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Key developments
Keratinocytes were first reliably cultured in the laboratory about 30
years ago9,10. This developed into the production of small sheets of cells
two or three layers thick11 (known as cultured epithelial autografts,
CEAs) that were used to treat burns victims12,13. The Timeline shows
key events in the development of tissue-engineered skin. Evidence suggests that cultured skin cells returned to a patient retain the capacity to
self-renew for a lifetime. Patients who received skin in the mid-1980s13
are alive today and have not required further treatment. Research
1
The Tissue Engineering Group, Department of Engineering Materials and Division of Biomedical Sciences and Medicine, Kroto Research Institute, North Campus, University of Sheffield, Broad
Lane, Sheffield S3 7HQ, UK.
874
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Sweat gland
pore
Hair shaft
Epidermis
Dermis
Sweat gland
duct
Subcutaneous
layer
Capillary
Touch receptor
Figure 1 | The structure of human skin. This diagram of human skin shows
the two main layers of skin the upper epidermal barrier layer and the
lower, much thicker, dermis. The epidermal barrier layer is relatively thin
(0.10.2 mm in depth) and securely attached to the underlying dermis by
a specialized basement membrane zone. This consists of several different
types of collagen fibre, which attach cells securely to the underlying
dermis, and is visible at the electron-microscope level. The dermis varies
in thickness depending on its site in the body and is composed primarily
of collagen I, with dermal inclusions of hair shafts and sweat glands, which
are lined with epidermal keratinocytes. The dermis is well vascularized and
also contains receptors for touch, temperature and pain. Keratinocytes in
the epidermis rely solely on diffusion from the adjacent dermal capillary
network. These cells progressively differentiate from cells in the basal layer,
which is located on the basement membrane and gives rise to daughter
keratinocytes, which are pushed upwards. These stratify, lose their nuclei
and eventually become an integrated sheet of keratin, which is later shed.
The upper keratinized epidermal layers provide the barrier layer, which
resists bacterial entry and prevents fluid and electrolyte loss. (Image
adapted, with permission, from ref. 79.)
in vitro has indicated that skin is the most accessible of all adult stemcell populations1416 (see page 834).
Without a well-vascularized dermal wound bed, cultured keratinocytes alone are of limited value in treating full-thickness burns17. In
such cases it is necessary to replace both epidermal and dermal layers
of skin. Accepted practice18 in these cases is to debride the burned tissue
(often to the muscle fascia), provide a dermal equivalent17,19 and, once
this has become well vascularized, provide an epidermal layer. This need
to provide a dermis prompted the evaluation of a range of biomaterials, both natural and synthetic. Donor (cadaveric) skin is commonly
used for managing serious burns and treating chronic wounds. It can
be grafted onto full-thickness wounds and will become vascularized.
The upper epidermal layer (which is not immune-system compatible)
is removed after about 3 weeks, leaving behind a vascularized donor
dermis, which provides an ideal wound bed for subsequent skin grafts
or tissue-engineered skin graft substitutes17. Donor skin is currently an
underused resource that could be especially valuable in the developing
world. Skin banks can be established at much lower cost than that of
purchasing other dermal substitutes. With good banking practices and
sterilization20 the risks of using donor skin can be reduced to that of
using blood.
Another useful dermal substitute is Integra21. This was designed for the
management of major burns and comprises bovine collagen and shark
chondroitin sulphate with a silicone membrane that functions as a temporary barrier. The material is grafted onto the wound bed and under
this membrane vasculogenesis occurs. After vascularization, the silicone
barrier membrane is removed and replaced with a barrier of the patients
own cells either a split-thickness skin graft or tissue-engineered material. Direct application of cultured cells to an Integra wound bed was
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Timeline
Understanding of the
co-dependence of keratinocytes
and fibroblasts8183
Keratinocyte culture
established9,10
1970
1975
1980
1985
1990
Determination of the
replicative capacity
of keratinocytes14
1995
2000
Development of a synthetic
dermal alternative (Integra)19
and rapidly expanding field (see ref. 42 for a review). In skin tissue engineering the ideal bioscaffold for in vivo use must not induce a toxic or
immune response or result in excessive inflammation. It should have
an acceptably low (preferably no) level of disease risk, be slowly biodegradable, support the reconstruction of normal tissue and have similar
mechanical and physical properties to the skin it replaces. It should
also be repairable, readily available and capable of being prepared and
stored with a long shelf life. Both natural materials (such as extracted
FDA-approved bovine collagen or hyaluronic acid, or acellular natural
human or porcine matrices) and synthetic FDA-approved biodegradable
scaffolds (such as poly-l-lactide, used in dissolvable sutures and related
polymers) are in use.
Cell delivery approaches
Improving ease of use for both clinicians and patients is essential if
skin repair products are to achieve their full potential. A number of
different strategies have been explored. Keratinocytes can be grown
into integrated cell sheets (CEAs, now available as Epicel43), made into
suspensions and sprayed onto wound sites with44 or without fibrin (for
example, CellSpray45) or delivered on various carrier dressings ranging
from bovine collagen46 to a chemically defined polymer carrier dressing
(such as Myskin47). Cells have also been cultured from hair follicles and
delivered as small sheets (for example EpiDex48). So far, there have been
few studies comparing one method with another49. Irrespective of how
keratinocytes are delivered to the wound bed, healing success depends
mostly on the condition of the wound bed the condition of keratinocytes is less important. Consequently, the use of any one particular
form of cell delivery will ultimately be determined by factors such as
ease of use, cost, transportation and variables other than the biological
health of the cells.
An example of this has been provided by work at Sheffield University
in the UK, where cultured cells have been delivered to burns patients
since 1992. A 10-year audit revealed that there was considerable waste
during the use of CEA sheets, as this method does not allow much flexibility in the timing of cell delivery to the patient50. Accordingly, the
preference now is for subconfluent cells to be delivered on a chemically
defined carrier dressing (for both chronic wounds51 and burns injuries52), from which the cells transfer to the wound bed. The inert carrier
dressing is coated with an extremely thin polymer containing acid functional groups using the technique of plasma polymerization53. Repeated
applications can promote wound healing in chronic wounds51. As with
other such studies, this technique requires regular repeated applications
of cells (either autologous or allogeneic) to achieve healing in wounds
that may have been stuck for a long period of time54.
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Pancytokeratin
Skin contraction
Unexpected results have been obtained in relation to the contraction of
human skin grafts. The data from work using reconstructed skin from
sterilized mature crosslinked dermis (Fig. 4d) show that keratinocytes
rather than fibroblasts contract this dermis6971. Both keratinocytes72
and fibroblasts73 can contract dilute collagen gels, but the finding that
keratinocytes have a marked ability to contract human dermis sheds
new light on the problem of skin-graft contracture. This model is
being used to identify new pharmacological targets to block contraction. Recent evidence shows that inhibitors of the collagen crosslinking
enzyme lysyl oxidase can block contraction to a large degree without
having adverse effects on skin morphology71.
Other uses
Reconstructed skin has also been used to develop models of human
skin diseases such as psoriasis74 and the genetic disorder epidermolysis
bullosa a severe, often lethal blistering problem. Here it is being
used to develop therapeutic gene expression to improve keratinocyte
HBL melanoma
cells
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