TISSUE ENGINEERING: Part B

Volume 14, Number 1, 2008

# Mary Ann Liebert, Inc.
DOI: 10.1089/teb.2007.0318

Skin Tissue Engineering for Tissue Repair and Regeneration

S. GEETHA PRIYA, M.S.,1 HANS JUNGVID, Ph.D.,2 and ASHOK KUMAR, Ph.D.1

ABSTRACT

Tissue-engineered skin is a significant advance in the field of wound healing. It has mainly been developed
because of limitations associated with the use of autografts and allografts where the donor site suffers
from pain, infection, and scarring. Recently, tissue-engineered skin replacements have been finding
widespread application, especially in the case of burns, where the major limiting factor is the availability
of autologous skin. The development of a bioartificial skin facilitates the treatment of patients with deep
burns and various skin-related disorders. The present review gives a comprehensive overview of the
developments and future prospects of scaffolds as skin substitutes for tissue repair and regeneration.

INTRODUCTION engineered skin replacements such as cultured autologous

T ISSUE ENGINEERING is the application of principles and
methods of engineering and life sciences to the de-
velopment of biological substitutes to restore, maintain, or
improve tissue function.1 It is a rapidly expanding, multi-
disciplinary field in biomedicine. Scientists have en-
gineered various tissues in the body, from skin substitutes
to artificial nerves to heart tissues, with varying degrees of
success.2 The application of tissue engineering progress in
medicine is discussed according to the origin of germinal
layers during embryonic development. Thus engineered
tissues could reduce the need for organ replacements.3
Skin tissue engineering
Skin, the largest organ in the body, primarily serves as a
protective barrier against the environment. Loss of integrity
of large portions of the skin due to injury or illness may
result in significant disability or even death. Adult skin con-
sists of 2 tissue layers: a keratinized, stratified epidermis and
an underlying thick layer of collagen-rich dermal connec-
tive tissue providing support and nourishment. Appendages
such as hair and glands are derived from the epidermis, but
they project deep into the dermal layer. Thus skin replace-
ment has been a challenging task for surgeons ever since
the introduction of skin grafts by Reverdin in 1871. Tissue-
and allogenic keratinocytes grafts, autologous or allogenic
composites, acellular biological matrices, and cellular ma-
trices including biological substances such as fibrin sealant
and various types of collagen and hyaluronic acid (HA) have
opened new options to treat such massive skin loss.
An experimental set-up of testing the performance of
tissue-engineered skin substitutes in full-thickness nude
mouse skin wounds is shown in Fig 1. A study was also
conducted to compare cultured, stratified epithelial sheet
grafts and cultured, non-stratified keratinocyte single-cell
suspensions in fibrin sealant with xenogenic meshed allo-
graft skin with control wounds. The keratinocyte fibrin
suspension can be available in 10 days because no epidermal
differentiation is required, whereas cultured epidermal
grafts need at least 21 days for grafting. Human autografts
are expensive and limited and therefore cannot be used in
case of extensive third-degree burns. Human allografts pro-
vide temporary skin coverage and prevent infection until
autografts are available and overlaid. Again, one possi-
ble disadvantage in the case of allografts are their limited
availability. Xenografts (tissues from other species) are
more accessible to allografts but they do not lead to per-
manent revascularization. In preliminary trials of assessing
burn injuries, keratinocyte single-cell suspensions in fibrin
sealant with xenogenic-meshed allograft skin provided

1
Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, India.
2
Protista Biotechnology AB, Ideon, Lund, Sweden.

105
106
enzymatic cell separation
Epidermins (human)
Dermis (rejected)
cell sheets
C A
control sheet grafts
FIG. 1. Experimental set-up to test the performance of tissue-engineered skin substitutes in full-thickness nude mouse skin wounds:
comparison of cultured and stratified epithelial sheet grafts after calcium addition (A) and subconfluently cultured non-stratified
keratinocyte single-cell suspensions in fibrin sealant (B) with xenogenic meshed allograft skin as an overlay versus control wounds (C).
Reproduced from Horch et al.4 with permission.
results similar to those of cultured epithelial sheet grafts.
Thus, even with limited donor sites, extensive burn areas can
be covered efficiently (Fig. 1).4 These models indicate the
clinical reality of burn wounds. In addition to traditional
approaches such as split- or full-thickness skin grafts, skin
bioengineering in vitro or in vivo has been developing over
the past decades.5 Cultured epidermal autografts can provide
permanent coverage of large areas from a skin biopsy.
However, 3 weeks is needed for graft cultivation. Cultured
epidermal allografts are available immediately, and no bi-
opsy is necessary. They can be cryopreserved and banked
but are not currently commercially available.6 Current re-
search in the field of tissue engineering continues to evolve,
with the ultimate goal being tissue-engineered skin that
matches the quality of the autologous skin graft.7
Wound healing
Skin repair is an important area of research in the field of
tissue engineering, especially in the case of extended third-
degree burns, where current treatments for promoting sat-
isfying skin regeneration are still in their infancy.8 During
the initial stages of wound healing, a temporary repair is
usually achieved in the form of a blood clot. Inflammatory
cells, fibroblasts, and capillaries later invade the clot to
form a contractile granulation tissue that draws the wound
margins together; meanwhile, the cut epidermal edges mi-
grate to cover the degenerated wound surface.
The tissue oxygen level and oxygen gradient are criti-
cal factors in wound healing. The gradient creates a high-
PRIYA ET AL.
cell culture
A B
Ca
B
Keratinocyte Fibrin suspension
+ glycerolized xenografts
oxygen environment in the wound to allow fibroblasts at the
wound margin to produce collagen and forms a structure that
helps in angiogenesis. The low-oxygen environment created
by the gradient in the wound center is essential for the
macrophages to release cytokines that stimulate chemotaxis,
mitosis, and other steps in the healing process. The diffusion
of oxygen from vessels at the wound margin creates this
gradient. Experimental evidence suggests that hyperbaric
oxygen cannot accelerate wound healing; rather it can only
act as a mild antiseptic agent and provides no advantage in
the treatment of burns.9
In the case of wound healing, tissue-engineered constructs
have already been applied successfully in burns and chronic
wounds, although improvements in these biomaterials
need to be optimized. In general, it is more promising to
substitute deficient components in vivo than to create com-
plex organs ex vivo. The most common approach is to create
3-dimensional (3-D) biodegradable scaffolds in the shape
of the tissue.10 Long-term function of 3-D tissue constructs
depends on adequate vascularization after implantation. To
achieve vascularization of tissue constructs, modification
of biomaterial properties of scaffolds is essential by creating
microvascular networks within 3-D tissue constructs in vitro
before implantation.11 Thus, an ideal therapy for wound
healing must promote rapid healing with minimum scar
formation.12
Wound healing has become the first field to see the benefit
of a new technology called tissue engineering. In this field, a
number of biomaterials are used as scaffolds that may use
natural or artificial materials. Generally, synthetic polymers
SKIN TISSUE ENGINEERING
such as Tegaderm and OpSite that are semi-permeable
polyurethane dressings are used as wound dressings for sim-
ple and shallow wounds. Their main function is to protect
from water loss, drying, and mechanical injury. OpSite is a
fine, transparent, elastic, self-adhesive polyurethane film. It
is impermeable to bacteria and water but permeable to gas.
Recent studies have indicated that a moist environment
provides optimum conditions for wound repair. Healing
under OpSite is said to be quicker because the serous exu-
date permits unhindered migration of new cells across the
wound bed and prevents cellular dehydration. In contrast,
under dry conditions, healing is delayed because the new
skin cells must cleave a path through dehydrated dermis
before migrating across the wound. With Op-site, the skin
remains dry and the wound moist, providing the ideal en-
vironment for rapid healing. The film does not adhere to the
moist wound and can therefore be removed without damage
to the newly formed epidermis. The adhesive is low allergic.
Finally, the wound can be assessed without removing the
transparent OpSite.13 Tegaderm is also a vapor-permeable,
transparent dressing. It supports the attachment and growth
of cell types. Because it is a transparent polymer, it allows
the observation of cell growth before re-epithelialization
after grafting to the wound. It is an inexpensive and easily
available polyurethane-based wound dressing for the treat-
ment of burn and chronic wounds.14 In a study, these
dressings were tested for their in vitro ability to kill potential
bacterial pathogens. In bacterial killing tests, OpSite was
found to be superior to Tegaderm.15 More-complex dress-
ings vary from dermal replacements made from collagen and
chondroitin sulfate backed by a polymer layer such as In-
tegra to the complex Apligraft, which contains collagen and
seeded cells. Ultimately, an engineered skin should contain
all the components necessary to modulate wound healing
and retain the characteristics of a natural skin with limited
scar tissue.16 Wound care is an essential step in promoting
wound healing. One of the significant factors causing wound
care pain is that the dressing adheres to the wound bed. Thus
it is possible that particular dressings with low adhesion can
result in painless removal.17
Tissue-engineered bioartificial skin
Artificial skin research is an active field in tissue engi-
neering, and it has encouraging prospects for clinicians to
restore various skin defects.18 It is preferable that the arti-
ficial skin possess a bilayer structure that imitates the nat-
ural skin, wherein the upper layer functions as a temporary
epidermis that is permeable to moisture and not to water
and a lower layer acts as a skin regeneration template.
Tissue-engineered skin is one of the most advanced tissue
constructs, yet it lacks several important functions; hair
follicles, sebaceous glands, sweat glands, and dendritic
cells. Although it is difficult to recreate human skin en-
tirely, better functions can be provided by genetic modifi-
cation of the cells that make up the tissues. Gene therapy
107
can also be used in wound healing to promote tissue re-
generation and to prevent scar formation.19
Multipotent skin stem cells have been identified in the hair
follicle bulge, which is at the base of the epithelial com-
partment. Bulge cells contribute to the epidermis during only
wound healing, but after isolation, when combined with
neonatal dermal cells, they regenerate new hair follicles,
epidermis, and sebaceous glands. In addition bulge cells
retain their stem cell characteristics even after propagation
in vitro and thus can find applications in tissue engineer-
ing.20 One such application of hair follicle bulge cells
(HFBCs) and dermal papilla cells (DPCs) was illustrated in
an experiment. The cells were isolated from human fetal hair
follicles using collagenase digestion and then cultured.
Polyglycolic acid (PGA)–collagen scaffolds as bioengi-
neered dermis were randomly divided into A and B groups.
The HFBCs and DPCs were seeded at a ratio of 1:2 in
scaffolds of group A, and equal amounts of DPCs were
seeded in scaffolds of group B as a control. The keratinocyte
sheets were then seeded onto the surfaces of the scaffolds as
bioengineered epidermis. The tissue-engineered skin grafts
were then transplanted to repair the full-thickness wound on
the back of nude mice. The full-thickness defect of nude
mice in A and B groups could be repaired using the bioen-
gineered skins. From the composite skin engineered with
PGA–collagen hybrid scaffolds and keratinocytes, HFBCs
and DPCs could effectively repair the full-thickness skin
defect in nude mice. Thus hair follicle stem cells participate
in the process of anatomic repair of wound and might be
able to induce the repair of skin structure and function.21
The application of autologous or allogenic stem cells in
combination with specific growth factors in bioartificial skin
may also help in tissue reconstruction. When the skin suffers
from trauma such as burn injury, it is thought that the bulge
cells migrate to the surface to aid re-epithelialization. Thus,
the regenerative role of bulge cells (or dissociated cells)
from the skin is multiple, and it leads to the development of
sebaceous glands, epidermis, and hair follicles.22
IMPORTANCE OF SCAFFOLD
BIOMATERIALS THAT MAINTAIN
TISSUE ARCHITECTURE
Isolated cells have a limited capacity to reform their
respective tissue architecture, because they lack a template
that guides in restructuring. Moreover, transplantation of
large volumes of tissues cannot be done because of diffu-
sion limitations that restrict interaction with the host en-
vironment for nutrients, gas exchange, and elimination of
waste products. Therefore, implanted cells survive poorly
under such conditions.23 Hence, an ideal scaffold that has
the capacity to act as a template for the construction of
neonatal skin is essential, provided that the template is bio-
degradable. A successful 3-D tissue-engineering scaffold
108
must have a highly porous structure and good mechanical
stability. High porosity and optimally designed pore size
provides structural space for cell accommodation and mi-
gration and enables exchange of nutrients between the
scaffold and environment.24
A porous scaffold composed of chitosan (Cs), gelatin
(Gel), and HA prepared using the freeze-drying method by
Liu et al.25 had an interconnected pore structure. The in-
corporation of HA greatly increased the water uptake
ability, flexibility, and biocompatibility of the scaffold. To
construct artificial skin in vitro, fibroblasts and keratino-
cytes were co-cultured in Cs-Gel-HA scaffolds at an air–
liquid interface. Thus, a Cs-Gel-HA scaffold can be a
promising matrix for skin tissue engineering.25 To alleviate
the extensive contraction of fibroblasts, cultured human
fibroblast sheets in combination with 3-D matrices, knitted
poly(lactic-co-glycolic acid) (PLGA) mesh, and collagen-
HA (CHA) sponge were used to form a bilayered skin
equivalent. When implanted into full-thickness wounds in
nude rats, the cultured skin equivalents based on PLGA
meshes had an uptake rate of 100% and showed wound
contraction similar to that of autografts, whereas wounds
grafted with PLGA meshes without cell sheets contracted
more than did autografts (Fig. 2).26
A biodegradable scaffold designed using collagen and Cs,
with glutaraldehyde (GA) as a cross-linker, was prepared.
The mean pore size of the scaffold was 100 to 200 mm, and
the porosity was greater than 90%. Cs can function as a
bridge to increase the cross-linking efficiency of GA in the
presence of collagen owing to the large number of amino
cell sheet
Matrix
petri- 1st fold
A dish
B C
FIG. 2. (A) Schematic representation of the process of folding a cell sheet over a matrix. Digital images showing (B) peeling of cell
sheet from a petri dish, (C) a poly(lactic-co-glycolic acid) (PLGA) mesh integrated within a cell sheet, (D) PLGA, and (E) collagen
hyaluronic acid-specimens after 7 days co-culture of fibroblasts and keratinocytes (stage 3). Reproduced from Ng et al.26 with
permission. Color images available online at www.liebertpub.com /ten.
PRIYA ET AL.
groups in its molecular chain. Hence, less GA can be used in
the presence of Cs, and its potential cytotoxicity can be re-
duced. It was found that GA-treated scaffold had greater
biostability and excellent biocompatibility. Further combi-
nation of Cs with GA preserves the original cytocompat-
ibility of collagen. Potential cytotoxicity of GA was not
evidenced at lower percentages of 0.25% in the scaffold.
Thus a collagen/Cs scaffold cross-linked with GA can be
a potential candidate for dermal equivalents in skin tissue
engineering.27
Collagen–polycaprolactone (PCL) composites were used
for the manufacture of tissue-engineered skin substitutes.
Films with collagen-PCL ratios of 1:4, 1:8, and 1:20 were
prepared using impregnation of lyophilized collagen mats by
PCL solutions followed by solvent evaporation. The prep-
aration of fibroblast–composite constructs was investigated
using cell culture, thereby mimicking the dermal–epidermal
structure of skin. Fibroblasts were found to attach to and
proliferate on all the composites investigated, reaching a
maximum of 1:20 collagen–PCL materials, with cell num-
bers declining thereafter. Keratinocyte growth rates were
similar on all types of collagen- materials investigated.
Thus, composite films of collagen and PCL are found to be
favorable substrates for growth of fibroblasts and keratino-
cytes and may find utility in skin repair (Fig. 3).28 A thin
biodegradable hybrid mesh composed of synthetic PLGA
and collagen was prepared such that web-like collagen mi-
crosponges were formed in the openings of a PLGA knitted
mesh for 3-D culture of human skin fibroblasts. Cells grew
quickly and secreted extracellular matrices (ECMs) that
2nd fold 3rd fold
4th fold
D E
SKIN TISSUE ENGINEERING
FIG. 3. The morphology of 3T3 fibroblasts after cell culture on
collagen–PCL composites: (A) 1 day on 1:8 composite; (B) 3 days
on 1:8 composite, and (C) 1 day on 1:20 composite. Reproduced
from Dai et al.28 with permission.
were more uniformly distributed in the hybrid mesh than in
the PLGA mesh. Fibroblasts cultured in the hybrid mesh
were then implanted in the back of a nude mouse. Dermal
tissues were formed after 2 weeks and became epithelialized
after 4 weeks. Thus PLGA–collagen hybrid mesh may be
a promising tool for skin tissue engineering (Fig. 4).29
Cs, the deacetylated derivative of chitin, is a promising
tissue-engineering scaffold because it is biocompatible and
biodegradable and the degradation products are resorbable.
However, the rapid degradation rate of Cs and its low me-
chanical strength limits its use. Therefore Cs with 80%,
90%, and 100% degrees of deacetylation (DDAs) cross-
linked with dimethyl 3-30 dithiobispropionimidate (DTBP)
was used and compared with uncross-linked scaffolds. The
tensile strength of scaffolds made from 100% DDA Cs was
significantly higher than for scaffolds made from 80% and
90% DDA Cs. Cross-linking of scaffolds with DTBP in-
creased the tensile strength. Hence, 100% DDA Cs can be an
effective, biocompatible, biodegradable scaffold for tissue-
engineering applications.30
Whey proteins, an inexpensive renewable resource, can
be used as a substrate for a variety of biomedical applica-
109
tions, including tissue engineering. Whey protein–based
biofilms were used with different plasticizers to prepare
suitable scaffolds for the growth of keratinocytes and fi-
broblasts. Keratinocytes and fibroblasts were grown in
plastic tissue culture dishes or on whey protein–based
biofilm surfaces. Whey protein isolate (WPI) was plasti-
cized with diethylene glycol (DEG), ethylene glycol (EG),
and glycerol (GLY) and another major whey protein,
b-lactoglobulin (BLG), was plasticized with DEG. Thus,
there were five different sets: control, WPI–EG, WPI–
DEG, WPI–GLY, and BLG–DEG. Keratinocytes growing
on the WPI–EG biofilm appeared to be bigger than control
cells. The cells were less elongated on the WPI–GLY and
BLG–DEG biofilms and were clustered on the WPI–GLY
biofilm. Thus the films toxicity was assessed by evaluating
cell adherence, growth and proliferation. Microscopic ob-
servation demonstrated that keratinocytes and fibroblasts
adhered well to biofilms. Structural analysis revealed that
keratinocytes stratified when cultured on these whey
protein–based biofilms and gave rise to multi-layered epi-
dermal structures (Fig. 5A, B).31
In an experimental group, the bi-layered Gel–C6S–HA
skin substitute seeded with or without cells was grafted onto
the dorsal muscular bed with a wound area of 1.5 cm in di-
ameter. Silicone gauze with antibiotics was applied on the
outermost skin equivalent to prevent from infections. Com-
mercialized framycin gauze dressings were used as controls.
The bi-layered Gel-CS-HA skin substitute had a positive
effect on promoting wound healing and also had a high rate
of graft intake. This reconstructed skin equivalent would
have the potential to be applied to extensive burns in patients
(Fig. 6).32 Tissue-engineering scaffolds are used extensively
as 3-D analogs of the ECM. Collagen-glycosaminoglycan
FIG. 4. Scanning electron microscopy photomicrographs of fi-
broblasts cultured in poly(lactic-co-glycolic acid) (PLGA) knitted
mesh (a, b) and PLGA–collagen hybrid mesh (c, d) after culture
for 30 min (a, c) and 5 days (b, d) at original magnification
200.
Reproduced from Chen et al.29 with permission.
110
FIG. 5. (A) Keratinocyte morphology after growth on whey
protein–based biofilms: (a) control, (b) whey protein isolate–
ethylene glycol (WPI–EG), (c) WPI–diethylene glycol (DEG),
(d) WPI–glycerol (GLY), and (e) b-lactoglobulin (BLG)–DEG.
Magnification 250
. Reproduced from Gilbert et al.31 with per-
mission. (B) Fibroblast morphology after growth on whey protein–
based biofilms: (a) control, (b) WPI–EG, (c) WPI–DEG, (d) WPI–
GLY, and (e) BLG–DEG. Magnification 250
. Reproduced from
Gilbert et al.31 with permission.
PRIYA ET AL.
(CG) scaffolds have long been used as ECM analogs for the
regeneration of skin.33
Tissues such as skin, blood vessels, and cartilage have
multi-layered structures with different cells in each layer. It
is important to control cell growth within these artificial
scaffolds. Using micro-fabrication technology, Ryu et al.34
designed a novel 3-D multi-layer micro-fluidic tissue scaf-
folds for biodegradable polymers such as polylactic acid,
PGA, and the copolymers of PLGA. This method empha-
sizes micro-fluidic interconnections (i.e., micro-holes of
10–100 mm in size) between layers within the scaffolds.
Micro-channels and micro-cavities were all created using
micro-molding processes. The interconnection of layers
within multi-layer scaffolds is critical, because micro-fluidic
connection across the layers is necessary for cell-to-cell
communication and the supply of oxygen and nutrients.
Culturing of human coronary artery endothelial cells on the
multi-layer scaffolds demonstrated that the cells had a pref-
erential growth over micro-holes of certain sizes (Fig. 7).
The biocompatibility and efficacy in wound healing of
a Gel-based interpenetrating polymer network (IPN) con-
taining poly(ethylene glycol) (PEG)-ylated arginine-glycine-
aspartic acid (RGD) and soluble keratinocyte growth factor
(KGF) (RGD–IPN þ KGF) was examined. IPNs were ap-
plied to full-thickness wounds on a rat model, and wound
healing was then assessed. A control IPN containing un-
modified Gel (unmod-IPN) and a conventional clinical ban-
dage were also applied and evaluated. The unmod-IPN and
conventional dressing wound showed a greater amount of
contraction than that of RGD–IPN þ KGF. The RGD–IPN þ
KGF–treated wound had lower macrophage and fibroblast
densities than unmod-IPN–treated wounds. The extent of
cellularity and ECM organization was also higher for
wounds healed with RGD–IPN þ KGF than those healed
with unmod-IPN. These results indicate that soluble and
immobilized bioactive factors can be incorporated into IPN
platforms to enhance the rate and quality of dermal wound
healing.35
Natural and synthetic biodegradable fibers are used ex-
tensively in biomedical applications and tissue engineer-
ing. The widely used polymers for skin preparations are
cellulose derivatives, Cs, carrageenan, polyacrylates, poly-
vinyl alcohol, polyvinylpyrrolidone, and silicones.36 In-
corporation of collagen into Cs resulted in the creation of
an ideal tissue-engineering scaffold that enhanced cell at-
tachment ability.37 The 3-D architecture of the collagen–Cs
tissue-engineered skin sponge has been shown to encourage
nerve growth.38 Similarly, in another study, it was observed
that biopolymer blends of collagen with low-molecular-
weight Cs had a high potential to be applied as a new ma-
terial for skin tissue engineering.39 A novel biodegradable
PCL and collagen nanofiber matrix supported the attachment
and proliferation of human dermal fibroblasts and have po-
tential in tissue engineering as a dermal substitute for skin
regeneration.40 Fibrin-coated PCL films also support the
attachment and proliferation of human keratinocytes and
SKIN TISSUE ENGINEERING
FIG. 6. The grafting process of bi-layer gelatin–C6S–hyaluronic acid skin substitute onto the dorsum of severe combined immu-
nodeficient (SCID) mice. (A) A wound size of 1.5 cm in diameter was created on the dorsum of a SCID mouse. (B) Commercialized
framycin gauze dressing was used as a control. Reproduced from Wang et al.32 with permission. Color images available online at
www.liebertpub.com /ten.
have the potential to be applied as a matrix material for
tissue engineering.41
Recombinant human-like collagen (RHLC) was added to
fibroin solution to prepare a novel hybrid scaffold material
for skin tissue engineering. Fourier transform infrared spec-
troscopy analysis indicated intermolecular cross-linkages
between fibroin and RHLC, and the viscosity of the blend
solution was also greater because of their interactions. After
methanol treatment, the fibroin–RHLC scaffold became
water-stable. The porosity of scaffolds was greater than
90%, with greater compressive strength and modulus. Fibro-
blasts cultured within fibroin–RHLC scaffolds were inves-
tigated using scanning electron microscopy, laser scanning
confocal microscopy, and 3-(4,5-dimethylthiazol-2-Yl)-2,5-
diphenyltetrazolium bromide assay, which showed that ad-
dition of RHLC resulted in significantly greater cell adhesion,
proliferation, and viability than with fibroin scaffolds.42
Alginates were also used for wound healing purposes.
Sterilized, pure, highly viscous alginates from seaweeds
have been found to inhibit scar formation by presenting a
physical barrier to the invading fibroblasts and thereby en-
hancing wound healing of the surrounding tissues.43 Scar-
free embryonic wounds show lower levels of transforming
growth factor (TGF)-b1, TGF-b2, and platelet-derived
growth factor (PDGF) and higher levels of TGF-b3. Ex-
perimental evidence also confirms that exogenous addition
of TGF-b3 or neutralizing the composition of TGF-b1, TGF-
b2, and PDGF results in markedly less or absent scarring.
Thus incorporation of appropriate growth factors in artificial
111
skin substitutes will enhance host intake and to minimize
scarring.22
Electrospun collagen promotes cell growth and penetra-
tion of cells into the engineered matrix.44 Using an elec-
trospinning method, chitin nanofibers were fabricated. The
morphology of spun chitin nanofibers, viewed using scan-
ning electron microscopy, indicated that chitin nanofibers
alone or with ECM proteins (particularly type I collagen) are
a potential candidate for cell attachment and spreading of
human keratinocytes and fibroblasts.45 The concept of using
an electrospinning array to form multi-component nanofi-
brous membranes will lead to the creation of novel scaffolds
for tissue-engineering applications. The application of na-
nofibrous composite substitutes such as PLGA and Cs–
polyvinyl alcohol prepared using an electrospinning method
has been found to positively mimic the structure of natural
ECMs and have the potential to be used as 3-D tissue-
engineering scaffolds.46
NATURALLY OCCURRING POLYMERS
FOR SKIN TISSUE ENGINEERING
Presently autografts are the gold standard for treatment
of extensive deep skin defects. Therefore, tissue engineers
have to develop cultured skin substitutes that match the
quality of auto skin grafts. To mimic the in vivo environ-
ment, a number of natural materials are widely preferred
for tissue-engineering applications.
112 PRIYA ET AL.

(a)

Hard Substrate

Soft Substrate

Blodegradeable Polymer

(b) (c)
Soft Substrate
Soft Substrate

FIG. 7. Micro-molding of biodegradable polymers: (a) set-up; (b) micro-molding of non-interconnecting micro-structures (cavities or
channels); (c) micro-molding of interconnecting micro-structures (holes). Reproduced from Ryu et al.34 with permission.

Silk, a natural biopolymer, promotes tissue repair
Silk, a naturally occurring biopolymer, has been used
clinically as sutures for centuries. It is composed of a fila-
ment core protein, termed fibroin, and a glue-like coating
consisting of sericin proteins. Silk fibroin in various forms
such as films, fibers, meshes, and sponges have been shown
to support stem cell adhesion, proliferation, and differenti-
ation in vitro and also promote tissue repair in vivo. In par-
ticular, stem cell–based tissue engineering using 3-D silk
fibroin scaffolds is promising for engineering a range of
skeletal tissues like bone, ligament, cartilage, and connec-
tive tissues like skin. The Bombyx mori silkworm has been
the dominant source for silk-based biomaterials.47 Silk fi-
broin displays a high rate of cell attachment and growth in
in vitro culture of most kinds of cells and is equivalent to
collagen. Thus, silk fibroin can be used as a scaffold material
in several fields, such as tissue engineering of skin, cartilage,
and blood vessels.48
Collagen: a biocompatible scaffold
for tissue regeneration
Collagen is the main structural protein in vertebrates and
the most useful of all biomaterials. As a scaffold, it enhances
cell attachment, migration, proliferation, and differentiation.
Because of its excellent biocompatibility, biodegradability,
and weak antigenicity, it is used as the primary resource in
medical applications. Development of recombinant sources
of human collagen has provided a reliable source of collagen
that is free of animal components. Recombinant human col-
lagens are an efficient scaffold for engineering skin, carti-
lage, and periodontal ligaments.49 Porous collagen matrices
with defined physical, chemical, and biological character-
istics are interesting materials for tissue engineering, and
introducing glycosaminoglycans may add to these charac-
teristics because they are constituents of ECM proteins.50
Fibrin glue: an adhesive and a growth factor
in tissue engineering
Fibrin glue has been widely used as an adhesive in plastic
and reconstructive surgery. It promotes hemostasis and also
has certain antibacterial properties. Furthermore, fibrin, as-
sociated with fibronectin, supports keratinocyte and fibro-
blast growth in vitro and in vivo and thereby enhances
cellular motility in the wound.51 In the initial phase of
wound healing, endogenous fibrin clots are known to form a
provisional matrix to promote angiogenesis. Growth factors
SKIN TISSUE ENGINEERING
such as vascular endothelial growth factor (VEGF) espe-
cially increase in wounds to stimulate angiogenesis. More-
over, fibrin, when used as a dermal substrate for cultured
skin substitute, increases the secretion of VEGF, thereby
promoting angiogenesis.52
Povidone and sugar stimulates wound healing
by activating keratinocytes and fibroblasts
Povidone, a complex of polyvinyl pyrrolidone and iodine is
a common antimicrobial agent and has been used as a surgical
scrub or a skin cleanser in various forms. The use of povidone
as a topical healing and stimulatory agent has been limited
because of its toxicity. At the same time, sugar and related
products from various natural sources have been shown to pro-
mote wound healing. Using these 2 substances, a compound
containing 70% to 80% sugar and povidone solution was used
that remarkably enhanced healing and at the same time re-
duced bacterial contamination in a wide variety of wounds,
burns, and ulcers.53 Similarly, in another study, it was shown
that a mixture of sugar and povidone activated the functions
of keratinocytes and fibroblasts more effectively than sugar
alone and has the potential to act as a powerful ameliorative
agent in the promotion of wound healing. Thus, it is likely that
a paste containing 70% sugar and 3% povidone acts on
wounds not only as an antibiotic agent, but also as a modulator
for keratinocytes and fibroblasts.54
HISTORICAL PERSPECTIVES OF TISSUE-
ENGINEERED PRODUCTS
Tissue-engineered biological dressings offer great prom-
ise in the treatment of burns, chronic ulcers, and a variety of
other dermatological conditions. Despite the large number
of potential beneficiaries, cellular tissue-engineered prod-
ucts have suffered setbacks in recent years. The reasons
may be that the mechanism of action of these products is
TABLE 1. FOOD AND DRUG ADMINISTRATION (FDA)–APPROVED COMMERCIALLY AVAILABLE SKIN SUBSTITUTES
FOR TISSUE ENGINEERING
Product FDA-Approved Indications (PMA, HDE)
Alloderm Burns/full-thickness wounds (allograft)
Apligraf Venous/diabetic ulcers (PMA)
Dermagraft Diabetic foot ulcers (PMA); ulcers secondary
to epidermolysis bullosa (HDE)
Epicel Deep partial-thickness and full-thickness burns
(HDE); congenital nevi (HDE)
Integra Deep partial-thickness and full-thickness burns
(PMA)
OrCel Split-thickness donor sites (PMA); mitten hand
deformity surgery of epidermolysis bullosa (HDE)
TransCyte Full- and partial-thickness burns (PMA)
PMA, pre-market approval; HDE, humanitarian device exemption.
113
not universally agreed upon and that these products do not
specifically match a clinical treatment to an underlying pa-
thology. Despite commercial setbacks, the first-approved
products, Dermagraft, Apligraf, and cultured epidermal
autograft (Epicel) are still being marketed.55 Tissue-
engineered products can cover wounds and provide an
environment that stimulates their repair. Graftskin (APLI-
GRAF) is the most advanced bioengineered skin product.
It is a bi-layered living skin construct consisting of a der-
mis and a well-differentiated epidermis. The epidermal
cells (keratinocytes) and dermal cells (fibroblasts) are ob-
tained from neonatal foreskin.56 Skin replacement products
are the most advanced, with a number of tissue-engineered
wound care materials in several international communities.
The potential effect of this field is that it offers novel so-
lutions to the medical field for drug screening, genetic
engineering, tissue and organ replacements.57 A number
of tissue-engineered medical products that are available in
the market are listed in Table 1.
Currently available products approved
by the Food and Drug Administration
as matrices for wound repair
Epicel (Genzyme Tissue Repair): A biopsy of the pa-
tient’s cells is grown into an integrated sheet and enzy-
matically detached for delivery to the patient.58
Integra (Integra Life Sciences): An alternative to donor
skin that provides a vascularized dermis for a subsequent
split-thickness skin graft. 59
Alloderm (Life Cell): Freeze-dried human donor dermis.60
Dermagraft (Advanced Biohealing): A synthetic material
conditioned with donor fibroblasts.61
Transcyte (Advanced Biohealing): Similar to Dermagraft
but with a silicone membrane to act as a temporary epi-
dermal barrier.62
Apligraf (Organogenesis): Combines allogenic kerati-
nocytes and fibroblasts with bovine collagen to provide a
Competitive Advantages
Not rejected; no cases of viral transmission
after > 100,000 product applications; 2-year shelf life
Mimics function of dermis; cryopreserved product
Mimics function of dermis; cryopreserved product
Autologous cells; no rejection, high incidence of
permanent take
Two layers; good barrier function; used in >10,000
patients; moderate shelf life
Mimics cytokine expression of healing skin; 9-month
shelf life cryopreserved
1.5-year shelf life frozen
114
temporary skin-replacement material suitable for use in
chronic wounds but not major burns.63
Orcel (Ortec International): Combines allogenic kerati-
nocytes and fibroblasts with bovine collagen to provide a
temporary skin-replacement material suitable for use in
chronic wounds.64
The main drawback of tissue-engineered skin products is
clinical failure due to delays in vascularization. Methods
for increasing vascularization and promoting wound heal-
ing have been evaluated and patented (United States Patent
6713079). The method consists of formation of a matrix
comprising Gel and a nitric oxide inhibitor. The Gel is
preferably denatured collagen. The nitric oxide inhibitor
may be an L-arginine analog such as N-nitro-L-arginine
and D-arginine. The matrix may further comprise a nitric
oxide scavenger, such as dextran, heparin, cysteine, or
cystine.
A better understanding of the factors influencing ECM
formation in tissue-engineering scaffolds is essential to
achieve further development in skin generation.
Clinical trials of certain skin grafts for burn injuries
TransCyte consists of a polymer membrane and neonatal
human fibroblast cells cultured in vitro on a nylon mesh.
Before cell growth, this nylon mesh is coated with porcine
dermal collagen and bonded to a polymer membrane (sil-
icone). The human fibroblast- temporary skin substitute
provides a temporary protective barrier. TransCyte has the
advantage of being transparent for direct visualization of
the wound bed. Biobrane is a biosynthetic wound dressing
constructed of a silicone film with a nylon fabric partially
imbedded into the film. The fabric presents to the wound
bed a complex 3-D structure of tri-filament thread to which
collagen has been chemically bound. Silvazine is a white
hydrophilic cream containing silver sulphadiazine and
chlorhexidine di gluconate, which is used in the treatment of
burns. The effectiveness of 3 burn dressings (TransCyte, a
bio-engineered skin substitute; Biobrane; and Silvazine
cream) were compared in treating children with partial-
thickness burns. Thirty-three patients with 58 wound sites
were used for the study (TransCyte, n ¼ 20; Biobrane,
n ¼ 17; Silvazine, n ¼ 21). The average time required for re-
epithelialization was 7.5 days for TransCyte, 9.5 days for
Biobrane, and 11.2 days for Silvazine. The number of au-
tografts required was found to be 5/21 (24%) for Silvazine,
3/17 (17%) for Biobrane, and 1/20 (5%) for TransCyte. Of
the wound dressings used for the study, TransCyte promoted
fastest re-epithelialization and required fewer overall
dressings than Biobrane or Silvazine. Patients who received
Silvazine or Biobrane required more autografting than those
treated with TransCyte.62
INTEGRA Dermal Regeneration Template is a bi-layer
membrane system made of a porous matrix of fibers of cross-
linked bovine tendon collagen and glycosaminoglycan
(chondroitin-6-sulfate). The temporary epidermal substitute
PRIYA ET AL.
layer is made of synthetic polysiloxane polymer (silicone)
and functions to control moisture loss from the wound. Upon
adequate vascularization, the temporary silicone layer is
removed, and a thin, layer of epidermal autograft is placed
over the ‘‘neodermis.’’ Cells from the epidermal autograft
grow and form a confluent stratum corneum, thereby clos-
ing the wound. In 31 patients who underwent INTEGRA
grafting for reconstructive surgery, 39 operational sites were
clinically observed. The length of follow-up ranged from 0.5
to 4 years. The final results in patients were considered to be
good in 28 cases, average in 6 cases, and poor in 3 cases.
Complications of silicone detachment and failure of the graft
were observed in 9 cases. Two patients (2 sites) were lost to
follow-up. The disadvantages of using INTEGRA in re-
constructive surgery are the necessity of 2 operations, the
risks of infection under the silicone layer, the silicone be-
coming detached, and recurrence of contraction. On the
other hand, INTEGRA has some advantages, including its
immediate availability in large quantities and the simplicity
and reliability of the technique.65
Dermagraft-TC (DG-TC, Advanced Tissue Sciences, Inc.
La Jolla, CA) is composed of human neonatal fibroblasts
cultured on a synthetic dressing (Biobrane; Dow B, Hickam,
Inc. Sugarland, TX). The material is stored frozen and
thawed immediately before use. DG-TC is semitransparent,
thus facilitating continuous observation of the underlying
wound surface. The surgeon removes the temporary cover-
ing when the patient’s own skin is ready to be grafted,
usually in 7 to 14 days. Biosynthetic human skin substitute
was compared with frozen human cadaver allograft for the
temporary closure of excised burn wounds. Burn wounds in
66 patients (mean age 36 years, mean burn size 44% of total
body surface area) were surgically excised. DG-TC was
equivalent or superior to allograft with regard to autograft
take at post-autograft day 14. DG-TC was also easier to
remove and resulted in less bleeding than did allograft.66 In
another study, burn wounds in 10 patients (mean age 33.5
years, mean burn size 39.9% of total body surface area) were
surgically excised. A control site on each patient received
cryopreserved human cadaver allograft skin (HCAS). Re-
sults showed that wound adherence and subsequent auto-
graft ‘‘take’’ were excellent with DG-TC and were at least
equivalent to HCAS. No evidence of immune rejection of
DG-TC was seen.67
The ability of an acellular allograft dermal matrix
to function as a permanent dermal transplant in full-
thickness and deep partial-thickness burns was evaluated.
The pilot phase included 24 patients to identify the opti-
mum protocol, and the study phase included 43 patients to
evaluate graft performance. After 14-day take periods, the
dermal matrix was statistically equivalent to the control
autografts. Histology of the dermal matrix showed neo-
vascularization and neoepithelialization. Assessment of
wounds showed that thin split-thickness autografts plus
allograft dermal matrix were equivalent to thicker split-
thickness autografts.60
SKIN TISSUE ENGINEERING
A cultured skin graft was created through successive
cultivation of fibroblasts and keratinocytes within a collagen
matrix. The collagen matrix has a honeycomb structure with
many holes; all holes through the sheet were filled with
collagen gel. This specific structure allows for the nourish-
ment of keratinocytes on the surface of the matrix. Auto-
logous cultured skin substitute was applied to a 51-year-old
man who had sustained a burn injury. Three sheets of the
cultured skin substitute (6
9.5 cm) were grafted onto the
full-thickness excised wound. One week after grafting, most
of the matrix disappeared, and stratified keratinocytes were
seen; 5 weeks after grafting, a cornified epidermal layer was
seen; and 10 months after grafting, a mature epidermis and a
well-differentiated papillary and reticular dermis replace-
ment were observed. The physical properties and color of
this grafted area resembled that of normal skin.68
Suprathel is produced from a synthetic copolymer based
mainly on dl-lactide (>70%); the other components are tri-
methylene carbonate and e-caprolactone. The monomers are
polymerized using a melting procedure and dissolved in or-
ganic solvents. The material was then processed using
modified phase-inversion and freeze-drying technique. The
final product is a porous membrane with a nearly symmet-
rical cross-section of interconnected pores. The pore sizes
vary between 2 and 50 mm, and the initial porosity of the
membrane is greater than 80%. Omiderm is a thin, trans-
parent polyurethane-based membrane. It is inelastic when
dry but highly flexible when wet. After removal of the burned
layers and cleansing of the wound, Omiderm is applied to the
wound. The membrane can be used wet and flexible to cover
uneven areas or dry on smooth ones. Suprathel was compared
with paraffin gauze (Omiderm) applied on split-skin donor
sites on second-degree burns. In both study parts, Suprathel
significantly reduced pain and could be handled more easily
than other materials. The Suprathel membrane adhered rap-
idly to the wound, thus protecting against infections and
promoting wound healing. Furthermore, no allergic reactions
were observed.69
Apligraf (Graftskin) (Organogenesis Inc., Canton, MA),
consists of living cells, proteins, and skin-healing sub-
stances. It is designed as a ‘‘living’’ skin patch that replicates
the healing function of healthy human skin. It is a unique and
advanced biological skin repair therapy for non-healing
sores. The potential of Apligraf when applied over meshed
split-thickness autografts was evaluated. Experimental
treatment sites had Apligraf placed over meshed autograft,
whereas control sites were treated with meshed autograft
covered with meshed allograft or meshed autograft not
covered by a biologic dressing. Thirty-eight patients were
evaluated for clinical studies. At the completion of the study,
the investigators rated 22 (58%) of the Apligraf sites supe-
rior to the control sites, 10 (26%) equivalent to the control,
and 6 (16%) worse than the control. By month 24, 18 (47%)
patients had normal vascularity at the Apligraf site, com-
pared with 6 (16%) patients at the control site. These results
indicate that Apligraf is suitable and clinically effective
115
in treatment for burn wounds when applied over meshed
autografts.70
CONCLUSION AND FUTURE PROSPECTS
Skin, the largest organ in the body, protects against toxins
and microorganisms in the environment and serves to pre-
vent dehydration of all non-aquatic animals. There are no
models of an artificial skin that completely replicates normal
uninjured skin. Therefore, the main goal of tissue engineers
is to produce a universal skin substitute that will provide
burn victims with the best quality skin in the shortest time
possible. Natural biopolymers such as collagen and fibro-
nectin have been investigated as potential sources of bio-
material. In our laboratory, we are focussing on preparation
of tissue-engineering scaffolds for skin tissue regeneration.
The scaffold is designed from cryogel materials that are
synthesized in moderately frozen conditions that provide
them with pore sizes as large as 100 mm. Cryogel materials
have been used in biomedical and biotechnological appli-
cations such as cell separation,71 immobilized biocatalysts,
cell culture scaffolds,72 and bioseparation and in high-
throughput screening and analysis.73 We have attempted to
use cryogels as tissue-engineering scaffolds because they
have a spongy and elastic morphology that is more resistant
to compressive load than conventional hydrogels; they retain
excess water content and prevent dehydration, thereby cre-
ating moist wound healing, because fluid balance has to be
maintained, especially in the case of severe burn injuries;
and in cryogels, cells are transported via convective mi-
gration through the matrices so that there is a uniform dis-
tribution of cell population.74
Successful tissue-engineering technique requires inten-
sive co-operation between clinicians, biologists, and engi-
neers. Tissue engineering has attracted much attention as a
new therapeutic means that may overcome the drawbacks
involved in current artificial organs and organ transplanta-
tion that have also been aiming at replacing lost or dam-
aged tissues and organs. However, the tissues regenerated
using this method are limited, including skin, bone, carti-
lage, capillary, and periodontal tissues.75 As engineered
skin substitutes improve in quality, they will become more
similar to native skin autografts. In this way, skin-substitute
materials may be able to stimulate regeneration rather than
repair.76 Understanding the biology of wound repair is es-
sential for the improvement of tissue-engineered medical
products.77 Thus tissue-engineering techniques bring a new
element of rational design and development to therapeutic
medicine.
ACKNOWLEDGMENTS
The work was financially supported by the Depart-
ment of Biotechnology (DBT), Ministry of Science and
116
Technology, Government of India. S. Geetha Priya grate-
fully acknowledges the institute assistantship received from
the Indian Institute of Technology—Kanpur for her PhD
program.
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Address reprint requests to:
Ashok Kumar, Ph.D.
Department of Biological Sciences and Bioengineering
Indian Institute of Technology Kanpur
208016—Kanpur
India
E-mail: ashokkum@iitk.ac.in