ward et al. 1989;
1999). The use of synthetic degradable gels is emerging Jahoda et al. 1996). It is these latter structures, along with
primarily as a way to deliver cells and/or molecules nerves and blood vessels, that are likely to prove the most
in situ; these are the so-called smart matrices. challenging since even with skin autografts these
A predominant approach, pioneered by Hubbell and structures are neither restored nor regenerated.
co-workers, is the formation of photopolymerizable gels To date, the approach has been to create a very simple
using polyethylene oxide-based macromer substrates skin substitute using cell populations within the skin.
(Desai & Hubbell 1991). Hubbell and colleagues have Disaggregated autologous keratinocytes and a highly
also explored a novel class of synthetic ECM analogues, porous, acellular, collagen–glycosaminoglycan matrix
which due to their homogeneous nanoscale micro- have been shown in a porcine model to regenerate a
structure were anticipated to restrict cell migration to dermis and epidermis in vivo (Butler & Orgill 2005).
proteolytic remodelling (Raeber et al. 2005). These During regeneration, a basement membrane of normal
molecularly engineered synthetic hydrogels are based appearance forms at the dermoepidermal junction and
on end-functionalized multiarm polyethylene glycol vascularization of the construct occurs (Butler & Orgill
(PEG) macromers, reacted via Michael-type addition 2005). A large number of parenchymal cells are required
under mild conditions with cysteine-containing peptide to repopulate wounds and restore skin structure and
sequences. This results in a hybrid network that can be populations of keratinocytes and fibroblasts can be grown
formed from aqueous precursors in the presence of cells. in relatively large numbers over a period of two to three
Such approaches are particularly amenable to inclusion weeks. A fundamental basis for generation of skin
of biologically active ligands (Seliktar et al. 2004; Van de substitutes comes about from the utilization of this
Wetering et al. 2005). PEG acts as an inert structural rapid growth of cultured cells. As soon as skin cells are
platform due to its hydrophilicity and resistance to prepared in sufficient quantity, they need organizing
protein adsorption. Hence, it exhibits the desired within the substitute to recreate the anatomical compo-
biological signals uniquely from incorporated peptides sition of normal skin. Currently, it is possible to create a
or proteins, with minimal structural or chemical back- polarized environment in which to do this with popu-
ground (Raeber et al. 2005). Similarly, these authors lations of skin cells. Human keratinocytes can be
have linked linear RGD-containing peptides as dangling co-cultured with a dermal substitute in vitro and exposed
ends and protease-sensitive oligopeptides as elastically to an air interface to create a stratified epithelium
active network chains to the PEG macromers to mimic (Kivinen et al. 1999). In culture, keratinocytes behave
the two essential biological functionalities of an ECM just as they would in vivo with cornified cells migrating
analogue: cell adhesion and degradability by proteases towards the air interface to form a squamous epithelial
(Raeber et al. 2005). surface. The fibroblasts forming the majority of the

Table 1. Examples of commercially created skin substitutes.
commercial
product name and
manufacturer epidermal component dermal component advantages disadvantages
epidermal Epicel cultured epidermal none large area of permanent wound coverage with three weeks required to produce fragile
substitutes Genzyme autograft grown from little risk of rejection confluent sheets, susceptible to blis-
Tissue repair patient skin biopsy tering post-grafting

Corporation
Laserskin cultured autologous kerati- none a less fragile delivery system for keratino- minimum of three weeks required to
Fidia nocytes from skin biopsy cytes. Hyaluronic acid/cell interaction expand keratinocyte population
Advanced in a perforated hyaluro- properties improve mechanical stability
Biopolmers nic acid membrane
EpiDex cultured autologous outer none cells have increased proliferative capacity and substitute takes up to six weeks after
Modex Thera- root sheath hair follicle can be cryopreserved for repeat appli- harvesting to produce. Product fragile
peutiques cells cations. Success in chronic ulcer treatment
Myskin cultured autologous Myskin with dermal fibro- PVC encourages keratinocyte attachment up to 14 days required for cell expansion.
CellTran keratinocytes on a PVC blasts under development and proliferation, providing a more stable Repeated application needed for good
polymer coated with a delivery platform. Keratinocytes can be clinical outcome
plasma-polymerized thawed for repeated application
surface
dermal sub- Alloderm none processed cadaver allograftprocessing helps reduce antigenic com- problems of graft rejection and disease
stitutes Life Cell skin ponents. Successful in resurfacing full- transfer
Corporation thickness burns
Dermagraft none allogeneic neonatal fibroblasts neonatal fibroblast rapidly proliferate to potential risk of rejection and disease risk
Advanced on a three-dimensional produce collagen, GAGs and growth from fibroblasts, although none
Biohealing, Inc. bioabsorbable scaffold factors to aid wound healing reported
Review. Tissue engineering of replacement skin
Integra synthetic polysiloxane bovine type I collagen and encourages ingrowth of fibroblasts and bovine collagen presents and antigenicity
Johnson & polymer GAGs epithelial cells. Epidermal equivalent and disease risk. Three weeks required
Johnson replaced after 14 days with an autograft to expand the dermal autograft
Transcyte thin silicone layer collagen-coated nylon mesh successfully used to treat second- and third- nylon mesh not biodegradable. Rejection
Advanced (Biobrane) seeded with neonatal degree burns. Dermal fibroblasts secrete and disease risk from fibroblasts
Biohealing, Inc. allogeneic fibroblasts collagen, GAGs and growth factors to aid
wound healing
Permacol none porcine-derived acellular non-immunogenic due to processing to revascularization sometimes inefficient to
Tissue Sciences dermal matrix remove non-collagenous and cellular support overlying epidermal graft
Laboratories material. Supports host fibroblast infiltra-
tion and revascularization
composite Apligraf human allogeneic neonatal human allogeneic neonatal graft take comparable to autografts with good risk of chronic graft rejection and disease
substitutes Organogenesis keratinocytes foreskin fibroblasts in cosmetic results. Improves granulation from allogeneic keratinocytes and
bovine type I collagen, tissue deposition and no signs of rejection fibroblasts. Requires repeated appli-
ECM proteins and observed cations
cytokines
OrCel human allogeneic neonatal human allogeneic neonatal provides a favourable environment for host not intended for use as a permanent skin
Ortec keratinocytes foreskin fibroblasts in a cell migration and provides a source of replacement. Designed as a biological
International bovine collagen sponge cytokines and growth factors dressing. Risk of rejection and disease
from bovine collagen and other cells.
A. D. Metcalfe and M. W. J. Ferguson 417
dermal substitute slowly degrade the biopolymer and lay current focus of research is towards developing a
down their own ECM. As the cell numbers increase, there tissue-engineered skin equivalent that combines living
is a concomitant increase in the number of soluble factors cells with natural or synthetic cellular components
released into the microenvironment. Keratinocytes and (Philips 1998; Auger et al. 2004). Removal of suitable
fibroblasts are known to produce a wide variety of skin for autologous grafting is a painful process and is
cytokines including various growth factors and inflam- not always possible if, for example, the patient has
matory mediators as well as different matrix polymers extensive burns. An engineered skin substitute would
and catabolic enzymes (Boyce et al. 1996). Allowing such need to fulfil specific criteria to replace the many
cell types to interact may stimulate paracrine functions of human skin (Boyce 2001). It should act as a
mechanisms to initiate production of cell proliferation barrier to micro-organisms, control fluid loss and have
factors, such as insulin-like growth factors and platelet- long-term elastic durability. It must also be histocom-
derived growth factor (PDGF), transforming growth patible, support wound healing, lack antigenicity and
factor-a (TGF-a) and -b (TGF-b) and basic fibroblast toxicity, yet also be cost effective (Harlan et al. 2002;
growth factor (bFGF), known to be important not only Ahsan & Nerem 2005; Donohue et al. 2005). Table 1
for cell competency but also for the process of wound summarizes skin substitutes that are available or have
healing and tissue regeneration. been available in the past for the treatment of skin
injuries in humans along with the advantages and
disadvantages associated with their use. At present, no
7. COMMERCIALLY AVAILABLE SKIN manufactured skin substitute has provided an outcome
SUBSTITUTES consistently comparable to an autograft.
Serious injury to the skin, such as burns, trauma or
chronic ulcers, requires immediate coverage to facilitate
repair and restore skin function. The gold standard for 8. PROBLEMS WITH EXISTING
skin replacement is the autologous skin graft in which an COMMERCIALLY AVAILABLE SKIN
area of suitable skin is separated from the tissue bed and SUBSTITUTES
transplanted to the recipient area on the same individual 8.1. Reduced vascularization
from which it must receive a new blood supply. Grafts
may be full thickness in which a complete section of When a skin graft is placed on a recipient wound bed, it
epidermis and dermis is transplanted, or split thickness must acquire a blood supply to maintain long-term
that includes only part of the dermis. Full thickness grafts survival and integrate with the host tissue. Some
are less likely to take than split skin, but provide existing skin substitutes do allow angiogenesis to
improved coverage and decreased wound contraction occur, but there it is still an area for improvement,
(Rudolph 1976). Syngeneic grafts, performed between particularly in cases where vascularization is not rapid
genetically identical individuals such as monozygotic enough. The repeated failure of fabricated skin replace-
twins or mice of the same inbred strain, take equally well ments to adequately vascularize has led to renewed
as autologous grafts. Xenogeneic skin grafting involves efforts to understand autologous skin graft revascular-
the transfer of tissue between species, but like allogeneic ization (O’Ceallaigh et al. 2006). This inability for
transplantation (from a non-genetically identical indi- substitutes to ‘take’ leads to cells in the replacement
vidual of the same species) there are problems with graft dying and ultimately the construct sloughs away from
rejection. Allografts of cadaver skin are used as a the host. Until recently, revascularization of skin
temporary cover for full thickness burns but are subject autografts was thought to occur either by direct
to rejection, because antigens present in the donor tissue anastomosis between graft vessels and bed vessels or
may elicit an immune reaction in the recipient (Burd & by ingrowth of bed vessels (angiogenesis) into the graft
Chiu 2005). Cadaver skin can be chemically treated using (O’Ceallaigh et al. 2006). These authors have recently
a three-step process that includes epidermis removal, cell shown that the initial onset of revascularization is
solubilization and dry preservation, in order to decrease attributable to early anastomoses between graft and
the antigenic components. This leaves an immuno- bed vessels, mainly within the central area of the graft
logically inert acellular dermal matrix (Alloderm, Lifecell (O’Ceallaigh et al. 2006). An obvious implication of this
Corporation, Branchburg, NJ), which aids the regen- work is that bioengineered skin substitutes incorporat-
eration of the underlying dermis. Alloderm has been ing prefabricated vessels may vascularize more rapidly
successfully used in the resurfacing of full-thickness burn in a fashion similar to autologous skin grafts.
wounds in combination with an ultra-thin autograft
which replaces the epidermis (Tsai et al. 1999). Other
8.2. Scarring
clinically useful products include Integra, Dermagraft,
Apligraf and Epicel and although they do not fully The process of scarring is discussed extensively later in
recreate the functions and aesthetics of skin, they have this review. In the context of skin grafting and
established milestones in treatments of skin problems and substitution, scarring at the graft margins is proble-
disorders (extensively reviewed by Supp & Boyce 2005; matic, functionally, mechanically and aesthetically.
Ehrenreich & Ruszczak 2006). Scar tissue is not identical to the tissue which it replaces
Early studies carried out by Rheinwald & Green and is usually of inferior functional quality. For
(1975), Bell et al. (1979) and Yannas & Burke (1980) example, scars in the skin are less resistant to
formed the basis for the development of future dermal, ultraviolet radiation, and sweat glands and hair follicles
epidermal and composite skin replacements. The do not grow back within scar tissue. Presently available

skin substitutes that integrate well often suffer from (allogeneic) have been used to develop commercial skin
scarring problems at the graft margins. The next substitutes such as Apligraf and have demonstrated
generation of skin substitute should incorporate anti- that the presence of skin cells can aid wound healing
scarring technologies to address this problem. (Falanga & Sabolinski 1999). The use of allogeneic skin
cells offers a means to develop an ‘off-the-shelf’ skin
replacement therapy for immediate application to injured
8.3. Absence of differentiated structures
skin. It is thought that cell age may affect the duration
Bioengineered skin substitutes are often relatively simple that allogeneic cells survive in vivo. Male allogeneic skin
single layer or bilayered structures. If ‘take’ is successful, cells shown to persist for up to 2.5 years were from a
then the substitute offers a barrier function similar to neonatal source (Otto et al. 1995). Foetal allogeneic
normal skin. The absence of complexity with regard to fibroblasts have also been shown to exhibit a similar
differentiated structures means that presently available spatial and temporal pattern of persistence to that seen
treatments offer none of the many other characteristics of with syngeneic cells and were shown to migrate and
functioning skin. There is a lack of temperature control proliferate within a wound environment (Sandulache
provided in normal skin by sweat and sebaceous glands, as et al. 2003). Cell persistence is therefore a key charac-
well as hair follicles. Additionally, in presently available teristic to understand when developing the next gener-
substitutes, insulation and an adequate vascular supply ation skin replacement. Cell persistence may be desirable
from adipose tissue do not exist. Skin substitutes often when covering a large area of missing skin but brings with
lack melanocytes and thus skin pigmentation, they also it long-term safety challenges, e.g. demonstrating lack of
do not have nerve supplies and so suffer from a lack of subsequent transformation.
sensation, both temperature and pressure. Critically, skin
substitutes have no resident Langerhans cells which play
an important function in immune regulation in the skin. 8.6. Biocompatibility, mechanical and handling
A key development to enhance present skin replacement properties
therapy would be to develop strategies to incorporate or
One of the prerequisites of bioengineered skin is that it
induce differentiated structures into skin constructs.
should be biocompatible, i.e. it will support appropriate
There are, however, extensive studies that have incor-
cellular activity, including the facilitation of molecular
porated melanocytes and Langerhans cells in skin
and mechanical signalling cascades, in order to
substitutes for testing purposes and melanocytes have
optimize tissue regeneration. Additionally, the bioen-
also been incorporated in therapeutic products for the
gineered product will not induce any undesirable local
treatment of vitiligo. A recent study by Hachiya et al.
or systemic responses in the host, e.g. chronic inflam-
(2005) used mixed cell slurries containing keratinocytes
mation. Skin replacements should also have appro-
and fibroblasts with melanocytes on the backs of severe
priate mechanical and handling properties that make it
immunodeficient mice to produce a skin substitute with
as mechanically durable as skin but with handling
spontaneously sorted melanocytes. This may offer a
properties that allow clinicians to manipulate it in a
means of treating both structural and cosmetic aspects of
surgical setting. As discussed earlier, currently avail-
skin conditions. One of the most remarkable examples of
able skin substitutes do not mimic normal skin
true organogenesis from adult tissue in culture is
composition. The three-dimensional architecture and
described by Zheng et al. (2005). Here, the authors inject
mechanical properties of skin replacements are there-
a mixture of isolated neonatal dermal cells with epidermal
fore completely different to normal skin. This is in part
aggregates into the dermis of nude mice. These are then
explained by the fact that the manufacturing processes
able to interact and undergo relatively normal hair
employed are not sophisticated enough to recapitulate
morphogenesis to give rise to cycling hair follicles within
the developmental morphogenesis used to create skin
8–12 days. Such approaches hold great hope for the future
naturally. In designing the next generation substitute,
of incorporating differentiated structures in a new
greater attention must be paid to overcoming some of
generation of skin substitutes.
these fundamental problems.
8.4. Delay involved with cell culture

Cells for the epidermal and dermal components can take 8.7. Cell source
between two and three weeks to expand to sufficient In sourcing cells for a skin substitute, there are
numbers for grafting purposes. This problem is high- essentially three locations from which cell types can
lighted in many of the skin substitutes considered in be derived: local; systemic; and progenitor cell popu-
table 1. New developments of protocols for rapid cell lations. Cells sourced locally include fibroblasts, kera-
expansion would help alleviate such problems, e.g. in tinocytes, melanocytes, adipocytes and hair follicle
severely injured patients. cells. Systemic cells are populations of cells that are
resident in the blood or bone marrow system, an
example of which would be fibrocytes, known to play a
8.5. Persistence of cells in heterologous grafts
key role in skin wound healing (Bucala et al. 1994; Abe
Ideally, skin cells are obtained from the patient (auto- et al. 2001). Progenitor cells are also resident locally in
logous) but in some cases, such as large surface area burns stem cell niches such as the hair follicle; they are
and with some skin disorders, this is not always possible. resident in the bone marrow or could arise from
Epidermal and dermal cells isolated from donor skin embryonic stem (ES) cell lines cultured in vitro.

8.8. Development, safety and product costs resume a basal cell phenotype upon contact inhibition
and differentiate into a stratified squamous keratinizing
The early stages of bringing a new tissue-engineered
epidermis (Schaffer & Nanney 1996).
product to the market place can be a costly exercise and
The final phases of the inflammatory response and
this has resulted in many of the smaller companies that
epithelialization coincide with the migration of fibro-
have developed skin replacements having to file for
blasts and endothelial cells and the formation of
bankruptcy. A final consideration for an off-the-shelf
granulation tissue. Angiogenesis and fibroplasia then
skin replacement must be the cost of the research and
take place, with fibroblasts becoming the dominant cell
development process, product safety, clinical trials,
type, laying down collagen and ECM. Remodelling of
storage, shelf-life and ultimately the cost of producing,
collagen occurs using matrix metalloproteinases pro-
marketing and selling the product.
duced by the fibroblasts and macrophages, and this is a
The components of bioengineered skin replacements
phase that can last several months and, in the adult, is
are continually being developed and improved. Hope-
characterized by scar formation (Chettibi & Ferguson
fully, many of the above problems will be overcome
1999). The balance of newly formed collagen with the
with advances in research and development tech-
destruction of old collagen establishes the final physical
nologies. The next generation of skin replacements
characteristics of the scar.
will probably evolve from lessons learned from our
Scars are the end point of the normal continuum of
understanding of wound repair and regeneration.
mammalian tissue repair and arise after almost every
dermal injury (Bayat et al. 2003). One of the key aims of
regenerative medicine is to produce novel replacement
9. WOUND REPAIR, SCAR-FREE HEALING AND skin which not only has the structural and functional
FOETAL REGENERATION IN MAMMALS properties of the original but also incorporates into the
In order to fully understand the differences between host tissue without any scarring. Scarring can be
regeneration and the normal outcome of tissue repair, assessed clinically using the Vancouver scar scale
namely fibrosis and scarring, it is useful at this point to (Powers et al. 1999) or the Manchester scar proforma
briefly review the processes of normal wound repair, the (Beausang et al. 1998). There are three different types
scarring process and to consider some examples of of scars: atrophic; hypertrophic; and keloidal. Atrophic
mammalian tissues, which undergo scar-free healing. In scars are depressed and cause a valley or depression in
designing a smart skin replacement for grafting, it is the skin. Hypertrophic scars are elevated and may
obvious that the mechanisms of both wound healing subside with time. Keloids are scar tissue which
and regeneration must be at the forefront of the tissue behaves like a benign tumour. Keloidal scars are
engineers’ mind. elevated, expansive and continue to grow beyond the
Tissue repair is normally a rapid process that has margin of the original wound. In addition to appear-
been devised through evolution to allow animals to ance, location and orientation are also important
escape danger and rapidly recover tissue integrity considerations in determining scar type. All types of
using scarring to join the wound edges or to fill tissue scarring can occur on all areas of the body, but some
voids (Caplan 2003). Wound healing has often been areas such as the chest, knees and elbows are more
described as a sequential mechanism and it is more susceptible to scarring.
specifically an event-driven process, whereby signals Foetal wound repair, however, is essentially a
from one cell type set off cascades in other cell types, regenerative process, characterized by an absence of
which propel the wound through the phases of healing scarring and fibrosis (see reviews by McCallion &
(Sweitzer et al. 2006). Adult wound healing is Ferguson 1996; Ferguson et al. 1996; Garg & Longaker
essentially a repair process, which normally exhibits 2000; Ferguson & O’Kane 2004). These differences in
scarring. Tissue repair begins immediately with fibrin the healing processes have sparked great interest and
clot deposition at the site of injury, preventing have lead to the development of several animal models
haemorrhage from damaged blood vessels. Circulating in which foetal scarless healing has been described.
platelets then aggregate at the site of injury and various These include the sheep, pig, rabbit, mouse, rat, guinea-
inflammatory mediators, such as PDGF, TGF-a and pig, chicken, opossum and monkey (Adzick & Longaker
TGF-b, epidermal growth factor (EGF) and FGFs, are 1991; reviewed in Chettibi & Ferguson 1999). Wounds
released. These molecules are also believed to play made in foetal tissues heal via different mechanisms and
major roles downstream in the wound repair process result in much less scarring than an equivalent wound
(Chettibi & Ferguson 1999). The inflammatory in adult tissues (Whitby & Ferguson 1991a,b).
response of adult tissues to wounding is characterized The characteristics of scar-free healing after inci-
by an early influx of neutrophils whose numbers sional wounding have been shown in various studies
steadily increase and reach a maximum 24–48 h (Ferguson & O’Kane 2004). They include minimal
post-wounding (reviewed in Chettibi & Ferguson inflammation and complete restoration of normal skin
1999). As the neutrophil numbers begin to decline, structure, with normal collagen deposition and reg-
macrophages take over and repopulate the wound site. ularly distributed hair follicles, capillaries and glands.
Re-epithelialization also occurs at the same time, with Such healing is believed only to occur through a
keratinocytes migrating across the granulation tissue gestational age equivalent to the first third of human
from deep within the dermis and the basal cells of the development; after that time, normal scarring as
wound edge. As soon as the keratinocytes have evident in the adult occurs (extensively reviewed in
re-established the barrier property of the skin, they Ferguson & O’Kane 2004).

Originally, it was thought that the sterile aqueous regeneration (Michalopoulos & DeFrances 1997; Fausto
environment provided by the amniotic fluid was 2000), rabbit ear regeneration (Goss & Grimes 1975),
important in scar-free healing; however, studies on axolotl and Xenopus limb regeneration (Goss & Holt
marsupials such as the opossum which are raised in 1992; Gardiner & Bryant 1996), antler regeneration
the mothers pouch proved otherwise (Armstrong & (Goss 1995; Allen et al. 2002), regeneration of the digit tip
Ferguson 1995). In this model, incisional wounds were in a child (Douglas 1972; Illingworth 1974; reviewed in
made at an equivalent time to the other incisional Han et al. 2005) or interdental papilla regeneration
wounding studies and newborn opossums were found to (reviewed in Chai & Slavkin 2003).
heal without scarring despite not being kept within a Regeneration is usually described as comprising one
sterile amniotic environment. In another study by or both of two processes:
Longaker et al. (1994), sheep skin from an adult or late
foetus was grafted onto a young foetus in utero and then (i) Epimorphic regeneration where replacement cells
incisionally wounded; the newborn lamb had scar tissue arise from undifferentiated cells (either stem cells
formation. Repair and regeneration of the skin there- or dedifferentiated cells) that form a blastema
fore appear to be correlated with the degree of skin from which the new structures derive.
differentiation and the inflammatory response to (ii) Morphallactic regeneration where new cells are
wounding (McCallion & Ferguson 1995). derived from existing tissues by cell differentiation
Key to the process of scar-free healing is the correct and/or migration.
deposition of ECM molecules such a hyaluronan,
various collagen types and tenascin-C (Whitby & Regeneration may have important links to develop-
Ferguson 1991a,b; Lindblad 1998; Chin et al. 2000). ment where undifferentiated stem cells are the source
Embryonic wounds which heal without scarring exhibit from which differentiated cell types arise in order to create
a number of major differences from adult wounds which (or recreate) functionally organized adult tissues.
scar including: Recently, a murine model for mammalian wound repair
and regeneration was discovered by Clark et al. (1998).
— lack of fibrin clots and platelet degranulation, Two millimetre through-and-through punch wounds
— markedly reduced inflammatory response, consisting made into the ears of MRL/MpJ mice closed with
of small numbers of poorly differentiated inflam- regeneration after 30 days, whereas they did not close in
matory cells, and the control strain, C57BL/6 mice. MRL/MpJ-Faslpr
— markedly elevated levels of molecules involved in mice, homozygous for the lymphoproliferation spon-
skin morphogenesis and growth. taneous mutation, show systemic autoimmunity, massive
lymphadenopathy associated with proliferation of
A consequence of this is that the growth factor aberrant T cells, arthritis and immune complex glomer-
profile of embryonic healing wounds is very different ulonephrosis. The lpr defect is due to a mutation in the fas
from that of an adult. Scar-free embryonic wounds gene, which leads to an inability to mediate apoptosis of
show reduced levels of TGF-b1, TGF-b2 and PDGF lymphocytes (Watanabe-Fukunaga et al. 1992). The
(released in the adult from degranulating platelets and MRL/MpJ strain, bred as a control for the MRL/
inflammatory cells) and elevated levels of TGF-b3 MpJ-Faslpr strain, also exhibits autoimmune disorders
(a skin morphogenetic molecule). Experimental manipu- but symptoms are manifested much later in life compared
lation of the growth factor profile of adult wounds by with those of the MRL/MpJ-Faslpr mice. The MRL/MpJ
exogenous addition of TGF-b3 or neutralization of mouse has the same regenerative capacity as the KFaslpr
TGF-b1, TGF-b2, PDGF, etc., results in markedly strain, and since the autoimmune symptoms occur
reduced or absent scarring (reviewed in Ferguson & later, it is often the strain chosen to observe the
O’Kane 2004). Such implications may be very import- regenerative mechanisms.
ant in artificial skin substitutes or grafts to enhance A further study by Rajnoch et al. (2003) details more
host take and minimize scarring at the perimeter and specifically how the MRL/MpJ mouse ear wound closes
base of the graft. in response to different traumas. These authors
compared the effects of two different types of punches
(a crude thumb punch and a clinical biopsy punch) on
10. HARNESSING REGENERATIVE three strains of mice. MRL/MpJ ear wounds healed
MECHANISMS TO TECHNOLOGICALLY faster than either C57BL/6 or Balb/c mice. The
ADVANCE CURRENT SKIN SUBSTITUTES MRL/MpJ mouse ears healed with enhanced blastema
Medical interest around regeneration has often focused on formation and markedly thickened tip epithelium.
the repair of damaged adult tissues. The challenge faced Rajnoch et al. (2003) postulated that the speed by
here would be to incorporate molecules associated with which a punched ear hole closes might be an important
the regeneration process into a smart matrix. There are factor before the onset of differentiation. The histo-
only a few examples in vertebrate species of tissues where logical organization of the regenerating MRL/MpJ ear
the initial phase of repair is followed by perfect functional edges closely resembles the blastema of a regenerating
and structural restoration of the organ. Regeneration has limb (figure 2).
captured scientists’ imaginations for well over 200 years Furthermore, the regenerative ability of this strain
since the early experiments carried out by Trembley in may be restricted to the ear owing to its structure: a
1744 on hydra (Fallon 2003). Classically, when we thin tissue covered on both sides by epithelium
consider regeneration in mammals, we think of liver (Rajnoch et al. 2003). The small dimensions of the ear

(a)
C
E
SG HF
C SG
(b)
C
C
Figure 2. Transverse sections through an MRL/MpJ ear 21 days post-biopsy punch wounding. The histological organization is
blastema-like in structure. (a) shows an ear section stained with Alcian Blue and fast red. The cut end of the cartilage (C) can be
seen clearly. Glycosaminoglycan deposits in the mesenchyme are stained blue. The apical epithelium (E) extends away from the
cut cartilage. (b) shows a similar ear section stained with anti-Aggrecan-TRITC, a cartilage precursor molecule. The section is
counterstained with the nuclear dye, DAPI. Note the formation of cartilage islands (I) in the mesenchyme and the infiltration of
Aggrecan into the mesenchyme. HF, hair follicle; SG, sebaceous gland. Scale bar, 100 mm.
were thought to allow for the diffusion of growth factors et al. 1998; Rajnoch et al. 2003; Metcalfe & Ferguson
and the establishment of appropriate gradients similar 2005). The tissue restoration process in the MRL/MpJ
to embryonic development, whereas in large thick and nude-nu mice resembles foetal-like healing and is a
tissues, e.g. skin, this may not be possible (Rajnoch rare example of regeneration among adult mammals.
et al. 2003). This was confirmed in studies involving Understanding and characterizing the molecules
wounding both the ear and the back skin of the involved in this regeneration from a tissue engineering
MRL/MpJ mouse, where the ear wounds completely perspective could lead to fundamental changes in skin
regenerate, whereas the back skin wounds heal with a substitute design.
scar (Metcalfe et al. 2006). In keeping with studies of
scar-free embryonic skin wound healing (Whitby &
11. REPAIR AND REGENERATION SHARE
Ferguson 1991a,b; Ferguson et al. 1996; Cowin et al.
MANY COMMON FACTORS
1998, 2001a) and the therapeutic manipulation of adult
dermal healing to reduce scarring (Shah et al. 1995), the A general problem in regeneration research is to
reduced inflammatory infiltrate seen with the biopsy as understand how the events of tissue injury, which
opposed to the crude thumb punch correlated with a would normally result in simple repair, are subtly
faster, more regenerative repair process with reduced coupled and able to diverge towards the activation of
scarring. Other studies on athymic nude-nu mice have plasticity in residual progenitor cells, resulting in tissue
also shown that wound healing of the ear resembles regeneration. The likelihood is that tissue repair and
regeneration (Gawronska-Kozak 2004). In this instance, regeneration are not that dissimilar and, in fact, they
it is thought that the absence of T-lymphocytes in share many common mechanisms that differ very
wounded ears provides a microenvironment conducive to subtly, a few examples of which will now be discussed.
regeneration of mesenchymal tissues (Gawronska-Kozak
2004; Gawronska-Kozak et al. 2006).
The process of regeneration involves the complex 11.1. Thrombin activation
tissue remodelling brought about by the formation of The regulation of the fibrinolytic system is of critical
a blastema during healing, after which time, cartilage, importance during haemostasis, wound repair, neoplasia,
sebaceous glands, hair follicles and blood networks inflammation and a variety of other biological processes.
reform. Thus, scarless healing accompanies blastema Successful haemostasis and wound repair is dependent on
formation and regrowth of various tissues such as platelet adhesion and aggregation. Prothrombin
cartilage and other differentiated structures (Clark activator converts plasma prothrombin into thrombin,

which is integral to the blood-clotting process. Platelets mediating the release of IL-6 from Kupffer cells
release fibrinogen which once converted to fibrin by (Mastellos et al. 2001). Additionally, C3 and C5 are
thrombin adds to the fibrin clot. In addition to activation expressed in the newt limb blastema and C5 in the
of fibrin, thrombin facilitates migration of inflammatory regenerating lens (Kimura et al. 2003; Tsonis et al.
cells to the site of injury by increasing vascular 2006). Positional memory is a critical aspect for the
permeability. By this mechanism, factors and cells autonomy of limb regeneration, because it specifies
necessary for wound healing flow from the intravascular the initial population of blastemal cells in relation to
space and into the extravascular space. the extent of the axis to be regenerated (Brockes &
In salamanders, it is thought that the local acti- Kumar 2005). An understanding of its molecular basis
vation of thrombin is a key to the regenerative is important for our appreciation of how stem cells are
processes observed. Apart from the role that it plays specified to give rise to different structures, rather than
in wound repair, thrombin is known to be an activator to different cell types. Studies on limb regeneration
of S-phase re-entry in differentiated cells in newt have led to the identification of Prod 1, a gene that is
myotubes (Imokawa & Brockes 2003; Imokawa et al. regulated by proximodistal axis and retinoic acid
2004; Brockes & Kumar 2005). A necessary aspect of (Brockes & Kumar 2005). Prod 1 is thought to act as
this response is the inactivation of Rb by phosphoryl- a cue for local cell identity that is expressed in the
ation allowing transit through the G1/S restriction normal limb and persists in blastemal cells. Prod 1 is
point (Imokawa et al. 2004). Cultured mouse myotubes apparently the newt orthologue of mammalian CD59,
missing both copies of the Rb gene can also be induced as evidenced by the prediction of secondary structure
to re-enter S-phase. From these and other studies, an (Brockes & Kumar 2005). Interestingly, CD59 protein
important parallel has been noted between newt and in mammals is associated with the inhibition of the
mouse myotubes—both are refractory to the familiar terminal phase of complement activation (Murray &
serum growth factors that act on mammalian myo- Robbins 1998).
blasts indicating that the post-mitotic arrest operates
in both contexts (Tanaka et al. 1999). Responsiveness
to thrombin-derived activity is a property of newt 11.3. TGF-b family isoforms
myotubes but not of mouse myotubes and the role of As a consequence of an altered inflammatory response
thrombin in dedifferentiation of newt myotubes is well and skin morphogenesis, the growth factor profile of a
documented (Tanaka et al. 1999). Lens regeneration is healing embryonic wound is very different quali-
also a thrombin-dependent response (Imokawa et al. tatively, quantitatively and temporally compared
2004). Lens regeneration in urodele amphibians such as with an adult wound (Whitby & Ferguson 1991b;
the newt proceeds from the dorsal margin of the iris, O’Kane & Ferguson 1997; Cowin et al. 2001a,b;
where pigment epithelial cells (PEC) re-enter the cell Ferguson & O’Kane 2004). Embryonic wounds express
cycle and transdifferentiate into lens (Imokawa et al. very high levels of TGF-b3 and very low levels of
2004). The axolotl, a related species that can regenerate TGF-b1 and TGF-b2. By contrast, adult wounds
its limb but not its lens, activates thrombin after contain predominantly TGF-b1 (and TGF-b2), which
amputation but not after lens removal. Apparently, all is derived initially from degranulating platelets and
vertebrates possess PECs that are capable of transdif- subsequently from inflammatory cells such as mono-
ferentiating into lens under appropriate conditions, but cytes and macrophages.
only certain species of adult newts and fishes can Application of neutralizing antibodies to TGF-b1
regenerate the lens (reviewed extensively by Tsonis and/or TGF-b2 (preferably both) to healing adult
2006). One theory for the newt’s ability to regenerate rodent wounds results in markedly improved scarring
the lens and the inability of the axolotl to do the same is (Shah et al. 1992, 1994a,b, 1995). Interestingly, pan-
that because the latter has lost the ability to activate neutralization of all the three TGF-b isoforms (TGF-
thrombin on the dorsal iris, it no longer expresses tissue b1, TGF-b2 and TGF-b3) does not improve scarring,
factor in this location (Imokawa & Brockes 2003). suggesting that neutralization of TGF-b3 may be
detrimental (Shah et al. 1994a,b, 1995). By contrast,
exogenous addition of TGF-b3 to healing adult wounds
11.2. Complement family members (to elevate levels similar to those seen in scar-free
The complement system plays an essential role in skin embryonic wounds) results in markedly improved or
repair acting as host defence against infectious agents absent scarring during adult wound healing (Shah et al.
and in the inflammatory process. The complement 1995). From these studies, it can be seen that subtle
pathway is activated during the inflammatory phase of alterations of the TGF-b isoform profile result in either
wound healing. C5a, C6 and C7 are chemotactic agents scarring repair or scar-free healing.
for neutrophil migration. C3a, C4a and C5a cause
degranulation of mast cells, leading to release of
11.4. The role of inflammation in wound repair
histamine and increased vascular permeability. The
and regeneration
membrane attack complex composed of C5b, C6, C7,
C8 and C9 is responsible for cytolysis. Skin is the primary structure of the body’s innate
Certain members of the complement family also immunity, acting as a barrier to micro-organisms. The
appear to play a role in regeneration. There is inflammatory response, initiated on injury to the
increasing evidence that C5 plays an important role skin as discussed earlier, is characterized by a series
in mammalian liver regeneration, probably by of events involving neutrophils, macrophages and

monocytes which are all important for proper wound regeneration using the scaffold for gene transfer or as a
closure and adequate scar-forming repair mechanisms. source of genetically altered cells (Vats 2003). Viral
Other innate receptors of the skin (dendritic cells) are gene delivery has, until recently, been one of the only
also activated on wounding. They are located through- ways to effectively transfer genes therapeutically.
out the epithelium of the skin, where in their immature Concerns over the safety of viruses as a gene carrier
form they are attached by long cytoplasmic processes. have led to the development of alternative gene delivery
The primary function of dendritic cells is to capture and strategies. Hubbell and colleagues used fibrin as a
present protein antigens to naive T-lymphocytes. surgical matrix to explore the potential of hypoxia-
Dendritic cells engulf micro-organisms and other inducible factor-1a (HIF-1a) gene therapy in stimulat-
materials and degrade them with their lysosomes. ing wound healing. Using a peptide-based gene delivery
Peptides from microbial proteins are then bound to a system, Trentin et al. (2006) expressed the HIF-1a
groove of MHC-II molecules produced by macrophages, dODD (oxygen-sensitive degradation domain) gene,
dendritic cells and B-lymphocytes. The peptide epi- translocated it to the nucleus under normoxic conditions
topes bound to the MHC-II molecules are then put on and consequently upregulated vascular endothelial
the surface of the dendritic cell, where they can be growth factor (VEGF)-A165 mRNA and protein levels
recognized by complementary shaped T-cell receptors in vitro. Peptide-DNA nanoparticles containing HIF-1a
and CD4 molecules on naive T4-lymphocytes. dODD entrapped in fibrin matrices were applied to full-
The involvement of the immune system in the thickness dermal wounds in the mouse. Angiogenesis was
response to tissue injury has also raised the possibility increased comparably strongly to that induced by VEGF-
that it might influence the outcome not only of tissue A165 protein alone (Trentin et al. 2006). These authors
repair but also of tissue regeneration. A common demonstrated that vessel maturity induced by HIF-1a
hypothesis is that the process of inflammation may dODD was significantly higher than that induced by
preclude the ability of a structure to regenerate. There VEGF-A165 protein, as shown by the stabilization of the
is, however, emerging evidence of the immune system neovessels with smooth muscle. This is one of many new
playing a more positive role in the regeneration of approaches being developed to deliver genes non-virally
immune privileged sites such as the lens (Godwin & and in a local way, representing a new therapeutic tool for
Brockes 2006). In various newt species, the ocular the tissue engineer.
tissues such as the lens are regenerative and it has been Recently, Larocca et al. (2002) have adapted
recently shown that the response to local injury of the filamentous phage vectors for targeted gene delivery
lens involves activation of antigen-presenting cells to mammalian cells, enabling the delivery of growth
which traffic to the spleen and return to displace and factors such as FGF-2 and EGF. Targeted phage
engulf the lens, inducing regeneration from the dorsal particles are specific for the appropriate target cell
iris (Godwin & Brockes 2006). Mammals have a very surface receptor and have distinct advantages over
highly developed adaptive immunity and a relatively animal viral vectors, because they are simple, econom-
poor capacity to regenerate, whereas urodeles regen- ical to produce at high titre, have no intrinsic trophism
erate structures more easily but have a less robust for mammalian cells and are relatively simple to
immune system (reviewed in Godwin & Brockes 2006). genetically modify and evolve (Larocca et al. 2002).
According to these authors, immunomodulation of the Other methodologies for targeted growth factor
injury response may play a positive or neutral role with transfer include plasmid and adenoviral vectors
respect to regeneration. The outcomes of wound repair immobilized in collagen–gelatin matrices. Such mix-
and regeneration are profoundly different, but may be tures containing FGF-2 and FGF-6 were delivered to
mechanistically linked by subtle differences in signal- excisional muscle wounds, producing early angiogenic
ling pathways. responses, increasing endothelial cell numbers that
Understanding these subtle alterations between the subsequently remodelled into arteriogenesis (Doukas
various molecules and their pathways using model et al. 2002). Muscle repair was also enhanced as FGF-
systems may begin to unravel the mechanisms under- treated wounds filled with regenerating myotubes.
lying repair and regeneration. The likelihood is that the These biomatrix-gene delivery approaches continue to
permissive environment necessary for regeneration to be developed and offer new approaches for tissue repair
occur is similar to that observed during embryonic and regeneration.
development. A more comprehensive appreciation of DNA array technologies provide rapid and cost-
these permissive conditions has major implications for effective methods of identifying gene expression and
further advances in tissue engineering. genetic variation. In the preparation of a skin sub-
stitute, it would be advantageous to know the genes
that are involved in the process of both integration and
12. ISOLATING AND CHARACTERIZING GENES
regeneration. Cole et al. (2001) compared gene
AND PROTEINS INVOLVED IN REPAIR AND
expression profiles in normal and injured skin from
REGENERATION
females undergoing breast reduction surgery. Speci-
Alterations in gene expression patterns or in a DNA mens were collected at 30 min and 1 h after the initial
sequence can have profound effects on biological injury. Representative cDNA from these samples was
functions such as regeneration. These variations in hybridized to microarray membranes containing
gene expression are at the core of altered physiological cDNAs from 4000 genes. At 30 min, injury resulted in
and pathological processes. To that end, tissue-engin- a consistent increase of 124 out of the 4000 genes and
eered scaffolds could provide a platform for enhanced these genes were primarily involved in transcription

and signalling (Cole et al. 2001). At 1 h, only 46 genes and amount of post-translational modifications necess-
showed an increase in expression but a further 264 ary for processes such as regeneration and wound
genes were significantly decreased, indicating silencing healing. The Human Genome Project identified about
of many structural genes. In another study by Li et al. 40 000 genes; these translate into approximately
(2001), the expression of 8734 sequence-verified genes 300 000 to 1 million proteins when alternative splicing
in response to ear punch was observed in MRL/MpJ- and post-translational modifications are considered.
Faslpr mice compared with the slow-wound-repair With the availability of DNA microarray analysis,
strain, C57BL/6J mice. Many genes of unknown permitting the expression of thousands of genes to be
function (expressed sequence tags; ESTs) exhibited a monitored simultaneously, it may be asked why
more than twofold increase in MRL/MpJ-Fas(lpr) or proteomics is so important. Basically, proteins provide
C57BL/6J mice, suggesting that current understanding the structural and functional framework for cellular life;
of the molecular events at the inflammatory stage of the genome in comparison is relatively static. However,
repair is still limited (Li et al. 2001). These authors there are several difficulties in the study of proteins that
concluded that fast-wound-repair in MRL/MpJ-Faslpr are not inherent in the study of nucleic acids. Proteins
mice is mediated by a metabolic shift towards a low cannot be amplified like DNA; therefore, less abundant
inflammatory response and an enhanced tissue repair. sequences are more difficult to detect. Additionally,
More recently, studies by Masinde et al. (2005) used proteins have secondary and tertiary structure that
restriction fragment differential display PCR to isolate must often be maintained during their analysis.
genes differentially expressed in the MRL (good healer) However, some studies have attempted an approach
mouse and the C57BL/6 (poor healer) mouse at to determine protein profiles in skin regeneration. Li
different stages of wound healing. They identified 36 et al. (2000) identify the temporal protein profile of soft-
genes that were differentially expressed in the regener- tissue healing processes in the ear-punched tissue of
ating tissue of good and poor healer strains of which regeneration strain, MRL/MpJ-Fas(lpr) mice, and
several genes are also genetically linked to wound compared it with the non-regeneration strain,
healing. Microarray expression data have also recently C57BL/6J mice, using surface-enhanced laser deso-
been reported from studies on the PU.1 mouse. This rption and ionization protein chip technology. Five
mouse is genetically incapable of raising a normal candidate proteins were identified in which responses of
inflammatory response, because it lacks macrophages MRL/MpJ-Fas(lpr) to the ear punch were two- to
and functioning neutrophils, but repairs skin wounds fourfold different compared with that of C57BL/6J
rapidly and with reduced fibrosis (Cooper et al. 2005). mice (Li et al. 2000). Of the five candidate proteins, the
Cluster analysis of genes expressed after wounding amount of a 23.5 kDa protein in the ear-punched tissue
wild-type mice versus PU.1 null mice distinguished was significantly correlated with the rate of ear healing
between tissue repair genes and genes associated with in six representative strains of mice, making it a
inflammation (Cooper et al. 2005). These authors potential candidate for fast-wound-repair/regenera-
described several pools of genes, giving an insight into tion. This protein was believed to be Ras-related
their likely functions during repair and hinting at protein RAB-8 (Li et al. 2000). Genomics and
potential therapeutic targets. proteomics are therefore complementary fields, with
The use of stem cells as a component of a proteomics defining the functional analysis. Although
bioengineered substitute will be discussed elsewhere in they constitute relatively new and under exploited
this review; however, it is useful at this point to disciplines, the future of bioengineering and regenera-
consider the power of DNA array technology when tive medicine will be impacted greatly by both.
defining the ‘stem cell molecular signature’. Ivanova
et al. (2002) and Ramalho-Santos et al. (2002) provided
the first genome-wide transcript analysis of ES cells, 13. INCORPORATION OF GROWTH FACTORS
AND CYTOKINES IN THE CREATION OF
haematopoietic stem cells and neural stem cells. Both
AN INTELLIGENT SKIN SUBSTITUTE
groups generated about 200 genes that were upregu-
lated in the tested stem cell populations. The analysis Regeneration is characterized by a constantly changing
involved the use of Affymetrix mouse genome chips environment in which cells are exposed to a complex
with up to 36 000 genes being able to be screened at pattern of molecular cues and signals, which impart
once. Gosiewska et al. (2001) assessed the differential positional information necessary for correct develop-
expression and regulation of ECM-associated genes in ment. These cell signals trigger a series of events that in
human foetal and neonatal fibroblasts. Foetal fibro- combination control cell proliferation, differentiation
blasts were found to secrete four- to tenfold more latent and cell death, such that a specific tissue can be
TGF-b1 and higher levels of collagen protein and delineated and its edges specifically defined. Since these
mRNA for most types of collagen (particularly type molecules are often major components of early develop-
III). mRNA for type V collagen was, however, mental pathways for cell specification, incorporating
significantly reduced in foetal cells. Approximately 20 them into a tissue-engineered product could produce
other mRNAs for various cytokines, matrix molecules major advancements in regenerating adult structures
and receptors were also found to be similar between the such as skin. The number of morphogens that exist and
two cell types (Gosiewska et al. 2001). are used in developmental and regenerative processes
However, it must be noted that DNA/RNA analysis are obviously too numerous to catalogue in full.
cannot predict the amount of a gene product that is However, in this review, a few key growth factors and
made, if and when a gene will be translated, or the type cell-signalling molecules will be considered, which if

integrated into a bioengineered material could be dorsal/ventral specification. BMP-4, for instance,
critical to creating a fully functional skin replacement. specifies the development of ventral structures, e.g.
Platelet-derived growth factor (PDGF) is a potent skin from ectoderm. In the developing limb bud, BMP-2
activator for cells of mesenchymal origin, and a interacts with sonic hedgehog and fibroblast growth
stimulator of chemotaxis, proliferation and new gene factor-4 (FGF-4) to allow chondrocyte and osteoclast
expression in monocytes, macrophages and fibroblasts, precursors to form (Niswander & Martin 1993).
accelerating ECM deposition (Pierce et al. 1991; Shure Fibroblast growth factors (FGFs) are a family of 21
et al. 1992). It has also been suggested that reduced isoforms with a broad spectrum of activities, including
levels of PDGF may play a role in the mechanism of regulation of cell proliferation, differentiation and
scarless cutaneous repair (Whitby & Ferguson 1991a; migration. FGFs 1, 2, 5, 7 and 10 are upregulated
Peled et al. 2001; Ferguson & O’Kane 2004). This during adult cutaneous wound healing (Werner et al.
family of growth factors exists in both homo- and 1992; Tagashira et al. 1997). Different FGFs are
heterodimeric forms and Pierce et al. (1989) reported expressed throughout embryogenesis where they act
that a single application of PDGF-BB to an incisional as morphogens (Niswander & Martin 1992; Heikin-
wound increased the neoangiogenesis through enhance- heimo et al. 1994; Ohuchi et al. 1994; Rappolee et al.
ment of endogenous PDGF-BB signalling (Kano et al. 1994). The FGFs are also necessary for proper limb
2005). Gu et al. (2004) adenovirally delivered dose- development. Inactivating both FGF-4 and FGF-8 in
related PDGF-B in a collagen-based biocompatible, the apical epidermal ridge (AER) produces severely
gene-activated matrix to a rabbit dermal wound model. deformed limb phenotypes. One hypothesis is that a
Dose-related increases in granulation tissue and cell principal function of AER–FGF signalling during
proliferation were observed along with concordant normal limb development is to ensure that enough
expression of PDGF-B RNA and protein. No treat- progenitor cells are available to form each element of
ment-related changes in haematology, serum chemistry the limb skeleton and perhaps in other tissues as well
or histopathology were observed and no immunological (Sun et al. 2002). FGF-1 and FGF-2 have also been
responses against collagen were observed. These implicated in limb regeneration in urodeles, since they
approaches for PDGF delivery have great potential in both have been shown to control blastema cell
a tissue engineering context, where such insights into proliferation (Zenjari et al. 1996; Ferretti et al. 2001).
the mechanisms underlying the effects of growth factor FGF-2 or basic FGF is released at the wound site by
co-stimulation could lead to a better design of damaged endothelial cells and by macrophages, but if
therapeutic angiogenic strategies. FGF-2 activity is blocked using monospecific
Cytokines of the transforming growth factor-b family antibodies raised against FGF-2, wound angiogenesis
(TGF-b) are multifunctional regulators of cell growth, is almost completely blocked (Broadley et al. 1989).
differentiation and ECM formation (Roberts et al. FGF-2 has also been implicated in scar-free healing
1990a,b). In mammals, there are three isoforms, TGF- (Spyrou & Naylor 2002). FGF-7 and FGF-10 are
b1, TGF-b2 and TGF-b3, and although they have up to secreted by fibroblasts but act on keratinocytes to
85% amino acid sequence homology, there are known stimulate migration and proliferation (Tagashira et al.
differences in their potencies and biological activities 1997). FGF-9 (glial-activating factor) on the other
in vivo. TGF-b is known to be the most potent growth hand is neurotrophic (Kanda et al. 1999). An initial
factor involved in wound healing throughout the body study by Kawai et al. (2000) has revealed that bFGF
(Whitby & Ferguson 1991a,b; Levine et al. 1993; Shah may have the ability to accelerate tissue regeneration in
et al. 1995; Ashcroft et al. 1997; Cordeiro 2002; Cordeiro artificial dermis. These authors describe a dermal
et al. 2003; Ferguson & O’Kane 2004). In particular, in substitute which had gelatin–bFGF microspheres
relation to wound healing in the skin, TGF-b1 and incorporated into its structure, the result of which
TGF-b2 are implicated in cutaneous scarring, whereas accelerated fibroblast proliferation and capillary forma-
TGF-b3 is known to have an anti-scarring effect (Shah tion in a dose-dependent manner (Kawai et al. 2000).
et al. 1995; O’Kane & Ferguson 1997; Ferguson 2002; However, the expression of FGFs during foetal
reviewed extensively in Ferguson & O’Kane 2004). One skin development and scarless wound healing has
study has been performed looking at the effect of TGF- not been properly characterized. Dang et al. (2003)
b1 delivered through a collagen scaffold (Pandit et al. hypothesized that differential expression of FGF iso-
1999). Here, three 3!3 cm, full-thickness defects were forms and receptors occurs during foetal skin develop-
created on the dorsi of 15 New Zealand White rabbits. ment and that this differential expression pattern may
Each rabbit had a control (no treatment), collagen regulate the transition from scarless repair to healing
scaffold and collagen scaffold with TGF-b1 (2 mg cmK2) with scar formation. After excisional wounding,
and was observed for up to three weeks. In summary, a expression of FGF-7 and FGF-10 was downregulated
greater inflammatory response was found in the in scarless wounds, whereas FGF receptor 2 expression
collagen scaffold-treated group, but the fastest epithe- decreased in both scarless and scar-forming wounds.
lialization and contraction rates were associated with Expression of FGF isoforms 5 and 9 did not change in
TGF-b and collagen (Pandit et al. 1999). scarless wounds (Dang et al. 2003). These workers
Bone morphogenetic proteins (BMPs) are members demonstrate an overall downregulation of FGF
of the TGF-b superfamily. There are 15 members and expression during scarless healing. Therefore, in an
although they are known for their role in bone and engineered substitute, it may be necessary to balance
cartilage formation, they have diverse roles in many the levels of FGFs to ensure that there is no scarring
other developmental processes such as neuronal and but sufficient angiogenesis.

Vascular endothelial growth factor (VEGF) is at high levels in saliva and may accelerate healing of
induced during the initial phase of skin grafting, skin lesions in animals when they lick their wounds
where endogenous fibrin clots are known to form a (Chettibi & Ferguson 1999). Topical application of
provisional matrix and to promote angiogenesis. EGF to human donor sites also appeared to accelerate
Growth factors such as VEGF increase in such wounds wound healing (Falanga et al. 1992) and is also believed
to stimulate angiogenesis. There is some evidence in to stimulate keratinocytes to produce hyaluronan
studies of diabetic mice that VEGF may promote (Pienimaki et al. 2001). Within the last few years, a
healing; these mice fail to produce VEGF at the wound new class of ligand, the matrikine or matricryptin, has
site and consequently healing is impaired (Frank et al. been characterized as subdomains of various ECM
1995). Fibrin rafts and fibrin glue have been used for proteins capable of signalling to the cell through
skin grafting for some time; however, it remains receptors, such as growth factor receptors (Tran et al.
unknown whether VEGF is induced when fibrin is 2005). The EGF-like repeats of tenascin-C and laminin-5
used as a dermal substrate for cultured skin substitutes signal to EGFR preferentially to upregulate migration
(Currie et al. 2001). during skin repair and tumour progression (Tran et al.
Recently, Hojo et al. (2003) investigated the effect of 2005). It is thought that this new class of matrikine ligand
fibrin gel as a dermal substrate for a cultured skin will have an impact on dermatology and tissue engin-
substitute, using human keratinocytes and dermal eering, as these proteins are altered in wound repair and
fibroblasts. Experiments were performed using 12 cul- skin diseases.
tured skin substitutes: four for histologic examination Hepatocyte growth factor/scatter factor (HGF/SF)
before transplantation; four for VEGF assay in vitro; and is a pleiotrophic growth factor produced principally by
four for the transplantation to athymic mice. With in vivo cells of mesenchymal origin. HGF/SF is an important
transplantation, the fibrin-type cultured skin substitute mitogen, morphogen and motogen known to play an
showed an excellent take on the wound bed and a important role in tumorigenesis and foetal development
normally proliferating keratinocyte layer with emergence through the c-Met receptor tyrosine kinase (Boros &
of vascular endothelial cells in the transplanted floor Miller 1995; Matsumoto et al. 1995; Zarnegar &
3 days after transplantation. It would seem from this Michalopoulos 1995). The association between HGF
study that using fibrin as a dermal substrate for cultured and c-Met receptor is known to be involved in
skin substitute increases the secretion of VEGF, improves morphogenesis and tissue organization during embryo-
regeneration of mature epidermal structure after in vivo nic development and regeneration following tissue
transplantation and promotes the migration of vascular injury. HGF was first thought to be involved in
endothelial cells. cutaneous physiology and wound healing based on the
Hypoxia is a potent stimulator of VEGF synthesis observation that HGF stimulates keratinocyte
(Shweiki et al. 1992). Subsequent angiogenesis leads to migration and proliferation in vitro (Matsumoto et al.
the migration of new capillaries into the provisional 1991; Sato et al. 1995). Transgenic expression of HGF in
matrix between the wound edges, initially a fibrin clot an epidermis-specific manner affects melanocyte
that is replaced by newly synthesized connective tissue. development, leading to dermal melanocytosis (Kunisada
Angiopoietin-1 (Ang-1), another endothelial-specific et al. 2000). Cowin et al. (2001b) showed that expression
growth factor, binds to and activates the receptor of HGF and c-Met receptor increased in response to
Tie-2, promotes endothelial cell survival and regulates cutaneous wounding and transgenic overexpression of
the responsiveness of the endothelium to other cyto- HGF resulted in increased vascularization and granula-
kines. Therapeutic angiogenesis requires an under- tion tissue expansion (Toyoda et al. 2001). Recently,
standing of how VEGF and Ang-1 interact to initiate HGF has been implicated in enhancing the cutaneous
neovascularization. Recently, Benest et al. (2006) have wound healing processes of re-epithelialization, neo-
used adenovirus-mediated gene transfer into the vascularization and granulation tissue formation
adjacent fat pad of the rat mesentery to characterize (Yoshida et al. 2003; Bevan et al. 2004).
induction of angiogenesis by VEGF and Ang-1. VEGF Retinoic acid (RA) induces the ‘super-regeneration’
gene transfer produced short, narrow, highly branched of organs that can already regenerate, such as the
and sprouting vessels, with normal pericyte coverage. urodele amphibian limb, achieved by respecifying
Ang-1 gene transfer induced broader, longer neovessels positional information in the limb (Maden & Hind
with no increase in branching or sprouting, yet a 2003). In organs that cannot normally regenerate such
significantly higher pericyte ensheathment. Com- as the adult mammalian lung, RA induces the complete
bination of both Ang-1 and VEGF generated a regeneration of alveoli that have been destroyed by
significantly higher degree of functionally perfused, various noxious treatments. Another tissue that fails to
larger, less branched and more mature microvessels, regenerate is the mammalian central nervous system
resulting from increased efficiency of sprout to vessel (CNS). RA does not induce neurite outgrowth as it does
formation (Benest et al. 2006). Thus, combinatorial in the embryonic CNS, because one of the retinoic acid
additions of these factors to a bioengineered skin receptors, RARb2, is not upregulated. However, when
replacement could have profound effects upon success- RARb2 is transfected into the adult spinal cord in vitro,
ful neovascularization and take upon grafting. neurite outgrowth is stimulated (Maden & Hind 2003).
Epidermal growth factor (EGF) has been implicated Recent studies by Dmetrichuk et al. (2005) assessed the
in wound healing and homeostasis in a number of role of RA and one of its receptors, RARb, in the
tissues including colon, skin, mammary gland and liver reciprocal neurotrophic interactions between regener-
(Wilcox & Derynck 1988). It is also known to be present ating limb blastemas and spinal cord explants from the

adult newt Notophthalmus viridescens. They found that 14. STEM CELLS AND THEIR APPLICATION
endogenous RA is one of the trophic factors produced TO AN ENGINEERED CONSTRUCT
by the blastema and it is thought that it may be capable
Adult mesenchymal stem cells (MSC) are believed to be
of guiding re-innervating axons to their targets. This
restricted in their ability to produce a wide range of
may have important implications for the understanding
replacement cells, but do nevertheless function as a
of re-innervation of skin substitutes. Interestingly, from
source of cells for tissue repair and homeostasis.
a skin engineering viewpoint in the diabetic mouse
Embryonic stem cells on the other hand are totipotent
model, db/db, all trans-RAs have been shown to
and are able to differentiate into many different cell
reverse the impaired wound healing displayed by this
types. The ES cell represents an attractive and viable
mouse (Kitano et al. 2001). RA therefore could be a
proposition for cell-replacement therapy. ES cells are
candidate molecule for incorporation in modern tissue-
derived from the inner cell mass of the embryonic
engineered skin substitutes where cellular positional
blastocyst. These cells can be maintained indefinitely
specification in addition to regeneration is necessary.
Homeobox (Hox) genes, although not strictly in vitro without loss of differentiation potential. To
morphogens, are an evolutionarily conserved family of date, many of the studies on ES cells have been limited
transcription factors and are attractive candidates for to mouse models, since human cell lines have only
inclusion in a bioengineered product. Hox protein recently become available (Tuan et al. 2002). There
activity is essential during embryogenesis for the are, however, many controversial ethical and technical
differentiation and specification of cell fate along the problems that need to be overcome before the full
body axes (Krumlauf 1994; Manak & Scott 1994) and potential of this type of cell can be realized (Ferrari
the Hox gene family have also been implicated as et al. 1998; Gussoni et al. 1999; Peterson 2002). Defining
important factors in limb regeneration (Brockes 1997; the similarities and differences between the MSCs and
Gardiner et al. 2002). Several Hox genes are expressed in ES cells will be especially relevant for applications of
mouse and human foetal and adult skin as well as nail regenerative medicine and bioengineering.
and hair follicles (Stelnicki et al. 1997; Godwin & Skin represents an ideal model system in which to
Capecchi 1998; Packer et al. 2000; Komuves et al. investigate the use of stem cells as a source of cell-
2002). Particular attention has been drawn to Hoxb13, replacement therapy, because it contains one of the few
known to be downregulated during foetal scarless wound well-characterized adult stem cell types, the keratino-
healing (Mack et al. 2003). This gene has been recently cytes. Adult somatic stem cells could resolve the
knocked out, the result of which is a mouse whose adult problems that ES cells potentially have, namely if
skin exhibits high levels of hyaluronan and enhanced adult stem cells are transplanted back into the same
wound healing (Mack et al. 2003). Hoxb13 overexpres- individual, then there should not be any inherent
sion in an adult organotypic epidermal model recapitu- problems with rejection. Treating patients with their
lates actions of Hoxb13 reported in embryonic own cells also avoids ethical and moral objections.
development (Mack et al. 2005). Epidermal cell prolifer- Unless they are responding to trauma, adult stem cells
ation is decreased, apoptosis increased and excessive typically divide infrequently to maintain homeostasis
terminal differentiation observed, as characterized by within their resident tissues (Alonso & Fuchs 2003).
enhanced transglutaminase activity and excessive Since they are responsible for all cell replacement
cornified envelope formation. Hoxb13 overexpression within a tissue, they are essential for tissue repair,
also produced abnormal phenotypes in the epidermal wound healing and regeneration. Adult stem cells reside
tissue that resembled certain pathological features of in specific niches and the niche exposes the stem cells to
dysplastic skin diseases. Mack et al. (2005) suggest that different differentiation cues—important in maintaining
Hoxb13 probably functions to promote epidermal the stem cell state. Each stem cell division produces one
differentiation, a critical process for skin regeneration replacement stem cell and one daughter cell. The latter is
and for the maintenance of normal barrier function. destined for differentiation and tissue regeneration.
The list of growth factors, cytokines and transcrip- One of the attractive features of skin from a
tion factors that could be incorporated into a skin bioengineering viewpoint is that skin keratinocytes
substitute as part of an intelligent design to facilitate can be maintained and propagated in the laboratory
skin regeneration are large; the above are just a few (Rheinwald & Green 1975, 1977). The cells that are
obvious examples. They do, however, represent an contained in these primary cultures have a remarkable
attractive group of factors, which if a suitable matrix ability to proliferate, because they are capable of being
could be found for defined release in both spatial and passaged for many hundreds of generations without
temporal dimensions could facilitate the rapid regen- undergoing senescence (Alonso & Fuchs 2003). From
eration and integration of skin. the work of Morris & Potten (1994), it is known that
Alternatively a minimalistic engineering approach slowly cycling cells in vivo are more clonogenic than
could be adopted to simulate embryonic development. actively dividing cells when placed into culture,
Here, normal cell–cell interactions, e.g. epithelial– suggesting that the less proliferative cells may be
mesenchymal interactions, occur which initiate signalling stem cells. The ability to maintain and grow such cells
cascades resulting in skin differentiation. If appropriate has lead to major advances in skin grafting tech-
cell types could be identified and allowed to interact, then nologies, such that burn patients routinely have
skin differentiation can result (Stenn 2003; Stenn & cultured skin keratinocytes engrafted. (Ronfard et al.
Cotsarelis 2005; Zheng et al. 2005). To this end, much 2000; Brouard & Barrandon 2003; Gambardella &
recent interest has focused on stem cells. Barrandon 2003). Unfortunately, at present, the skin

substitutes that are used are not fully functional in that Since bone marrow is a key to the process of
they lack hair follicles, sweat and sebaceous glands as haemopoiesis, it is also likely to contain hormones
well as nerve and blood supplies. Whether long-term such as granulocyte–monocyte colony-stimulating
keratinocyte cultures contain multipotent stem cells factor which is thought to accelerate wound repair
which could produce hair follicles remains a subject of (Gillitzer & Goebeler 2001; Lingen 2001).
debate (Alonso & Fuchs 2003). The problem for the Another potentially important source of MSCs,
tissue engineer is that although the existence of skin known to be derived from bone marrow, is found in the
stem cells is highly likely within such primary cell circulating peripheral blood (for review see Tuan et al.
cultures, their isolation and characterization are prov- 2002; Mori et al. 2005). Bucala et al. (1994) described a
ing to be a challenge. Fractionation by fluorescence- distinct population of blood-borne fibroblast-like cells
activated cell sorting has provided some insights. that rapidly entered sites of tissue injury in a wound
Jones & Watt (1993) found that some cells within the chamber model. The presence of fibroblasts in wound
primary keratinocyte culture showed great prolifera- chambers had mainly been thought to be attributable to
tive capacity in vitro and are enriched for b1 integrins, recruitment from surrounding subcutaneous tissue
and other studies by Li et al. (1998) and Kaur & Li (Dunphy 1967). However, the presence of such large
(2000) show that similar cells have upregulated a6 numbers of fibroblast-like cells coinciding with the entry
integrin. Further characterization of these cells has of circulating inflammatory cells suggested that the
shown that they adhere preferentially to ECM com- cell population was arising from peripheral blood and
ponents, fibronectin (a5b1 receptor) and collagen IV not exclusively by slow migration from adjacent
(a2b1 receptor), suggesting that the reason why stem connective tissue (Bucala et al. 1994). This new cell
cells are kept in their niche is due to the very strong type was termed fibrocyte with a distinct phenotype
interaction with and adherence to basement membrane originally described as collagenC/vimentinC/CD34C
components (Watt 1998; Segre et al. 1999). (Bucala et al. 1994). More recently, leucocyte-specific
Another source of multipotent skin stem cells resides protein-1 (lsp-1) has been added to this profile as a
in the hair follicle bulge (Stenn & Cotsarelis 2005). This fibrocyte marker in hypertrophic scars (Yang et al.
niche resides at the base of the permanent epithelial 2005). Although these cells make up only 0.5% of
portion of the hair follicle which is the deepest, most peripheral blood leukocytes, they constitute 10% of the
protected place within the contiguous epithelial com- cells infiltrating subcutaneously implanted wound
partment (Alonso & Fuchs 2003). Kobayashi & chambers in mice (Bucala et al. 1994). They are thought
Nishimura (1989) dissected dermal papillae cells and to have diverse roles in the wound healing process,
reconstituted them with fragments of hair follicle inducing angiogenesis in vivo and in vitro (Hartlapp et al.
containing the hair bulge region. On transplantation 2001), produce chemoattractants to recruit CD4C
under the kidney capsule of an athymic mouse, viable lymphocytes (Chesney et al. 1997) and express the
hair follicles formed. Other studies have shown that chemokine receptor, CCR7, involved in the process of
when the bulge cells of a normal mouse are surgically cell migration into the wound (Abe et al. 2001). Fibrocyte
replaced with cells from a lac-Z mouse, the new recruitment is also thought to be upregulated systemi-
chimeric hair follicles ubiquitously express lac-Z within cally in burn patients (Yang et al. 2002). The intercellular
all lineages of the hair follicle (Oshima et al. 2001). signals that modulate fibrocyte trafficking, proliferation
There are several studies which suggest that this and differentiation are only partially defined, but a better
compartment provides a source of multipotent stem understanding of these signals is likely to enable new
cells which could be used for bioengineering hair therapies to prevent pathologic fibrosis or to improve the
follicles (Stenn & Cotsarelis 2005). When the skin tissue repair response (Quan et al. 2006).
suffers trauma such as burn injury or wounding, it is The final consideration regarding cell sourcing is
thought that the bulge cells migrate to the surface to whether cells are autologous or heterologous. A rapid
aid re-epithelialization (Taylor et al. 2000). Thus, the off-the-shelf tissue engineering product favours heter-
regenerative role of bulge cells (or dissociated cells) ologous cell types and to date one of the most successful
from the skin is multiple; not only is it thought that bioengineered products has been Dermagraft. This has
they contribute to the production of sebaceous glands been a classic example of a bioengineered product
and epidermis, but also they are key to the formation of that has been both successful and, at the same time,
hair follicles (Zheng et al. 2005). distributed by three separate companies, Advanced
In defining cells for a bioengineered skin, apart from Tissue Sciences, Smith and Nephew and Advanced
the keratinocyte and hair bud follicular cells, bone Biohealing, Inc. Unlike many of the other skin
marrow is another logical candidate for sourcing cells to substitutes, Dermagraft has a reasonable shelf-life; the
seed in a skin substitute (Badiavas & Falanga 2003). cells cryopreserved within the construct survive for six
Bone marrow is known to contain inflammatory cell months (reviewed extensively by Mansbridge 2006).
progenitors and multipotent stem cells. In some This relatively long cell persistence, combined with a
wounds, e.g. chronic ulcers or non-healing burns, it is better understanding of cryopreservation techniques,
thought that the mesenchymal cells that fill the dermis bodes well for the persistence of stem cells in an
become phenotypically altered or senescent (Mendez engineered off-the-shelf product. The ultimate tissue
et al. 1998; Vande Berg et al. 1998; Raffetto et al. 1999). engineering goal would be to combine the various cell
The plasticity of bone marrow-derived stem cells means types discussed earlier, with or without a natural or
that they would have the inherent capacity to produce synthetic matrix in the presence of growth factors and
new skin cells if the conditions for growth were correct. cytokines known to promote regeneration (figure 3).

+/– natural/synthetic
matrix
keratinocytes
+ growth factors and

cytokines important
for regeneration
fibroblasts/fibrocytes
endothelial cells/pericytes
next generation, fully functional
preadipocytes skin substitute
progenitor cells hair follicle cells

Figure 3. A schematic of the requirements to create a fully functional skin substitute.
15. FUTURE PROSPECTS appropriate cells which when the carrier disappears in
the body (by non-inflammatory dissociation) allows the
The ultimate goal of tissue engineering of the skin is to
cells to interact so as to regenerate all of the skin
rapidly produce a construct that offers the complete
structures—such as that happens during embryonic
regeneration of functional skin, including all the skin
development or adult regeneration. In this case, the
appendages (hair follicles, sweat glands and sensory
substitute is minimally engineered so as to hold the cells
organs) and layers (epidermis, dermis and fatty
in an undifferentiated state prior to delivery and, for this,
subcutus) with rapid take (vascularization) and the
then to disappear rapidly. Understanding and exploiting
establishment of a functional vascular and nerve
the mechanisms of embryonic development, stem cell
network and scar-free integration with the surrounding
biology and biomaterial engineering will be the key to
host tissue. Such a construct should allow the skin to
achieving these next generation skin substitutes.
fulfil its many normal functions: barrier formation;
pigmentory defence against UV irradiation; thermo- The authors are grateful to the BBSRC, MRC and EPSRC for
regulation; and mechanical and aesthetic functions. their support of the UK Centre for Tissue Engineering.
Some but not all of the functions can be restored with
existing skin substitutes, e.g. cultured keratinocytes
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J. R. Soc.

Interface (2007) 4, 413–437

Tissue engineering of replacement skin: the

Keywords: skin; regeneration; stem cells; tissue engineering; scarring

Received 8 August 2006

J. R. Soc. Interface (2007)

J. R. Soc. Interface (2007)

available skin substitutes is their failure to vascularize. 6. CULTURING SKIN COMPONENTS

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8.4. Delay involved with cell culture

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+ growth factors and

progenitor cells hair follicle cells

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