BIOTECHNOLOGY, MOLECULAR BIOLOGY AND NANOMEDICINE
VOL.1 NO.2 DECEMBER 2013
ISSN: 2330-9318 (Print) ISSN: 2330-9326 (Online) http://www.researchpub.org/journal/bmbn/bmbn.html
Recent Advances in Medical Grafting Involving
the Transplantation of Skin
Meda Cosma, MD,

(SC). It must be noted that even if many potential skin therapies
are successful in rodent models they might not be applied to
humans because of major differences in wound healing
mechanisms in thin-skinned versus thick-skinned animals.
Future skin regeneration therapies should include biomaterials
in combination with cell therapy.
Abstract— Current therapies aim to restore the
cutaneous barrier to fluid loss and infection. This is hard to
achieve in large acute wounds or non-healing chronic
wounds as patients have an urgent need of epidermal
barrier. Large acute wounds are mainly treated focusing on
coverage, while chronic wounds require coverage and
convert a non-healing wound bed to an environment
conducive to healing. The challenges in restoring skin have
restricted treatments for large acute wounds to
significantly change in 30 years, and treatments for chronic
wounds have only developed in the past 10–15 years.
Keywords—
tissue
keratinocytes
regeneration,
skin
The prototypical biomaterial approach to skin restoration is
provided by Integra® is. The bilayered construct consists of
type I bovine collagen combined with chondroitin-6-sulfate and
an overlying silicone membrane.[4] The collagen/chondroitin
layer manages the growth of “neo-dermis”, while the silicone
membrane is a temporary epidermal barrier until the construct
is finally covered with an autograft. Integra® has been mostly
used in large burn wounds, being a front-line treatment ain this
medical department.[5] Many treatments have simulated its
dermal and epidermal structure and all the new approaches
must compete with these existing mechanisms.[6] Future skin
regeneration approaches employing biomaterials are to
manipulate the healing properties of the natural ECM. Collagen,
fibrin and hyaluronic acid are ECM hydrogels produced at
specific intervals in the normal wound healing process to
stimulate migration and proliferation of skin cells.[7, 8] The
knowledge of ECM-skin cell interactions is crucial when
employing ECM as a regenerative therapy for wounds. Control
of ECM composition by fibroblasts and keratinocytes has a
major impact on scarring and wound contraction.[9] Cell
migration facilitation is a well known feature of collagen and it
is the ground for many of the currently existing dermal
substitutes, such as Integra®. The initial sealant and scaffolding
promoter in normal wound healing is fibrin, which reduces
wound contraction if combined with skin grafts.[10]
Consequently, fibrin is mostly used as a delivery vehicle for
applying skin cells to a wound.[11] Despite the wide use of
collagen and fibrin in new skin therapies, hyaluronic acid is a
potential component of many future therapies. Hyaluronic acid
is the major component of the ECM in fetal wounds that heal
without scarring[12], thus having implications for adult
healing.[13] The choice of biomaterial for skin regeneration
clearly influences the clinical outcome. Future skin
regeneration therapies should incorporate more natural
scaffoldings in order to ease wound healing.
grafting,
I. SKIN GRAFTING
Nowadays, the gold standard for wound coverage is the
split-thickness autograft[1], impossible to be readily and
completely carried out in patients with limited skin for donor
sites.[2] Collateral therapies include Integra® (Integra Life
Sciences, Plainsboro, NJ) and cultured epithelial autografts,
commercially available since the 1990s, but unable to achieve
widespread use. These treatments which are available today can
restore the epidermal barrier with clinically satisfactory
cosmetic outcomes. But clinically satisfactory cosmetic
outcomes do not automatically include adnexal skin structures
such as hair and pigment, which are crucial for normal skin
functions.[3] Thus, the ultimate purpose of RM for the
integumentary system is the restoration of fully functional skin
which should be physiologically and cosmetically similar to the
normal skin of the patient.
The most promising regenerative therapies for skin restoration
can be divided into 2 main categories: artificial skin substitutes
and cell-based therapies. Artificial skin substitutes mainly
employ biomaterials for skin restoration, while cell-based
therapies employ the healing response of skin cells. Novel
therapies include new formulations of naturally occurring
extracellular matrix (ECM) or in situ delivery of stem cells
Biomaterials possess significant wound healing properties, but
they lack the full wound healing potential of skin cells. Cells
Gastroenterology Institute, Department of Nanomedicine, 19-21, Croitorilor
street, Cluj-Napoca, Romania.
*Correspondence to Meda Cosma (e-mail: [email protected]).
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close a wound and create the skin function structures. In the
USA, cultured epithelial autografts (CEA) are the prototypical
cell-based therapy,[14] where keratinocytes are grown in a
sheet and applied on a wound[15] CEA can be cultured from
autologous or allogeneic keratinocytes and can reconstruct the
epidermal barrier with clinically satisfactory cosmetic
outcomes.[14, 16] Nevertheless, cell-based therapies are
potentially restricted by cell survival and the decreased storage
abilities of living biological constructs.[17] However, these
limitations have not constituted a drawback for several
cell-based therapies, such as Apligraf® (Organogenesis Inc.,
Canton, MA), in becoming commercially applicable treatments
for chronic wounds. [18]
II. THE NEED
SUBSTITUTES
CEA are produced in cell sheets grown in culture and applied to
wounds as sheets, which can be very fragile and difficult to
handle. Therefore, researchers and clinicians have analized the
use of cell spraying to provide the same skin cells without
handling a fragile cell sheet.[19, 20] This method can basically
deliver any type of skin cell in a vehicle, mainly a fibrin
hydrogel. Cell sprays have been further used in non-cultured
autologous skin cells isolated in the operating room and directly
applied to a wound.[21] In the clinical trials in the United States,
this technology employed in burns, called ReCell ® (Avita
Medical, Woburn, MA), is part of the Department of Defense
funded Armed Forces Institute of Regenerative Medicine.
Cell therapies have been promising wound treatments for the
delivery of fibroblasts and keratinocytes. Nevertheless,
fibroblasts and keratinocytes do not accomplish all skin
functions, probably requiring a source of stem cells (SC) to
fully restore all integumentary structures in a major wound.
There are too many different cell types in fully functional skin
to allow for a realistic delivery of each different cell type in
specified locations within a wound. Skin SC sources have been
identified in the epidermis and hair follicles.[19] These highly
proliferative cells are crucial contributors to normal wound
healing, showing increased wound healing after culturing,
possibly due to proliferative progenitor selection throughout
the culturing process.[22] Non-scarring fetal wounds might
owe some of their unique properties to a high number of SCs.
[23] Various SC types, including bone-marrow mesenchymal
SC (MSC), adipose-derived SC and embryonic SC (ESC), have
been investigated for the treatment of cutaneous wounds.[24]
These cells can generate dermis and epidermis, but there is need
for more research before turning SC therapy into a viable
approach in the management of acute or chronic skin wounds.
FOR
TISSUE-ENGINEERED
SKIN
Patients with 50% total body surface area (TBSA)
full-thickness wounds have only 50% of undamaged skin left to
be used for split-thickness skin harvesting. The rest would be
obtained from donor sites and result in 100% wound area
covering. The combination between damaged epidermal barrier
and poorer immunity of heavily burned patients can lead to
bacterial sepsis, one of the main complications in deep
extensive burns. [27] The healing of donor sites can occur with
some scarring and can be quite painful. Therefore, there is need
for an additional analgesic pharmacological load. Additionally,
based on dermis thickness, it is only possible to harvest three to
four split-thickness skin samples from the same site and in
order to repeat this procedure, it is necessary to wait until
re-epithelialization. [28]
When the wound is more extensive and there are limited donor
sites, meshing techniques can be used, with a uniform
perforation and stretching of grafted skin to cover a larger
wound area. Even if this technique helps covering a larger area
and results in lower mortality rates, the cosmetic and functional
results are poorer than those of conventional split skin grafts
(SSG). This is due to dermis absence in the interstices of the
stretched meshed skin graft and slow epithelialization from
graft margins across interstices, determining greater graft
contraction, delayed healing, scar tissue formation and obvious
‘crocodile skin’ scar appearance. Even meshing techniques can
be incapable in near-total full-thickness skin wounds due to
unavailable donor sites. In this situation, wounds are covered
with temporary dressings or cadaver skin to form a mechanical
barrier to restrict fluid loss and microbial contamination. The
only solution to heal injured skin in these cases is delayed serial
autologous split skin grafting.[29, 29] These wounds are left
unhealed for a long period of time throughout the treatment,
waiting for epithelial regeneration, and severe complications
may occur, possibly resulting in death.
For the treatment of extensive full-thickness wounds lacking
donor sites for SSG harvesting, alternative life-saving
approaches include the use of cultured autologous
keratinocytes and/or bioengineered skin substitutes. Recently,
the development and clinical use of these products has known a
significant progress.[30-32]. These approaches might be used
for the treatment of extensive deep injuries due to their
‘off-the-shelf’ availability and quick production of adequate
quantities of epithelium able to permanently close the wound.
As indicated before, the future of skin regeneration lies in the
successful development of an approach combining the
biomaterial with cell-based therapy. Stratagraft® (Stratatech
Corporation, Madison, WI), one of the most promising options
[25, 26], is a full-thickness skin substitute with a fully-stratified
epidermal layer consisting of NIKS® cells grown atop a dermal
layer consisting of human fibroblasts embedded in a collagen
matrix. In 2011, StrataGraft® is going to enter a Phase I/II burn
clinical trial.
The great significance and request for skin-substitution
products has been the focus of many research groups around the
world, all trying to create biomaterials for skin replacement.
These biomaterials are sometimes confusingly termed. They
are referred to as bioengineered skin equivalents,
tissue-engineered skin, tissue-engineered skin constructs,
biological skin substitutes, bioengineered skin substitutes, skin
substitute bioconstructs, living skin replacements and
bioengineered alternative tissue.[33] Despite the slight
42
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difference from each other, most researchers consider these
biomaterials to be equal and interchangeable. Therefore, our
review will use these definitions to describe any skin substitute
products, produced or modified artificially in any way,
including modifications of naturally occurring substances, such
as dermis, to fully or partially, temporary or permanently
replace damaged skin, having some anatomical and functional
similarities with human skin.
The majority of these products is based on allogeneic skin cells
incorporated into a dermal scaffold. This method favors the
manufacture of large quantities of relatively ‘off-the-shelf’
available uniform product batches. On the other hand, these
biomaterials are mainly temporary biologically active wound
sheets[37], providing growth factors, cytokines and ECM for
host cells and stimulating and mediating wound healing. Host
immunogenic tolerance to allogeneic fibroblasts has been
reported in some reviews[38], as well as their survival in the
host for up to three weeks [39]. There have also been reports
regarding the preservation of allogeneic fibroblasts and their
proliferation for up to two months in the host, without
indications of immune rejection. [40-43] Nevertheless,
allogeneic fibroblast survival for more than 7 days could not be
confirmed in porcine studies[44], or in clinical studies
transplanting allogeneic fibroblasts onto burn wounds.[45]
There are three major requirements that all tissue-engineered
skin substitute bioconstructs have to comply with. They must
be safe for the patient, clinically effective and easy to handle
and apply. Several authors[34] have recently reviewed the
properties of the ‘ideal’ skin substitute for in vivo use.
Generally, these biomaterials must be non-toxic,
non-immunogenic and should not cause excessive
inflammation, having no or low risk of transmissible disease.
Skin reconstruction biomaterials should be biodegradable,
repairable and capable to support the reconstruction of normal
tissue, possessing similar physical and mechanical properties as
the original skin. It should offer remedy against pain, prevent
fluid and heat loss and protect the wound from infection. It
should be cost-effective, readily available, user-friendly and
long-lived.
On the one hand, allogeneic keratinocytes are effective pain
relievers, accelerating wound healing, but on the other hand,
they do not survive for more than a few weeks when applied to
the wound, as they face host rejection.[45, 46] The expression
of the human leukocyte antigen (HLA) complex might be
different in fibroblasts and keratinocytes, thus subjecting
allogeneic fibroblasts to tissue rejection determined by HLA.
Fibroblasts are not able to induce T-cell proliferation through
cytokine production when involving HLA class II
molecules.[47] Some of the suggestions to study the
immunologic tolerance to allogeneic fibroblasts in the host
have been in vivo models examining acute graft-versus-host
disease.[48] In conclusion, both allogeneic or autologous
fibroblasts can be used to obtain permanent dermo-epidermal
skin substitutes, but only the use of autologous keratinocytes
can help provide permanent wound closure.
Currently,
there
are
no
commercially
available
tissue-engineered skin replacement biomaterials possessing all
the above-mentioned properties, able to completely replace the
functional and anatomical properties of the skin they replace.
However, there are several bioengineered skin-replacement
products available to clinicians and used for wound-healing
purposes. Generally, these tissue substitutions only partly
address skin functional requirements and surgeons are likely to
use different products to attain certain purposes.[35, 35]
described four groups of functions that can be provided by
bioengineered skin-replacement products: protection, through
a mechanical barrier against micro-organisms and vapour
loss; procrastination, early wound cover until achieving
permanent wound closure by means of serial skin grafts or
cultured autologous cell applications, particularly in extensive
burns; promotion, delivery of dermal matrix components,
cytokines and growth factors, to boost natural host
wound-healing responses; provision, new structures (dermal
collagen or cultured cells) are administered inside the wound,
persisting during and/or after wound healing.
III. DERMO-EPIDERMAL
SUBSTITUTES
(COMPOSITE)
IV. EPIDERMAL SUBSTITUTES
The possible serial culture of human keratinocytes in vitro[49]
increased the number of patient keratinocytes ex vivo, quickly
transfering this technology into clinical applications[50],
determining an improvment in patient survival rates.[51] But
there are still disputes regarding the benefits of cultured
keratinocytes.
An essential step in the design and production of epidermal
substitutes is represented by the isolation of keratinocytes from
a donor, followed by in vitro cell culture to provide the required
number of keratinocytes for therapeutic purposes. Different
approaches in the production of epidermal substitutes are
related to: cell culture techniques (submerged or air–liquid
interface models), cell differentiation stage and epithelial
organization (confluent sheets, subconfluent cell layers and
suspensions), cell delivery methods (confluent sheets mounted
onto support layer, subconfluent dispersed keratinocytes
delivered by means of aerosol techniques or microcarrier
beads), as well as the use of additional substrates to improve
cell culture and delivery (both synthetic and biological).[52,
53]
SKIN
The purpose of dermo-epidermal or composite skin substitutes
is to imitate the histological structure of normal skin, with both
epidermal and dermal layers. This similarity has some
functional resemblance to normal skin. In terms of epidermal
and dermal substitutes, these are the most advanced and
sophisticated products, as well as the most expensive
tissue-engineered biological constructs for tissue repair. [36]
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Autologous cell culture is initiated by a skin biopsy of 2–5 cm2,
generally removed along with initial wound excision during
patient presention to the clinic. Epidermis and dermis are
separated from each other and single keratinocytes are
delivered from the sheet via exposure to enzymes. These
keratinocytes are plated into tissue culture vessels where single
cells divide to produce colonies in the presence of mitotically
inactivated mouse fibroblasts and culture medium containing
foetal calf serum and necessary supplements. Keratinocytes can
be expanded in xenogeneic-free conditions avoiding murine
fibroblasts and bovine serum[54], but with considerably
reduced proliferative lifespan of these cells.[55] Single colonies
of keratinocytes mingle and form stratified epithelial layers
which can be enzymatically detached from the culture flasks,
fitted onto backing supports (e.g. paraffin gauze) to maintain
basal–apical orientation, further applying the sheet to the
wound. [56]
keratinocyte attachment, determining blistering when exposed
to minor shearing forces, was obvious for several months after
grafting. [63]
V. CULTURING
LABORATORY
SKIN
COMPONENTS
IN
THE
Highly injured full-thickness skin cannot spontaneously
regenerate in mammals. A fully functional skin substitute can
only be created after completing recapitulation of ontogenesis,
which cannot be achieved in vitro.[64] Nevertheless, the
phenotype expressed by human skin cells in culture is very
similar to wound healing physiology [64, 65], including
cytogenesis, morphogenesis and histogenesis, but excluding
organogenesis. Cells should promote the wound healing
process and boost the developmental regeneration process.
Therefore, they might provide full function to an artificial skin
substitute produced like this. Individual skin components have
been successfully cultured and transplanted in many
experimental studies, but not as part of a fully functional skin
replacement process. Connective tissue cells are believed to
recolonize grafts from the wound bed, but many skin
substitutes consist of dermal fibroblasts facilitating such repair
processes.[66] Melanocytes have also been employed to
recolonize burn scars and to treat vitiligo, as well as to
supplement various polymer scaffolds. [67] Nevertheless, full
restoration of skin sensation could not be proved by either split
thickness skin grafts or engineered skin, whereas sweat and
sebaceous
glands
have
only
been
transplanted
experimentally.[68] These latter structures, together with
nerves and blood vessels, might be the most difficult to achieve,
as they are neither restored nor regenerated.
Cultured epithelial autografts (CEAs) have qualities which are
related to clonal cellular composition[57], which supposedly
influences graft survival and long-term performance in in vivo
applications. Basal keratinocytes producing holoclones (in
vitro colonies with the highest proliferative potential) are basic
tools in providing long-term graft survival. Meroclones,
containing transient amplifying cells, possess a fluctuating
proliferation potential and are only able to provide temporary
wound closure in in vivo applications. Paraclones, which are
committed keratinocytes, represent most of the normal
epithelial cell population but can only replicate a few times
before differentiation and senescence. Thus, CEAs containing
only paraclones are not able to serve as substrate for permanent
wound closure. Holoclone preservation is possible in current
culturing techniques if keratinocytes are grown in vitro for a
extensive period of time.[55]
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CEA sheets are produced by means of in vitro keratinocyte
expansion techniques, being large enough to cover all the body
surface area in three to four weeks using only a 3 cm2 skin
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al.[59] achieved 4.1 m2 of graftable epithelium from a 4.5
cm2 skin biopsy cultured for 15 days on a fibrin matrix,
compared to 1.4 m2 when cultured on plastic matrices. The use
of this technique has also improved material handling and
basement membrane formation.
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