A Review of Tissue-Engineered Skin Bioconstructs Available For
A Review of Tissue-Engineered Skin Bioconstructs Available For
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REVIEW
Situations where normal autografts cannot be used to replace damaged skin often lead to a
greater risk of mortality, prolonged hospital stay and increased expenditure for the National
Health Service. There is a substantial need for tissue-engineered skin bioconstructs and
research is active in this field. Significant progress has been made over the years in the devel-
opment and clinical use of bioengineered components of the various skin layers. Off-the-shelf
availability of such constructs, or production of sufficient quantities of biological materials to
aid rapid wound closure, are often the only means to help patients with major skin loss. The
aim of this review is to describe those materials already commercially available for clinical use
as well as to give a short insight to those under development. It seeks to provide skin
scientists/tissue engineers with the information required to not only develop in vitro
models of skin, but to move closer to achieving the ultimate goal of an off-the-shelf, complete
full-thickness skin replacement.
skin grafting as they cannot epithelialize on their own less contraction there will be at the site of application
and may lead to extensive scarring, resulting in limit- but the longer it will take to heal the donor site
ations in joint mobility and severe cosmetic (Andreassi et al. 2005).
deformities (Papini 2004).
In the case of major burn injuries, the currently
accepted treatment tactic requires an early excision of
a dry scab (eschar) to remove heat-denatured proteins
2. THE NEED FOR TISSUE-ENGINEERED
of the skin followed by wound closure (Burke et al.
SKIN SUBSTITUTES
1976; Papini 2004). This avoids triggering compli-
cations such as infection, multiple organ dysfunction Patients with 50 per cent TBSA full-thickness wounds
syndrome or hypertrophic scar formation. Heat- have only 50 per cent of undamaged skin left which
denatured proteins of the eschar may also cause an could be used for split-thickness skin harvesting.
uncontrolled inflammatory response and also serve as Donor sites would add to the total wound size resulting
a good source of nutrients for pathogenic micro- in a wound area covering 100 per cent of the body. An
organisms. This is of particular importance in heavily impaired epidermal barrier combined with reduced
burnt patients as the nature of the injury leads to a tem- immunity of heavily burned patients can result in bac-
porary suppression of cell-mediated and humoral terial sepsis which is the main complication in deep
immunity (Stoilova et al. 2007). extensive burns (Stoilova et al. 2007). Donor sites also
Early permanent wound closure results in minimal or heal with some scarring and may be very painful;
no scarring complications, whereas delayed treatment hence an additional analgesic pharmacological load is
leads to severe hypertrophic scarring directly proportional required. Moreover, depending on the thickness of the
to the wound closure delay time. One study of 337 scalded dermis, only three to four split-thickness skin harvests
children showed that demarcation time for the wound clo- are possible from the same site and re-cropping is
sure was 21 days, and after this time a much higher delayed by the time necessary for re-epithelialization
incidence of hypertrophic scarring occurred (Cubison (Atiyeh & Costagliola 2007).
et al. 2006). Earlier permanent wound closure is also In the case of a more extensive injury, donor sites are
associated with lower mortality and better functional extremely limited and in such cases, meshing techniques
long-term results (Wolfe et al. 1983). can be used where grafted skin is uniformly perforated
Wound size plays a major role in the outcome of the and stretched to cover greater areas of the wound.
injury. Current advances in anaesthesia, ventilation Although this method allows greater area coverage
and resuscitation as well as drug and nutrients support and reduces mortality rates, the cosmetic and func-
of burns patients, new dressings and topical wound- tional outcomes of such a treatment are inferior when
healing agents, as well as technical improvement of compared with the standard SSG application. This is
specialized burns units allow the successful treatment because of the lack of dermis in the interstices of the
of extensive burns which would have been considered stretched meshed skin graft, and slow epithelialization
lethal just half a century ago. According to Bull & from graft margins across interstices, resulting in a
Fisher (1954) between 1942 and 1952, the mortality greater graft contraction, delayed healing, scar tissue
rate of the age group 15– 44 years with 60 per cent formation and pronounced ‘crocodile skin’ appearance
burns of the total body surface area (TBSA) was 100 of the scar. In near-total full-thickness skin injuries
per cent. A study undertaken from 1998 to 2003 even meshing techniques are no help owing to the una-
revealed a reduction in the mortality rate of the same vailability of donor sites. In such cases wounds are
age group with 60 per cent TBSA, to only 41.4 per covered with temporary dressings or cadaver skin to
cent (Chua et al. 2007). Current advances in burns form a mechanical barrier in order to prevent fluid
treatment allow the successful treatment of patients loss and microbial contamination. Only delayed serial
with major extensive burns, although treatment of autologous split skin grafting can be used to heal
inhalation and deep extensive burns still remains a sub- injured skin in these cases (Papini 2004). Such
stantial challenge to the surgeon. wounds are left unhealed for a long time over the
Currently, the clinical ‘gold standard’ in full- course of treatment while awaiting epithelial regener-
thickness injuries treatment is split-thickness ation, and are prone to severe complications which
autologous skin grafting (Stanton & Billmire 2002; can result in death.
Andreassi et al. 2005; Supp & Boyce 2005). Epidermis Alternative life-saving approaches in the treatment
with a superficial part of the dermis is harvested with of extensive full-thickness wounds, where donor sites
a dermatome from an undamaged skin donor site and for SSG harvesting are not available, include the use
applied to the full-thickness wound. Being applied to of cultured autologous keratinocytes and/or bioengi-
the wound, capillaries of the split skin graft (SSG) neered skin substitutes. Significant progress has been
form anastamoses or ‘plug in’ into the existing capillary made recently in the development and clinical use of
network to provide nutrients for graft survival; this is these products (Horch et al. 2005; Clark et al. 2007;
referred to as graft ‘take’ (Converse et al. 1975). The MacNeil 2007; Pham et al. 2007). ‘Off-the-shelf’ avail-
donor site heals similarly to the superficial partial- ability or the possibility of producing, in a relatively
thickness wound by keratinocyte migration from short period of time, sufficient quantities of epithelium
hair follicles, sweat glands and edges of the wound. It capable of permanent wound closure sometimes make
heals within a week and can be used for further SSG these approaches the treatments available in extensive
re-harvesting. Generally, the thicker the SSG is, the deep injuries.
Because of the great importance and demand for structures, such as dermal collagen or cultured cells,
skin-replacement products, there is a long history of which are incorporated into the wound and persist
material development, and many research groups during wound healing and/or thereafter.
worldwide have focused on creating biomaterials for There are many different classifications of currently
skin substitution. Skin substitute biomaterials are com- available skin-substitute products ( Jones et al. 2002;
monly referred to by a variety of terms that can lead to Atiyeh et al. 2005; Horch et al. 2005; Atiyeh & Costagliola
confusion. They can be described as bioengineered skin 2007; Clark et al. 2007; MacNeil 2007; Patel & Fisher
equivalents, tissue-engineered skin, tissue-engineered 2008), and they can be summarized as follows.
skin constructs, biological skin substitutes, bioengi-
neered skin substitutes, skin substitute bioconstructs, (i) Anatomical structure:
living skin replacements and, more recently, as bioengi- — dermo-epidermal (composite),
neered alternative tissue (Kim et al. 2006). Although — epidermal,
these terms differ slightly from each other, and may — dermal.
not truly describe the product, they are considered to
be equal and interchangeable by the majority of inves- (ii) Duration of the cover:
tigators. For the purpose of this review we shall use — permanent,
these definitions to describe any skin substitute pro- — semi-permanent,
duct, produced or modified artificially in any way, — temporary.
including modifications of naturally occurring sub-
stances, such as dermis, for the purpose of damaged (iii) Type of the biomaterial:
skin replacement, fully or partially, temporary or per- — biological: autologous, allogeneic, xenogeneic,
manently, and possessing some similarities with — synthetic: biodegradable, non-biodegradable.
human skin, both anatomical and functional.
All tissue-engineered skin substitute bioconstructs (iv) Skin substitute composition regarding cellular
need to comply with three major requirements. They component:
must be safe for the patient, be clinically effective and — cellular,
be convenient in handling and application. Properties — acellular.
of the ‘ideal’ skin substitute for in vivo use have been
described elsewhere and recently reviewed by MacNeil (v) Primary biomaterial loading with cellular com-
(2007). In general, such biomaterials must not be ponent occurs:
toxic, immunogenic or cause excessive inflammation, — in vitro,
and should also have no or low level of transmissible dis- — in vivo.
ease risk. The biomaterial for skin reconstruction should
be biodegradable, repairable and able to support the Some of the currently marketed and clinically avail-
reconstruction of normal tissue, with similar physical able tissue-engineered skin-substitute products are
and mechanical properties to the skin it replaces. It reviewed in this paper (tables 1 – 3) and organized
should provide pain relief, prevent fluid and heat loss according to anatomical structure classification, which
from the wound surface and protect the wound from is the most commonly used, but many more are still
infection. It is also of great advantage if the skin substi- in the process of investigation (table 4) and are not dis-
tute bioconstruct is cost-effective, readily available, cussed in detail due either to the unavailability of
user-friendly and possesses a long shelf life. product information or lack of experimental or clinical
No currently commercially available tissue-engin- results on the materials’ performance.
eered skin replacement biomaterials possess all the
above-mentioned properties nor can they fully replace
3. DERMO-EPIDERMAL (COMPOSITE)
the functional and anatomical properties of the native
SKIN SUBSTITUTES
skin. There are, however, a number of bioengineered
skin-replacement products which are currently available Dermo-epidermal or composite skin substitutes aim to
to clinicians and are used for wound-healing purposes. mimic the histological structure of normal skin where
In general, these tissue replacements only partially both epidermal and dermal layers are present. This
address skin functional requirements and surgeons similarity also provides some functional resemblance
tend to use different products to achieve specific to the normal skin. These are not only the most
purposes. Shakespeare (2005) outlines four groups of advanced and sophisticated products, when compared
functions which bioengineered skin-replacement pro- with epidermal and dermal substitutes, but also the
ducts can offer: protection—by establishing a most expensive tissue-engineered biological constructs
mechanical barrier to micro-organisms and vapour for tissue repair ( Jones et al. 2002).
loss; procrastination—following early wound debride- Most of these products are based on allogeneic skin
ment some wound cover is needed until permanent cells, incorporated into a dermal scaffold. This approach
wound closure can be achieved with serial skin grafts allows the production of large quantities of uniform
or cultured autologous cell applications, especially in batches of the product, with a relative ‘off-the-shelf’
extensive burns; promotion—delivery to the wound availability. However, these biomaterials act rather
bed of dermal matrix components, cytokines and like temporary biologically active wound dressings
growth factors, which can promote and enhance natural (Supp & Boyce 2005), providing growth factors, cyto-
host wound-healing responses; provision—of new kines and ECM for host cells while initiating and
membrane (microperforated); auto, autologous; allo, allogeneic; xeno, xenogeneic; recomb, recombinant; synth, synthetic.
allograft (cadaveric) native native allo allo native human skin with dermal temporary
from not for profit skin banks and epidermal cells
OrCel cultured keratinocytes in vitro allo xeno bovine collagen sponge temporary
Ortec International, Inc., and fibroblasts
New York, NY, USA
The Netherlands
TissueTech Autograft System cultured keratinocytes in vitro auto recomb HAM permanent
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MySkin cultured keratinocytes (subconfluent in vitro auto synth silicone support layer with a permanent
CellTran Ltd, Sheffield, cell sheet) specially formulated
UK surface coating
Laserskin or Vivoderm cultured keratinocytes (confluent cell in vitro auto recomb HAM permanent
Fidia Advanced sheet)
Biopolymers, Padua,
Italy
Bioseed-S cultured keratinocytes (subconfluent in vitro auto allo fibrin sealant permanent
BioTissue Technologies cell suspension)
GmbH, Freiburg,
Review. Tissue-engineered skin bioconstructs
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Germany
fibroblasts; HAM, hyaluronic acid membrane (microperforated); HYAFF, a derivative of hyaluronan; GAG, glycosaminoglycan; auto, autologous; allo, allogeneic; xeno, xenogeneic;
recomb, recombinant, synth, synthetic.
Permacol Surgical Implant — in vivo — xeno porcine acellular diisocyanite cross- permanent
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(Continued.)
Table 3. (Continued.)
OASIS Wound Matrix — in vivo — xeno porcine acellular lyophilized small permanent
Cook Biotech Inc., West intestine submucosa
Lafayette, IN, USA
Pelnac Standard/Pelnac Fortified — in vivo — xenoþsynth silicone/silicone fortified with silicone semi-
Gunze Ltd, Medical Materials gauze TREX, atelocollagen derived permanent
Center, Kyoto, Japan from pig tendon
Review. Tissue-engineered skin bioconstructs
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TransCyte (DermagraftTC) cultured neonatal in vitro allo xenoþsynth silicon film, nylon mesh, porcine temporary
Advanced BioHealing, Inc., fibroblasts dermal collagen
New York, NY and La Jolla,
CA, USA
R. V. Shevchenko et al.
(Continued.)
235
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permanent
duration of immunogenic tolerance to allogeneic fibroblasts
permanent
temporary
the cover (Coulomb et al. 1998) and their survival in the host
semi-
up to three weeks (Morimoto et al. 2005). Long-term
preservation of allogeneic fibroblasts and their prolifer-
ation up to two months in the host without signs of
immune rejection have also been reported (Sher et al.
1983; Bell et al. 1984; Eaglstein et al. 1999; Hebda &
Dohar 1999; Sandulache et al. 2003; Griffiths et al.
2004). However, porcine studies could not confirm allo-
HYAFF layered on silicone
alloþsynth
in vitro
in vivo
fibroblasts
incorporated
human cells
listed in table 1.
—
Hyalograft 3D
CA, USA
primary
cellular
incorporated human loading cell scaffold duration of
name manufacturer or investigating group cells occurs source source scaffold material the cover
polycaprolactone collagen Nanoscience and Nanotechnology cultured dermal in vitro allo synth polycaprolactone-blended temporary
nanofibrous membrane Initiative, Division of Bioengineering, fibroblasts collagen electrospun
National University of Singapore, nanofibrous membrane
Singapore
Tegaderm-nanofibre Nanoscience and Nanotechnology cultured dermal in vitro allo xeno þ synth poly(e -caprolactone)/gelatin temporary
construct Initiative, Division of Bioengineering, fibroblasts nanofibrous scaffold
National University of Singapore, electrospun on
Singapore polyurethane dressing
(Continued.)
R. V. Shevchenko et al.
237
Table 4. (Continued.)
238
primary
cellular
incorporated human loading cell scaffold duration of
name manufacturer or investigating group cells occurs source source scaffold material the cover
collagen– INSERM, U553 and Université Paris 7, cultured dermal in vitro allo xeno bovine collagen I/ temporary
hybrid nanofibrous PLGA/ School of Materials Science and — in vivo — synth PLGA/chitosan hybrid permanent
chitosan membrane Engineering, Tianjin University, electrospun nanofibrous
Tianjin, China membrane
biodegradable polyurethane Department of Materials Science and — in vivo — synth biodegradable polyurethane permanent
microfibres Engineering, University of Delaware, microfibres
Newark, DE, USA
silk fibroin and alginate Department of Veterinary Physiology, — in vivo — xeno þ synth silk fibroin/alginate-blended permanent
College of Veterinary Medicine and sponge
School of Agricultural Biotechnology,
Seoul National University, Seoul,
South Korea
R. V. Shevchenko et al.
polyvinyl alcohol/chitosan/ Department of Sericulture and — in vivo — xeno þ synth polyvinyl alcohol/chitosan/ permanent
fibroin-blended sponge Entomology, National Institute of fibroin-blended sponge
Agriculture and Technology, Suwon,
Korea
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composite nano-titanium Department of Nursing, Cardinal Tien — in vivo — allo þ recomb composite nano-titanium permanent
oxide –chitosan artificial College of Healthcare and oxide –chitosan with
skin (NTCAS) Management, Taipei County, Taiwan gelatin and hyaluronic
acid
bacterial cellulose Vascular Engineering Center, Institution — in vivo — recomb cellulose nanofibrils permanent
of Surgical Disciplines, Sahlgrenska synthesized by
University Hospital, Göteborg, Acetobacter xylinum
Sweden
(Continued.)
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duration of
permanent
permanent
ocell Tissue Engineering AB, Karolinska University
the cover
Hospital, Stockholm, Sweden). However, the use of an
allograft is associated with some complexities such as
availability of skin banks, denial of application on reli-
gious grounds, and its safety for the patient. Rigorous
transglutaminase
scaffold material
3.2. Apligraf
microbial
matrix
Apligraf consists of viable allogeneic neonatal fibro-
blasts, grown in a bovine type I collagen gel matrix,
combined with viable allogeneic neonatal keratinocytes,
forming a confluent superficial layer of the construct,
thus mimicking the normal structure of human skin.
Although this product does not cause immunological
scaffold
xeno
xeno
loading
cellular
in vivo
in vivo
low-calcium culture medium that enhances the initial dermal replacement construct Hyalograft 3D and epi-
migration and proliferation of keratinocytes (Hernon dermal substitute Laserskin (Uccioli 2003). These are
et al. 2007). However, clinical studies now need to be based on autologous keratinocytes and fibroblasts,
undertaken. grown on microperforated hyaluronic acid membranes,
and described later in this article. According to avail-
3.3. OrCell able publications (Uccioli 2003), this system allowed
successful treatment of diabetic foot ulcers as estab-
This tissue-engineered skin construct includes cultured lished in randomized clinical trials, where the 70.3 per
allogeneic fibroblasts and keratinocytes obtained from cent rate of wound closure was achieved in neuroischae-
the same neonatal foreskin. Fibroblasts are seeded mic, ischaemic, neuropathic and post-surgical ulcers,
into a bovine type I collagen sponge, which has a many of which were full-thickness and with the area
non-porous collagen-gel coating, on top of which kerati- greater than 5 cm2 in 85 per cent of cases. Recurrence
nocytes are added to form a confluent layer. The rates were also low (not exceeding 8.2%) when the
product was licensed in 2001 to treat donor sites in TissueTech Autograft System was applied. Although
burns and recessive dystrophic epidermolysis bullosa. this system may allow for definitive wound closure, it
This bilayered product is reported to produce an array is not a ‘true’ bilayered skin substitute where both
of cytokines and growth factors such as fibroblast dermal and epidermal layers are present, as it requires
growth factor-1, keratinocyte growth factor-1, platelet- grafting of two products, and may be complicated to
derived growth factor, vascular endothelial growth use in a clinical setting.
factor and transforming growth factor-a, which are all The preceding literature therefore suggests that no
favourable for host cell migration and wound healing, commercially available true bilayered ‘skin substitute’
thus ‘conditioning’ the wound bed for further treatment for permanent deep wound closure exists yet.
with skin grafts. This artificial skin substitute product There are many reports describing combinations of
showed reduced scarring, and a shorter healing time cultured human keratinocytes and fibroblasts with allo-
was also reported when compared with the acellular geneic or xenogeneic decellularized dermis, but these
bioactive wound dressing Biobrane (Still et al. 2003). composites are mainly used for in vitro studies on
Being composed of allogeneic cells, the product per- cell– cell interactions rather than for clinical use
forms a temporary role, resorbs in 7 – 14 days (similar (Harrison et al. 2006). There have been attempts to
to Apligraf ) and no cellular DNA from the product combine commercially available dermal substitutes
can be found in the wound 14– 21 days post-application. with either cultured or non-cultured autologous cells
in pre-clinical studies (Compton et al. 1998; Boyce
3.4. PolyActive et al. 1999; Jones et al. 2003; Wood et al. 2007) with
This bilaminar product is based on autologous cultured promising results, but no follow-up clinical trial results
keratinocytes and fibroblasts seeded into a PolyActive are available yet. There is only one three-dimensional
matrix. This porous matrix consists of a soft polyethy- reconstructed skin substitute which has achieved clini-
lene oxide terephthalate component and a hard cal use and has been found to be very promising—
polybutylene terephthalate component, which prevents the so-called Cincinnati Shriners Skin Substitute or
contraction of this polymer (IsoTis NV, Bilthoven, The PermaDerm—which was designed by Boyce and
Netherlands; Xiao et al. 1999; El Ghalbzouri et al. 2004). colleagues (Supp & Boyce 2005; Boyce et al. 2006). It
This polymer is commonly used for bone reconstruction is based on the collagen sponge, seeded with autologous
and its use for skin repair is poorly elucidated in the lit- fibroblasts and keratinocytes. It therefore delivers per-
erature. The product uses autologous cells and therefore manent wound closure and can be viewed as a ‘true
does not pose the same potential risks as those associ- skin substitute’. Although this skin substitute product
ated with allogeneic material such as cross- has won award from the American Burns Association
contamination by infective agents or immune rejection, for its clinical performances, the product is not yet
suggesting potential benefits over allogeneic-based bio- commercially available.
constructs. However, the use of autologous cells may Currently available composite skin substitutes use
limit the product’s ‘off-the-shelf’ availability and only two cell types—keratinocytes and fibroblasts—
increase its costs when compared with competitive allo- therefore they cannot perform all the functions of the
geneic-based products (e.g. Apligraf, OrCell). Perhaps skin owing to the lack of innervation, and lack of
the PolyActive tissue-engineered skin construct may immune cells, sweat glands and hair follicles. There
find use as a biologically active dressing in the treat- are sparce reports regarding improvements of these
ment of partial-thickness wounds and also skin graft types of skin substitutes where additional cell types,
donor sites providing growth factors necessary to such as endothelial cells, are incorporated for improved
enhance wound healing. The fact that this product fea- functionality of the constructs (Ponec et al. 2004;
tures a non-biodegradable synthetic dermal component Tonello et al. 2005), Langerhans cells (Regnier et al.
precludes its use as a permanent skin substitute. 1998; Dezutter-Dambuyant et al. 2006). Melanocytes,
although normally present in fresh keratinocyte cultures
and dermo-epidermal constructs (Rehder et al. 2004)
3.5. TissueTech Autograft System
were specifically investigated in the work of Hedley
This system combines two tissue-engineered biomater- and co-workers (Hedley et al. 2002) where the regulat-
ials designed by Fidia Advanced Biopolymers (Abano ory role for fibroblasts in skin pigmentation was
Terme, Italy) and applied consecutively to the wound: revealed since, when added to in vitro skin constructs,
fibroblasts downregulated spontaneous pigmentation. of keratinocytes merge together and form stratified epi-
Melanocyte-containing skin constructs are used exten- thelial layers which can be enzymatically detached from
sively for in vitro studies of ultraviolet light effects on the culture flasks, mounted onto backing supports (such
the skin such as phototoxicity and photoageing as paraffin gauze) to maintain basal–apical orientation
(Marrot et al. 1998; Lee et al. 2007). and the sheet then applied to the wound (Atiyeh &
It was reported recently that murine skin stem cells, Costagliola 2007).
found in hair follicle bulges and isolated for in vitro cul- The quality of such stratified cultured epithelial
ture, retain their stem cell characteristics and are capable autografts (CEAs) depends on the clonal cellular com-
of producing multiple cell types including keratinocytes, position (Barrandon & Green 1987; Rochat et al.
hair follicles and functionally active sebaceous glands, 1994; Papini et al. 2003), which putatively determines
when returned to an in vivo situation (Blanpain et al. graft survival and long-term performance when applied
2004). There is a significant pool of knowledge generated in vivo. Basal keratinocytes, cells which give rise to
on human skin stem cells to date (Jahoda 2003; holoclones (in vitro colonies with the highest prolifera-
Blanpain et al. 2007; Waters et al. 2007) and this may tive potential), are essential for successful long-term
allow in vivo work to proceed to the same endpoint. If graft survival. Meroclones, consisting of transient
so, this will give a considerable potential to produce his- amplifying cells, have a variable potential for prolifer-
tologically similar and fully functional true skin ation and can provide only temporary wound closure
equivalents for the treatment of extensively burnt if applied in vivo. Committed keratinocytes, or para-
patients and other acute and chronic skin defects. clones, form the majority of a normal epithelial cell
population but are able to replicate only a few times
before differentiation and senescence. Therefore CEA,
consisting exclusively of paraclones, cannot serve as a
4. EPIDERMAL SUBSTITUTES
substrate for permanent wound closure. Current cultur-
As it became possible to cultivate human keratinocytes ing techniques allow for holoclone preservation when
serially in vitro (Rheinwald & Green 1975) and to keratinocytes are cultured in vitro over long periods of
rapidly expand the number of patient keratinocytes time (Papini et al. 2003).
ex vivo, this technology was rapidly transferred into In vitro keratinocyte expansion techniques produce
clinical applications (Gallico III et al. 1984) where CEA sheets large enough to cover the entire surface of
it contributed to improve patients’ survival rates the body in three to four weeks from only a 3 cm2
(Carsin et al. 2000). However, the value of cultured skin biopsy (Chester et al. 2004). If additional support
keratinocytes remains disputed and controversial to substances, such as a fibrin matrix, are used to culture
the present day. keratinocytes, it is possible to further expand the area of
A key step in the designing and production of epider- CEAs in shorter periods of time. Ronfard and col-
mal substitutes is the isolation of keratinocytes from a leagues (Ronfard et al. 2000) obtained 4.1 m2 of
donor and the subsequent in vitro culture of these graftable epithelium from a 4.5 cm2 skin biopsy cultured
cells, to obtain the necessary number of keratinocytes for 15 days on a fibrin matrix, compared with 1.4 m2
for therapeutic needs. Differences in approach to the when cultured on plastic surfaces. Material handling
production of epidermal substitutes are dependant on: and basement membrane formation were also improved
cell culture techniques (submerged or air– liquid inter- when this technique was employed.
face models); the stage of cell differentiation and Clinical ‘take’ or integration of such cultured epi-
epithelial organization (confluent sheets, subconfluent thelial autografts in a sheet form varies significantly
cell layers and suspensions); the methods of cell delivery from ‘excellent’ to ‘poor’ (Pandya et al. 1998; Horch
to the patient (confluent sheets mounted onto support et al. 2005; Wood et al. 2006a,b; Atiyeh & Costagliola
layer, subconfluent dispersed keratinocytes delivered 2007). This can be partly attributed to the fact that
via aerosol techniques or via microcarrier beads); as CEAs contain terminally differentiated keratinocytes
well as the use of additional substrates to enhance cell in which integrin expression, responsible for attachment
culture and delivery (synthetic and biological; Chester to the underlaying matrix, is altered (Chester et al.
et al. 2004; Atiyeh & Costagliola 2007; MacNeil 2007). 2004). Among other disadvantages of CEA sheets are:
To initiate a culture of autologous cells, a skin biopsy the long culture time; friability of the grafts; and com-
of 2 – 5 cm2 is usually taken along with initial wound plicated handling and application procedures. There is
debridement upon the patient’s arrival at the clinic. also a need for precise coordination between the tissue
The epidermis is separated from the dermis and single culture facility and the clinic. A major disadvantage
keratinocytes are released from the sheet by exposure of sheet application is the unpredictable clinical out-
to enzymes. These keratinocytes are plated into tissue comes with varied take rates of 15– 85% (Williamson
culture vessels where single cells start to divide to et al. 1995; Atiyeh & Costagliola 2007). Poor keratino-
form colonies in the presence of mitotically inactivated cyte attachment, resulting in blistering when exposed
mouse fibroblasts and culture medium containing foetal to minor shearing forces, could be seen months post-
calf serum, and necessary supplements. It is possible to grafting in patients treated with confluent CEAs
expand keratinocytes in xenogeneic-free conditions (Gallico III et al. 1984).
where murine fibroblasts and bovine serum are avoided It could be postulated that the very nature of the
(Notara et al. 2007) but the proliferative lifespan of cells confluent, layered cell culture system is responsible for
cultured under these conditions is noticeably reduced the unpredictable clinical outcome. As the cell layers
(Ronfard et al. 2000; Papini et al. 2003). Single colonies build up in the culture vessel, the proliferating basal
cells start to be isolated from the nutrients in the cell of cells derived from a small skin biopsy (Carsin et al.
culture medium. The differentiating, keratinizing cells 2000; Vacher 2003), whereas EpiDex is cultured from
in the upper layers of the culture are tightly packed keratinocytes obtained from the outer root sheath of
together via desmosomal junctions forming a barrier scalp hair follicles (Tausche et al. 2003).
between nutrients and the basal cells. The more cell This is the oldest approach in keratinocyte delivery
layers, the easier it is to handle the cell sheet, but the and shares the previously described disadvantages of
greater the chances of starving the basal cells and there- long culture time; difficulties in handling and appli-
fore the greater the chances of a poor ‘take’ or survival cation; variable take rate; poor long-term results;
on the wound bed. These shortcomings led to the inves- necessity for dermal support; high cost; and a short
tigation of the use of subconfluent keratinocytes, which (24 h) shelf-life (Horch et al. 2005). Despite these diffi-
have a greater in vivo proliferative activity, can be har- culties, as well as a declining interest and rising doubt
vested much earlier (after 5 – 7 days in culture) and have in the usefulness of CEA products, they still remain a
a degree of flexibility in the coordination of cell propa- valuable life-saving treatment in cases of extensively
gation, harvesting and clinical application processes burned patients (Atiyeh & Costagliola 2007).
(MacNeil 2007).
Subconfluent keratinocytes can be applied to the 4.2. MySkin
wound bed via an aerosol of cell suspension (Navarro
et al. 2000). They can be delivered resuspended either This product uses subconfluent autologous living kera-
in cell culture medium or fibrin glue (Grant 1999; tinocytes which are grown on a silicone support layer
Grant et al. 2002). The fibrin glue improved cell attach- with a specially formulated surface coating (Moustafa
ment to the wound bed and helped to control bleeding et al. 2004). Such an approach allows not only easier
but did not affect keratinocyte take rate or the resulting handling and application of keratinocyte grafts, but a
epithelial cover area (Currie et al. 2003). Subconfluent decreased time for cell culture. Another advantage is
keratinocyte suspensions contributed to an earlier that proliferatively active keratinocytes could be deliv-
basement membrane formation with a mature dermal– ered to the patient with greater time flexibility
epidermal junction region when compared with CEA (Hernon et al. 2006) that cannot be achieved with con-
sheets (Andree et al. 2001). As the developed basement fluent cultured epithelial sheet grafts. This product is
membrane is crucial for the strong bonding between indicated for the treatment of neuropathic, pressure
the epithelial layer and the underlying tissue this may and diabetic foot ulcers, superficial burns and skin
also explain the poor take levels and long-term results graft donor sites with reported positive clinical out-
when using CEA sheets (Woodley & Chen 2001). comes (Zhu et al. 2005; Moustafa et al. 2007). It can
Another approach to deliver subconfluent keratino- also be applied to full-thickness wounds in combination
cytes to the wound bed is to culture a monolayer of with meshed skin grafts but cannot be used alone for
subconfluent keratinocytes on delivery membranes deep-wound treatment and in this way is similar to
which can be either mechanically peeled off the culture other epithelial bioengineered constructs.
vessel (Ronfard et al. 2000) or can be applied with the
cultured cells directly to the wounded site (Hernon 4.3. Laserskin (Vivoderm)
et al. 2006). Both techniques obviate the need for Laserskin was designed and manufactured by Fidia
enzymatic detachment of cells as enzymatic treatment Advanced Biopolymers (Italy) with rights to manufac-
can alter the structure of anchoring fibrils responsible ture and distribute this product granted to ConvaTec, a
for the graft attachment to the underlying tissue division of Bristol-Myers Squibb Company under the
(Compton et al. 1989; Hernon et al. 2006). Delivery Vivoderm trade name. The product consists of autolo-
membranes can be made of synthetic materials such gous keratinocytes cultured on a hyaluronic acid
as a silicone support membrane with a specially formu- membrane which is laser-microperforated. This allows
lated surface coating (MySkin); polyurethane; or based the keratinocyte migration from a support material
on biological materials such as collagen, fibrin glue, down to the wound bed (Ramos-e-Silva & Ribeiro de
hyaluronic acid or decellularized dermis (Chester Castro 2002). Hyaluronic acid is a naturally synthesized
et al. 2004). The use of these delivery systems allows polymer of the human skin ECM which is reported to
for an earlier clinical cell application by reducing the promote fibroblast and keratinocyte migration and pro-
culture, preparation and application times, with the liferation. It is also reported to participate in scarless
added benefits of convenient material handling as foetal wound healing (Price et al. 2007). Preliminary
well as the biological properties of some of the delivery studies with Laserskin have shown the promising poten-
membranes which may affect and improve wound tial of the product giving good graft take rate,
healing. biocompatibility as well as low infection rates in a
Some commercially produced epidermal substitutes, pre-clinical animal model (Lam et al. 1999; Myers
which have been approved and marketed for clinical et al. 2007) and small clinical trials (Price et al. 2006).
use, are listed in table 2.
4.4. Bioseed-S
4.1. Epicel, EPIBASE, EpiDex
This product consists of 3 – 6 106 ml21 cultivated sub-
These products are manufactured using a patient’s own confluent autologous keratinocytes resuspended in a
keratinocytes which are grown to confluency within 15 fibrin sealant (Tissucol Duo S Immuno, Baxter). To
days to form CEA sheets. Epicel and EPIBASE consist date it has mainly been used to treat therapy-resistant
chronic venous leg ulcers ( Johnsen et al. 2005) and wound epithelialization by sprayed keratinocytes
multinational randomized controlled clinical trials (Wood et al. 2007).
suggest almost 50 per cent increase in wound-healing The majority of products for dermal substitutions
efficiency when compared with standard treatment are acellular, based either on allogeneic, xenogeneic or
(Vanscheidt et al. 2007). No information regarding synthetic materials (Anthony et al. 2006). It is much
the use of this material in burns patients is available, easier to manufacture these and to obtain a licence for
although there is potential for its use in this area. clinical application when compared with cell-containing
An animal study with analogous material, where bilayered skin substitute constructs. The ability to pro-
autologous keratinocytes were resuspended in autolo- duce large batches associated with rigorous quality
gous fibrin sealant, applied to full-thickness wounds, control and reduced costs has resulted in the array of
revealed the usefulness of such application methods products which have found their way to the clinic,
resulting in a good epithelialization (Grant et al. where some of them have been widely adopted. Cur-
2002). The fibrin did not improve the take rate of kera- rently commercially available or marketed dermal
tinocytes when compared with sprayed keratinocytes bioengineered constructs are listed in table 3.
without fibrin glue, although improved handling, cell
attachment, haemostasis and wound healing were
5.1. AlloDerm, Karoderm, SureDerm,
noted.
GraftJacket
4.5. CellSpray AlloDerm, Karoderm, SureDerm and GraftJacket all
represent human accellular dermal matrix products.
CellSpray products, provided by Clinical Cell Culture AlloDerm is a freeze-dried human accellular dermal
company (C3, Perth, Australia), use either cultured or matrix, with preserved basement membrane, acting
non-cultured autologous keratinocytes. This technology similarly to cadaver allograft, readily incorporates into
is based on the possibility of harvesting subconfluent the wound without rejection and does not cause immu-
keratinocytes in their most active proliferating state fol- nogenic response owing to the lack of a cellular
lowed by their application to the wound bed component. Initially intended as a dermal replacement
by spraying. This allows further in vivo proliferation material, it showed uncertain rates of vascularization
to confluency (wound closure), and cell differentia- (Shakespeare 2005) and has therefore recently gained
tion to form a recognizable epithelial structure (Navarro more popularity for abdominal wall hernia reconstruc-
et al. 2000; Chester et al. 2004). Such an approach tion (Espinosa-de-los-Monteros et al. 2007; Patton
results in a reduced cell culture time with earlier wound et al. 2007; Lipman et al. 2008), subcutaneous mastect-
coverage by viable activated proliferating keratinocytes omy (Ashikari et al. 2008), rhinoplasty and
(Atiyeh & Costagliola 2007). Although this method temporomandibular joint reconstruction (Khariwala
allows a more convenient way of delivering keratinocytes et al. 2007), periodontal surgery (Zigdon & Horwitz
to the wound bed at earlier stages post-wounding, such 2006), and rectovaginal, rectourethral or tracheoeso-
an application is limited to partial-thickness and graft phageal fistulae reconstruction (Shelton & Welton
donor site wounds. Full-thickness wounds still require a 2006; Lesser et al. 2008; Su et al. 2008), where immedi-
dermal element to achieve functional permanent skin ate revascularization is of less importance. It is still used
restoration (Wood et al. 2006a,b). for acute thermal injury treatment with very promising
The listed epidermal substitute products provide results. It is being used in one-stage operating pro-
permanent wound closure. They are effective in treating cedures in combination with extra-thin split-thickness
chronic ulcers and improving the quality of life of these skin grafts (Tsai et al. 1999; Callcut et al. 2006), but
patients, although their efficiency and long-term out- longer follow-up and more clinical trials are needed. It
comes for burns treatment are still questioned by is reported to produce acceptable functional and cos-
many. It is also widely appreciated that combination metic results but, being a human-derived biomaterial,
with some sort of dermal substitute is needed in order it is associated with potential safety and ethical issues
to achieve effective full-thickness wound healing. and avoided on moral grounds by some clinicians and
patients.
GraftJacket is a similar product, 0.4 – 0.8 mm thick,
5. DERMAL SUBSTITUTES
pre-meshed for the convenience of application, often
Wound bed preparation and the resultant recipient sur- used for tendon (Valentin et al. 2006; Furukawa et al.
face are very important for an effective graft take. It is 2007) and low extremity wounds repair (Brigido
reported that cultured epithelial autografts would take 2006). Successful treatment of superficial and deep
in only 15 per cent of cases when grafted onto chronic wounds has been reported (Kim et al. 2006) but infor-
granulation tissue, in 28– 47% of cases if grafted onto mation regarding thermal injuries treatment is limited
early granulation tissue or a freshly debrided wound, because of the novelty of this biomaterial.
but would have a 45– 75% chance of integration when SureDerm is produced by HansBiomed (Korea) and
applied to the wound with a dermal or neodermal bed uses human allogeneic acellular lyophilized dermis
(Orgill et al. 1998). Other clinical trials also outline (Kim et al. 2003). It is indicated for the replacement
the importance of dermal pregraftment for the success- or repair of damaged soft tissue including hypertrophic
ful take of cultured autologous keratinocytes (Travia scar revision and burns wounds. The material can be
et al. 2003). In vivo studies have also shown the impor- stored up to 2 years, requires 10 min rehydration
tance of a dermal bed for successful full-thickness before application, is permanently incorporated into
the wound bed and provides a dermal bed for 5.5. Integra Dermal Regeneration Template,
subsequent skin grafting. Terudermis, Pelnac Standard Type/Pelnac
Fortified With Mesh Type
Integra Dermal Regeneration Template, initially
5.2. Permacol Surgical Implant, Matriderm designed by Yannas & Burke (1980), consists of
These decellularized dermal products are similar to a porous dermal component made of bovine type I
AlloDerm but of animal origin. This reduces risks collagen and shark chondroitin-6-sulphate glycosamino-
associated with transferable human viral diseases, glycan which is bonded to a silicone pseudo-epidermis
such as HIV and HepB. The wide availability of raw (Yannas & Burke 1980). The dermal component of
materials makes these easier and cheaper to produce. the bioconstruct becomes populated with host cells,
Permacol Surgical Implant is a decellularized porcine including fibroblasts, which contribute towards neoder-
dermal layer containing collagen and elastin fibres. mis formation while the material’s scaffold degrades
Material is cross-linked with diisocyanate by a patented and the pseudo-epidermal component protects wounds
technology. It is used mainly for abdominal wall hernia from vapour loss and bacterial contamination. When
reconstruction (Parker et al. 2006), especially when Integra vascularization and neodermis formation are
microbial contamination is present (Catena et al. complete, usually within 15 – 20 days, the silicone
2007). Its use for dermal reconstruction is limited layer is peeled off and the wound can be closed perma-
owing to the slow biointegration and vascularization nently with an SSG. This material was successfully
(MacLeod et al. 2004, 2005). clinically tested in managing burn wounds in 1981
Matriderm is of bovine origin, consists of 1 mm-thick (Burke et al. 1981) and since then has become widely
structurally intact native collagen matrix coated with adopted for full-thickness burns treatment (Heimbach
a-elastin hydrolysate from the ligament, freeze-dried et al. 1988, 2003; Heitland et al. 2004) becoming clini-
and non-cross-linked. In small clinical trials to treat cally a ‘gold standard’ dermal substitute biomaterial.
full-thickness burns it has shown promising results It is also used for chronic ulcer treatment (Silverstein
when applied simultaneously with split-thickness skin 2006) and full-thickness non-thermal skin wound man-
grafts in a single-stage operative procedure (van Zuijlen agement (Violas et al. 2005). Advantages of the
et al. 2000; Haslik et al. 2007; Ryssel et al. 2008). product include its long shelf life, simple handling, low
risks of immunogenic response and disease trans-
mission, good cosmetic outcomes with reduced rates of
contraction and scarring (Anthony et al. 2006; Kim
5.3. OASIS Wound Matrix et al. 2006). Meticulous surgical preparation of the
OASIS Wound Matrix is produced from porcine small- wound bed is required to guarantee a good take of Inte-
intestine submucosa and intended for wound closure gra. It cannot be used on infected wounds, it requires a
stimulation in acute, chronic and burns wounds. It is relatively long time of 10– 14 days for vascularization
freeze-dried and decellularized to prevent immunologi- and also requires a second operative procedure to
cal responses. Positive results have been obtained in achieve permanent wound closure with an SSG. In
randomized prospective controlled multicentre trials attempts to achieve a single-stage surgical procedure,
for chronic leg ulcer treatment where faster healing the product has been seeded with disaggregated cul-
time and less ulcer recurrence was achieved (Mostow tured ( Jones et al. 2003) or non-cultured (Wood et al.
et al. 2005). OASIS Wound Matrix has also been 2007) autologous keratinocytes, using in vivo exper-
shown to support in vitro epidermal differentiation imental models. Results were promising, but further
and basement membrane formation (Lindberg & clinical follow-up is required.
Badylak 2001). It was also evaluated in vivo as a Terudermis consists of a layer of lyophilized collagen
wound dressing in rodent full-thickness wounds where sponge reconstituted from a mixture of fibrous and
it contributed towards contraction minimization and heat-denatured bovine collagen which is cross-linked by
had no effect on epithelialization (Prevel et al. 1995). No dehydrothermal treatment. The collagen layer
results of clinical trials regarding its use in full-thickness is bonded to the silicone membrane which controls
wound management have been published yet. bacterial contamination and vapour loss during engraft-
ment, similar to Integra. The material is designed for
deep burns treatment, where bone, muscle or ligament
exposure is present (Choi et al. 1999). It is also reported
5.4. EZ Derm
to be useful for skin flap donor site regeneration (Lee et al.
EZ Derm is a reconstituted collagen of porcine origin 2005), post-traumatic deformity corrections (Yurugi et al.
which is cross-linked with aldehyde to increase its ten- 2002) and in otological surgery (Bessho et al. 1998).
sile strength. The product does not incorporate into Terudermis, when loaded with cultured fibroblasts,
the wound and has to be removed (Bello et al. 2001). endothelial cells, platelet-derived growth factor and
It is therefore marketed as a bioactive wound dressing. then applied to rodent in vivo models, showed not only
Comparison of this dressing with petrolatum non- angiogenesis enhancement but also the potential to use
adherent gauze dressing for partial-thickness burns the material simultaneously with an SSG for a one-step
care revealed no differences in bacterial colonization operative procedure (Soejima et al. 2006).
rate, healing time, pain relief and frequency of dressing Pelnac Standard Type/Fortified With Mesh Type
change procedures (Healy & Boorman 1989). are produced by Gunze Ltd, Medical Materials
enhanced keratinocyte take, and reduced hypertrophy state it can only be considered as a temporary bioabsorb-
and wound contracture rates when compared with exclu- able synthetic wound dressing.
sive application of keratinocyte cultures (Travia et al.
2003). Hyalograft 3D also contributes towards rapid
basement membrane formation (Stark et al. 2004). Hya- 7. BIOMECHANICAL CHARACTERISTICS
lomatrix PA has been investigated with favourable OF SKIN SUBSTITUTES
outcomes in a porcine preclinical model of full-thickness In comparison with the literature addressing clinical
wounds (Myers et al. 2007), and it has also been used behaviour, and support of cell growth in tissue engineer-
clinically for the treatment of deep partial-thickness ing skin substitutes, surprisingly few reports consider
burns. The material served as a temporary dressing to the mechanical properties of such skin substitutes. In
stimulate wound regeneration after dermabrasion and our view, a number of problems need to be addressed
was reported to be a good and feasible approach for before an objective and coherent approach to both
such wound treatments (Gravante et al. 2007). It is design and testing of the mechanical properties of skin
also reported to give favourable outcomes in deep paedia- substitutes can be considered.
tric burns treatment (Tamisani 2004). Both products are
also appealing from the safety point of view—they do not (i) How closely should skin substitutes’ properties
contain any animal or allogeneic human-derived mimic those of natural skin at the time of clinical
components. application? Other properties such as ease of hand-
ling by the surgeon, deformability to follow
anatomical contours or resistance to tearing may
6. POTENTIAL BIOMATERIALS FOR be more important immediately upon application.
SKIN SUBSTITUTION (ii) After a period of cell invasion and remodelling,
mechanical properties closer to those of natural
Besides the above listed biomaterials, the majority of skin would probably be more appropriate. If so,
which are based on collagen as the most studied, tra- those variables affecting changes in mechanical
ditional and convenient component of ECM known for properties during cell invasion and remodelling
its biocompatibility and bioconductivity and therefore need to be identified.
used for bioengineering of skin substitutes (Cen et al. (iii) The most appropriate mechanical properties pre-
2008), there is a variety of different skin and dermal substi- dicting skin substitute behaviour need to be
tute constructs that are currently under investigation. identified. For instance, should we be measuring
Some of them are still in the process of in vitro investi- elasticity, stiffness, viscosity, viscoelasticity, fail-
gation; however, some have entered stage II–III clinical ure, brittleness, etc.? Also under what kind of
trials and could possibly be on the market shortly available loading should these variables be measured: static
for patients and health practitioners. Novel potential skin tension, compression, creep, dynamic oscillatory
substitute biomaterials and scaffolds include human hair testing, etc.?
keratin-collagen sponge (Chen et al. 2006), hyaluronan (iv) More pragmatically, what mode of measurement of
coupled with fibronectin functional domains (Ghosh these properties should be employed? Does simple
et al. 2006), poly(lactic-co-glycolic acid)/chitosan hybrid extension or compression to obtain a Young’s mod-
nanofibrous membrane (Duan et al. 2007), biodegradable ulus in a moving beam instrument tell us enough, or
polyurethane microfibres (Rockwood et al. 2007), polyca- do we need to resort to rheological methods such as
prolactone (PCL) collagen nanofibrous membrane cone–plate or plate–plate oscillatory or creep
(Venugopal et al. 2006), silk fibroin and alginate (Roh testing?
et al. 2006), polyvinyl alcohol/chitosan/fibroin blended (v) If we are to compare the mechanical properties of
sponge (Yeo et al. 2000), Tegaderm-nanofibre construct skin substitutes with those of native skin, how
(Chong et al. 2007), bacterial cellulose (Helenius et al. should the latter be measured? Should we use
2006), ICX-SKN skin graft replacement (Boyd et al. values produced in vivo or ex vivo, should we
2007), porcine collagen paste (Shevchenko et al. 2008), measure full- or partial-thickness skin samples,
bovine collagen cross-linked with microbial transglutami- and if an in vivo measurement is attempted,
nase (Garcia et al. 2008), collagen–glycosaminoglycan– how do we know how much underlying tissue
chitosan dermal matrix seeded with fibroblasts (Kellouche contributes to the values obtained?
et al. 2007), composite nano-titanium oxide–chitosan arti- (vi) Many reports in the literature present data
ficial skin (Peng et al. 2008), keratinocytes and fibroblasts obtained from in vivo tests on the human forearm.
grown on Collatamp, deacetylated chitin or plant cellulose The question remains as to how representative
transfer membranes (Johnen et al. 2008) and many others these data are of skin at sites elsewhere in the body.
(table 4). Some of these experimental biomaterials, like
PermaDerm, have produced promising clinical results
7.1. The basic engineering principles
(Boyce et al. 1999, 2006) and have a potential to be
licensed and marketed for clinical use. However, with Both skin and skin substitutes such as cross-linked
such variety of available biomaterials for skin substitution collagen gels display viscoelastic (VE) properties
a differentiated evaluative approach should be employed, (Edwards & Marks 1995; Sheu et al. 2001). Viscoelastic
especially by medical practitioners. Although some bioma- behaviour implies that energy used in deforming a
terials like Suprathel are described as skin substitutes material is partly stored (elasticity) and partly dissi-
(Schwarze et al. 2008), the material’s performances clearly pated (viscosity). In skin, the elastic components are
60 per cent in the elderly. However, all of these values believed to affect cellular population of the matrix. Fur-
display wide ranges over the population studied. thermore, this model suggested that the mechanical
In vivo static testing includes uniaxial extensometry, properties of the matrix are independent of pore size,
where tabs are glued to the skin and pulled apart, the but that post-production covalent cross-linking
resulting stress or strain being measured and modifi- increased stiffness dramatically.
cations to allow biaxial or torsional testing to be In terms of the effects of such cross-linking,
achieved. Testing of skin hardness has been attempted Powell & Boyce (2006) have cross-linked collagen –
by measuring the deformation caused by a stylus glycosaminoglycan matrices with increasing amounts
pressed into the skin (so-called indentometry). How- of the water-soluble carbodiimide 1-ethyl-3-(3-
ever, for the reasons outlined above, a dynamic dimethylaminopropyl)carbodiimide (EDC). The stiff-
approach will yield more informative data, and a wide ness of the matrix increases to a point where
variety of methodologies exist in the literature. Such brittleness renders the matrix mechanically unsuita-
techniques include transforming the static tests into ble for tissue replacement, but when tested for the
an oscillatory mode, but also by the measurement of growth of human fibroblasts and keratinocytes, cyto-
shear wave propagation, and suction cup techniques, toxicity was observed at higher EDC concentrations,
often with the use of ultrasound scanning to identify an optimum between increased mechanical stability
movements in underlying tissues. More recently, con- and reduced cytotoxicity existing at 5 mM EDC.
siderable progress has been made in the modelling of An alternative approach has been adopted by
the viscoelastic properties of skin; for instance a model Garcia et al. (2008) by cross-linking the matrix to
using only four parameters has been developed by improve its mechanical stability using an enzymic
Khatyr et al. (2004) which describes the behaviour of approach with microbial transglutaminase. The introduc-
skin well, and sets of important material parameters tion of the glutamyl-lysine cross-links into the collagen
which should be considered in modelling skin have matrix modified its properties such that wound contrac-
been identified by Kvistedal & Nielsen (2009). Of par- tion in an in vivo model was significantly reduced. This
ticular interest when considering the interface of approach to mechanical modification also avoids potential
biomaterials with native skin are the findings of Holt cytotoxicity contributed by chemical cross-linkers.
et al. (2008) while measuring the viscoelasticity of The mechanical behaviour of these collagen
skin under low shear and low frequency conditions. matrices, but measured on the nanoscale, has been
They found that the epidermis appeared to contribute investigated by Chaudhry et al. (2009). Using a
rigidity and elasticity, while the dermis was far more nanoindentation approach, they were able to discrimi-
viscoelastic in nature, suggesting that the skin is nate between surface and bulk properties, and
mechanically a two-phase system. This finding could reported values for the storage modulus of 0.71 GPa,
pose serious problems when designing skin substitutes and for the loss modulus of 0.40 GPa. Constantinides
which interface with a wound bed and healing tissue. et al. (2008) report a value for the elastic modulus of
porcine skin of 222 kPa also using a nanoindentation
approach, but this was derived from a creep compli-
7.3. Biomechanical properties of skin substitutes
ance approach. Nevertheless, this does suggest at
Investigations into the biomechanical characteristics of least some discrepancy between collagen matrix and
skin substitutes have concerned themselves chiefly with skin in terms of mechanical properties at the nano-
either elucidating the properties of the fresh matrix, or metre scale. This is important as cell attachment is
attempting to follow changes in matrix properties as likely to be influenced more at dimensions similar to
they become populated with cells, and start to degrade. those of focal adhesions than bulk dimensions, and
Many of the matrices investigated have been of the cells are known to respond to the mechanical stiffness
collagen – glycosaminoglycan type, because these are of their substrate (Discher et al. 2005). A direct com-
commonly used clinically, but some others are presently parison of ultimate tensile strength, stress –strain
under investigation. behaviour, stress relaxation and creep parameters
An interesting approach has been adopted by Harley between native excised human skin and four tissue
et al. (2007) in modelling such a matrix. They adopt a engineering scaffolds was attempted by Zhang et al.
cellular solids description, treating the matrix as a (2007). Unfortunately, the scaffolds used are not well
foam, consisting of a network of interconnected struts. characterized in the report, but are two cross-linked
Under compression, this shows three phases of collapse: dermal matrices from human and pig, and two non-
(i) a linear elastic region as the struts bend; (ii) a col- cross-linked matrices from pig and goat. The par-
lapse bending region as the struts buckle; and (iii) a ameters for the non-cross-linked matrices
densification region as the pores have collapsed and approximated those of the natural excised skin, while
the bulk properties of the material predominate. The the gluteraldehyde-fixed matrices gave results signifi-
experimental results, both in terms of the properties cantly higher than the natural skin.
of individual struts tested by atomic force microscopy In an effort to improve the mechanical properties of
and the properties of the whole material in compression skin substitutes at the bulk level, several workers have
and tension, agree rather well with this model. This attempted to introduce synthetic polymers into natural
modelling has importance when the ingrowth of cells polymer matrices such as collagen. Interestingly, Powell &
into the material is considered as fibroblasts, for Boyce (2009) have added increasing amounts of PCL to
instance, are known to collapse such struts, and the an electrospun collagen scaffold, and have shown that
mechanical properties of the material at this scale are increasing PCL ratios increase the stiffness of the
material, making it easier to handle for the surgeon. wound healing while the patient’s own skin regenerates
However, after growth of human keratinocytes and to be used for serial autografting. Products based on
fibroblasts into the scaffolds, the mechanical properties autologous cultured keratinocytes and fibroblasts are
were either not significantly different, or even worse more likely to contribute to actual skin substitution
than those of the collagen alone. This suggests and results of clinical trials are encouraging (Boyce
that investigation of the mechanical properties of et al. 2006); however, no one will agree that these
tissue-engineering matrices both before and during products at the current level of sophistication can fully
the invasion of cellular components is critical in replace damaged skin (MacNeil 2007; Metcalfe &
the design of artificial skin. Ferguson 2007b).
The estimation of changes in mechanical properties There are many challenges faced by bioengineers
of skin substitutes after population by cells has been supplying live cell products that should also be taken
the subject of a number of recent studies. For instance, into account. There are long, complicated and expens-
Saddiq et al. (2009) have measured the ultimate tensile ive cultivation procedures, specific (and expensive)
strength and stiffness of four collagen gel matrices transport and storage conditions, a limited shelf-life,
before and after the growth of either 3T3 mouse the friable nature of cell-containing biomaterials,
fibroblasts or primary human fibroblasts into the especially for products based on live cells, a need for
matrix. The matrices used were collagen; precise coordination between the tissue culture facility
collagen – glycosaminoglycan (GAG); collagen cross- and the clinic if autologous cells are used just to name
linked with carbodiimide and putrescine, and col- a few. All the above reasons as well as an unattractive
lagen – GAG cross-linked with carbodiimide and cost-effectiveness of cell-based biomaterials which
putrescence. While the addition of GAGs and cross-lin- are only partially effective at fulfilling skin functions
kers increased both the ultimate tensile strength and make it also very difficult for any cell-based skin
the stiffness initially, after 6 days of fibroblast growth substitute product to reach the clinic.
(of both types) into the matrices, these values dropped Currently available products for permanent skin
significantly. Perhaps, even more surprisingly, the substitution can only partially replace the protective
values to which these parameters fell appeared indepen- barrier function of skin, but other functions, including
dent of the original gel treatment, with cross-linking touch and temperature sensation, excretion, perspira-
offering no protection from matrix degradation. tion, thermoregulation, protection from ultraviolet
A number of workers have adopted a different rays, synthetic function, not to mention the aesthetic
approach for obtaining a mechanically acceptable skin function, are not restored by the existing skin tissue-
substitute matrix, which is to create a cell-derived engineered products (MacNeil 2007; Metcalfe &
matrix directly from cell culture, rather than developing Ferguson 2007b). The simultaneous combination of
a collagen, or similar, matrix separately. For instance, different skin cell types including keratinocytes, mela-
Ahlfors & Billiar (2007) have developed a rapid nocytes, fibroblasts and endothelial cells derived from
method for producing a fibroblast-derived matrix that postnatal skin is aiming to create a functional skin
promotes further organized growth of cells. Mechanical replacement (Regnier et al. 1998; Hedley et al. 2002;
testing of the final product using an inflation method Ponec et al. 2004; Tonello et al. 2005; Dezutter-
similar to that of Billiar et al. (2005), in terms of ulti- Dambuyant et al. 2006). Attempts are being made to
mate tensile strength showed it to be superior restore skin appendages, such as hair follicles and
(313 kPa) to collagen and fibrin gels, but still not as sebaceous glands to maximally functionalize skin-
strong as native skin (713 kPa). replacement bioconstructs (Blanpain et al. 2004).
Recently, a novel approach to measuring skin substi- Bone marrow-derived cells have also been looked at as
tute mechanical properties in vivo has been reported by a potential cell source for skin-substitute products
Kim et al. (2008) using ultrasound elasticity measure- (Fioretti et al. 2008; Yoshikawa et al. 2008). Another
ments. This report describes the use with a tissue approach to even further functionalize artificial scaf-
phantom, but suggests this method as a useful approach folds for skin substitution is the addition of signalling
to non-invasive measurement of the properties of skin molecules for the regulation of cell – cell and cell–
substitutes as they become populated with cells matrix interactions either to accelerate biointegration
during the wound-healing process. (Tsuji-Saso et al. 2007; Wilcke et al. 2007; Garcia
et al. 2008; Ma 2008) or to adjust it according to the
phases of the wound-healing process. Such ‘intelligent’
biomimetic hybrid materials are termed ‘smart’ with
8. FUTURE PERSPECTIVES
the aim of producing a more natural skin restoration
Currently available tissue-engineered products for skin (Rosso et al. 2005; Metcalfe & Ferguson 2007b).
substitution, including dermal and epidermal con- No matter how complicated a strategy is adopted to
structs, although not perfect, occupy a specific niche create a skin replacement product that is based on post-
within a complex approach to treat full-thickness exten- natal cellular material, it is unlikely to succeed. Such
sive burns, improving patients’ survival rates and their products use mechanisms of tissue repair rather than
quality of life after injury (MacNeil 2007). Such tissue regeneration (Metcalfe & Ferguson 2007a).
products target only limited specific roles in the Normal wound-healing processes, required to repair
wound-healing process. Predominantly, they serve as wounded sites, have evolved over thousands of years
temporary biologically active dressings, donators of of human evolution to provide an efficient way to
cytokines and structural molecules necessary for cope with skin injuries, most of which resulted from
bites and mechanical trauma. These wounds were Prevention of scarring is therefore the second major pro-
contaminated with saliva, blood, dirt and micro- blem to be addressed after the restoration of the
organisms. There was no pharmacological means of damaged skin. Insights into foetal wound healing and
decontamination, and therefore natural non-specific understanding that scarless healing is partly attributed
and specific immunities evolved. Fibrin clots with a pro- to a decreased inflammatory response (Martin &
visional fibrin matrix helped to stop bleeding; an acute Leibovich 2005), reduced fibrin clot formation and
inflammatory response dealt with bacteria and any platelet degranulation has already allowed production
wound contamination with non-biological materials; of therapeutic measures directed at scar-free wound
and a rapid expansion of fibrovascular granulation healing (Ferguson & O’Kane 2004; Metcalfe & Fergu-
tissue allowed filling-in and closing-up of any wound son 2007b). Growth factors from the TGF-b family
defects within a relatively short period of time in have been closely studied in relation to scar-free healing.
order to preserve the individual life and the continu- It was established that during foetal wound healing
ation of the given species (Clark 1993; Li et al. 2007). TGF-b1 and -b2 isoforms are low or absent, whereas
Skin wounds and the clinical strategies to treat them the concentration of TGF-b3 isoform is significantly
have significantly changed over the past century: higher. During adult wound healing, the amount of
sharp or laser aseptic surgical dissection of the damaged TGF-b3 is insignificant, but amounts of TGF-b1 and
tissues; meticulous wound bed preparation -b2 are elevated by platelet degranulation and synthesis
(Panuncialman & Falanga 2007); pharmacological by cells of monocytic lineage during the inflammatory
medications to avoid wound bacterial colonization phase of wound healing (Ferguson & O’Kane 2004).
(Khan & Naqvi 2006; White et al. 2006; Landis 2008); Manipulation with adult wound healing by immuno-
and biomaterials to replace damaged tissue ( Jones genic blocking of TGF-b1 and -b2 isoforms and
et al. 2002; Horch et al. 2005; Ehrenreich & Ruszczak exogenous addition of the TGF-b3 isoform to the
2006; Clark et al. 2007) significantly obviated the wound in rodent, pig and human studies resulted in sig-
need for the natural protective cascades involved in nificantly reduced scarring (Shah et al. 1995; O’Kane &
wound healing. Apart from function restoration, Ferguson 1997; Ferguson & O’Kane 2004). Interest-
accepted by our ancestors as appropriate results of ingly, neutralization of all three TGF-b isoforms did
wound healing, issues of cosmesis, improved functional- not result in reduced scarring confirming complexity
ity and quality of life are now of great importance of the molecular interplay and alternative pathways
during skin-restoration treatment. The inflammatory during wound healing. Not only particular signalling
response, the production of cytokines to promote fibro- molecules, but their concentrations, time-dependent
vascular tissue proliferation, is considered a normal release cascades and pathways of their interaction
process to cope with wounds to deliver their ‘repair’. with cells and ECM need to be fully exposed and under-
However, in the light of current advances in medical stood to use this knowledge in creating clinically safe
science, these are now more likely to hinder optimal and efficient products to control scarring in postnatal
tissue ‘regeneration’ as they nearly always result in scar- organisms.
ring (Martin & Leibovich 2005). Any bioengineered Another approach where both true skin regeneration
product based on ‘natural’ mechanisms of wound heal- and avoidance of scarring may be successfully achieved
ing will result in scarring as well as limited is the use of either embryonic or adult stem cells. While
functionality, rather than giving fully functional skin embryonic stem cell research is delayed by ethical and
regeneration (Metcalfe & Ferguson 2007a). political debate, the number of investigations in the
True regeneration in vertebrates is seen in Xenopus use of adult stem cells is constantly growing, including
and axolotl where the entire limb is restored (Goss & skin research. It is well established that the hair follicle
Holt 1992; Gardiner & Bryant 1996). Examples of outer root sheath contains pluripotent epithelial stem
tissue regeneration in humans consist of the regeneration cells capable of self-renewal (Blanpain et al. 2004;
of the digit tip in a child (Illingworth 1974); regeneration Tumbar et al. 2004; Tumbar 2006). Although many
of the liver (Fausto 2000); and teeth regeneration (Chai & in vitro and in vivo assays exist to label, isolate and ana-
Slavkin 2003). Human skin does not regenerate post- lyse skin stem cells, it is still impossible to identify a
natally. However, during the early antenatal period, stem cell within an intact human skin tissue without
when injured, all skin layers and appendages are regener- ambiguity (Tumbar 2006). Nevertheless, pluripotent
ated. Foetal wound healing does not result in scar epithelial stem cells are isolated from hair follicles and
formation after injury (Lorenz & Adzick 1993; Yannas grown into epithelial sheets to be used clinically for epi-
2005). Understanding the mechanisms by which foetal thelial restoration (Limat et al. 2003; Tausche et al.
wounds heal could result in a real breakthrough in adult 2003). Although positive results regarding wound
wound healing with the possibility of real skin regener- epithelialization were reported, no restoration of hair
ation rather than defective and inferior scar-like skin follicles or sweat glands was seen; such stem cells exhib-
repair (Ferguson & O’Kane 2004). ited stable self-renewal potential, but their plasticity
Scars are the outcomes of any postnatal healing of was limited (Lemoli et al. 2005). Varying microenviron-
wounds caused by burns, trauma, surgical intervention, mental stimuli may affect stem cell plasticity, and result
or indeed anywhere tissue damage occurs, including in acquiring stem cell functions by more differentiated
integration of tissue-engineered skin-substitute pro- cells and limiting stem capacity by pushing stem cells
ducts. Uncontrolled scarring may result in defective into a more differentiated state (Lemoli et al. 2005).
aesthetics and the possible loss of function where exces- Hence, unsuitable biochemical and mechanical con-
sive tissue production and contraction occurs. ditions in a wound may limit plasticity and
proliferative activity of implanted stem cells. Multipo- Anthony, E. T., Syed, M., Myers, S., Moir, G. & Navsaria, H.
tent adult stem cells isolated from rodent dermis have 2006 The development of novel dermal matrices for
been shown to transdifferentiate into cells of several cutaneous wound repair. Drug Discov. Today: Therapeut.
embryonic lineages such as adipocytes, smooth muscle Strat. 3, 81–86. (doi:10.1016/j.ddstr.2006.03.001)
Ashikari, R. H., Ashikari, A. Y., Kelemen, P. R. & Salzberg,
cells, glial and neuron cells retaining their regenerative
C. A. 2008 Subcutaneous mastectomy and immediate
capacity (Toma et al. 2001). Studies have shown that reconstruction for prevention of breast cancer for high-
these cells can be subcultivated as distinct diverse cell risk patients. Breast Cancer 15, 185–191. (doi:10.1007/
types for at least 1 year without the loss of regenerative s12282-008-0059-7)
capacity (Toma et al. 2001). Novel therapeutical Atiyeh, B. S. & Costagliola, M. 2007 Cultured epithelial auto-
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stem cells of hair follicle bulges, isolated for in vitro cul- ogies for burn wound closure and healing—review of the
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