Wound healing in the 21st century

Stephan Schreml, MD,a Rolf-Markus Szeimies, MD, PhD,a Lukas Prantl, MD, PhD,b
Michael Landthaler, MD, PhD,a and Philipp Babilas, MD, PhDa
Regensburg, Germany
Delayed wound healing is one of the major therapeutic and economic issues in medicine today. Cutaneous
wound healing is an extremely well-regulated and complex process basically divided into 3 phases:
inflammation, proliferation, and tissue remodeling. Unfortunately, we still do not understand this process
precisely enough to give direction effectively to impaired healing processes. There have been many new
developments in wound healing that provide fascinating insights and may improve our ability to manage
clinical problems. Our goal is to acquaint the reader with selected major novel findings about cutaneous
wound healing that have been published since the beginning of the new millennium. We discuss advances
in areas such as genetics, proteases, cytokines, chemokines, and regulatory peptides, as well as therapeutic
strategies, all set in the framework of the different phases of wound healing. ( J Am Acad Dermatol
2010;63:866-81.)
Key words: cellular; molecular; novel findings; signal transduction; pH value; skin wound.

utaneous wounds are the result of disrupted

skin integrity. The healing process depends
on local wound factors, systemic mediators,
the underlying disease, and the type of injury. These
factors combine to determine if physiologic or acute
wound healing occurs, or if there is an abnormal
healing process, also called chronic wound healing.
Chronic wounds are the result of an inadequate
repair process that is unable to restore anatomic and
functional integrity in an appropriate length of time.
Chronic wounds affect about 1% of the European
population and are frequently a management challenge, even with an interdisciplinary approach. In
addition to having an adverse effect on the quality of
life of the affected individuals, chronic wounds also
create a significant economic burden: nearly 2% of
health budgets are devoted to the care of chronic
wounds.1
Our understanding of the mechanisms involved in
cutaneous wound healing has dramatically increased
From the Departments of Dermatologya and Plastic Surgery,b
Regensburg University Hospital.
Supported by grants of the German Research Foundation (Deutsche Forschungsgemeinschaft DFG, BA 3410/3-1) and the
Novartis Foundation (S.S., Novartis Graduate Scholarship).
Conflicts of interest: None declared.
Reprint requests: Stephan Schreml, MD, Department of
Dermatology, Regensburg University Hospital, Franz-JosefStrauss-Allee 11, 93053 Regensburg, Germany. E-mail:
stephan.schreml@klinik.uni-regensburg.de.
Published online June 24, 2010.
0190-9622/$36.00
2009 by the American Academy of Dermatology, Inc.
doi:10.1016/j.jaad.2009.10.048

866

Abbreviations used:
AGE:
Cyr61:
ECM:
EGF:
ERK:
FGF:
HGF:
HSP:
IL:
IL-1ra:
KGF:
LacZ:
LL-37:

advanced glycation end product

cysteine-rich angiogenic inducer-61
extracellular matrix
epidermal growth factor
extracellular regulated kinase
fibroblast growth factor
hepatocyte growth factor
heat shock protein
interleukin
interleukin-1 receptor antagonist
keratinocyte growth factor
lactose operon Z
C-terminal fragment of human
cathelicidin antimicrobial peptide-18
MMP:
matrix metalloproteinase
mTOR: mammalian target of rapamycin
NF:
nuclear factor
NPY:
neuropeptide Y
Nramp1: natural resistanceeassociated macrophage protein-1
Nrf:
nuclear factor-E2-related factor
PI3K:
phosphatidyl inositol-3 kinase
RE:
response element
Shh:
sonic hedgehog
SPARC: secreted protein acidic and rich in
cysteine
TF:
tissue factor
TGF:
transforming growth factor
TIMP:
tissue inhibitor of matrix
metalloproteinase
TLR:
toll-like receptor
VEGF:
vascular endothelial growth factor

in the past few years. Keeping up to date with the

current literature is sometimes difficult. Our aim is to
provide researchers and clinicians working in the field
of wound healing with selected new insights into
wound pathogenesis and cutaneous repair

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mechanisms. We consider topics such as genetics,

Intercellular adhesion molecule-1 interacts with leuproteases, cytokines, chemokines, and regulatory
kocytes via CD11a (together with CD18 = lymphopeptides, as well as therapeutic strategies. All are
cyte function-associated antigen-1). Nagaoka et al4
found that intercellular adhesion molecule-1edefiviewed in the context of the intertwined phases of
cient mice showed impaired wound healing because
wound healing: (1) inflammatory phase, (2) proliferof a lack of leukocyte and macrophage infiltration
ative phase (neoangiogenesis, granulation, re-epitheinto the wound site. These cells are required to
lialization), and (3) remodeling phase (extracellular
establish an inflammatory rematrix [ECM] remodeling).
action, which is a major mileDetailed figures are provided
CAPSULE SUMMARY
stone on the way to
to facilitate the understanding
organized wound repair.
of the rather complex pathoSince the beginning of the new
Coordinated inflammagenetic mechanisms.
millennium a large number of articles
tory phases require a
Chronic wounds are dehave been published dealing with
subtle balance of proinfined as wounds that do not
cutaneous wound healing.
flammatory cytokines and
follow the well-defined stepThis article reviews novel findings related
chemokines and their antagwise process of physiologic
to the major phases of cutaneous wound
onists. Although interleukin
healing. Instead, they are
healing: inflammation, proliferation, and
(IL)-1 is known as a key factrapped in an uncoordinated
tissue remodeling.
tor, little is known about the
and self-sustaining phase of
functions of the IL-1 receptor
inflammation that impairs
Newly discovered molecular targets and
antagonist (IL-1ra). Ishida
the restoration of anatomic
pathways provide the basis for further
et al5 found that IL-1ra e/e
and functional integrity in
research and future clinical studies.
mice showed an interruption
the normal period of time.
in transforming growth
Many of the pathophysiofactor (TGF)-b1 signaling, which resulted in reduced
logic factors (hypoxia, pH changes, and bacterial
collagen deposition and vascular endothelial growth
colonization) that contribute to delayed wound
factor (VEGF) expression. IL-1ra is only temporarily
healing are well known. However, the exact pathoup-regulated until 10 days after skin injury. IL-1ra
genesis of chronic wounds remains unclear.
deficiency induces prolonged nuclear factor (NF)-kB
Rather than giving a comprehensive overview on
p65 nuclear translocation. The prolonged inflammawound healing, the following sections will focus on
tory phase leads to delayed wound healing in these
newly discovered molecular mechanisms and their
mice.
importance in wound healing.
Ishida et al6 further studied the role of chemokine
receptors in wound pathogenesis in a full-thickness
THE INFLAMMATORY PHASE
excisional skin mouse model. The chemokine cheThe initial phase after cutaneous injury is domimokine C-X3-C motif ligand-1 (CX3CL1) (nomenclanated by inflammatory reactions mediated by cytoture according to patterns of conserved cysteines:
kines, chemokines, growth factors, and their actions
chemokine C-X3-C motif ligand-1; fractalkine) and
on cellular receptors (Fig 1). Intracellular signaling
its receptor chemokine C-X3-C motif receptor-1
cascades are activated, contributing to cell prolifer(CX3CR1) are up-regulated at wound sites. Their
ation, migration, and differentiation. In addition,
role in wound healing was elucidated in an excichemoattractant factors recruit different cell types,
sional wound CX3CR1 e/e mouse model that
such as granulocytes and macrophages, to the
showed reduced macrophage infiltration and then
wound site, thus initiating wound repair. The
reduced TGF-b1 and VEGF signaling (because both
wound milieueconsisting of various proteinases,
are released by macrophages), which in turn led to
cytokines, chemokines, pH gradients, and pO2
decreased collagen deposition and neoangiogenegradientsehas a major impact on cellular functions.
sis, inevitably resulting in delayed wound healing.
The importance of wound fluid in regulating the
When IL-1ra is knocked out, different chemokines
responsiveness of fibroblasts to proliferation signals
are also up-regulated.
mediated by cytokines has been shown by Nedelec
Furthermore, the shift in balance between proinet al.2
flammatory and anti-inflammatory factors is one of
During the inflammatory phase of wound healing,
the central reasons for persistent inflammation in
a variety of membrane-bound receptors play a role in
chronic wound healing. An anti-inflammatory factor
recruiting leukocytes and other cells. One receptor
involved in regulating the balance is secretory leumediating leukocyte-endothelial cell interaction3
kocyte protein inhibitor-1. Its gene expression is
is intercellular adhesion molecule-1 (CD54).
d

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Fig 1. Inflammatory phase. Cells communicate via connexin-43 (Cx43) and proinflammatory
cytokines and chemokines, such as CC chemokine ligand (CCL)-2, tumor necrosis factor (TNF ),
and interleukin (IL)-1 trigger inflammation. Vascular endothelial growth factor (VEGF ),
transforming growth factor (TGF )-b, and keratinocyte growth factor (KGF )-1 are induced
and facilitate the next stage of wound healing (proliferative phase). Reactive oxygen species
(ROS ) are being produced, degraded by peroxiredoxin-6, and their effects are reduced by
LL-37. asODN, Antisense oligodeoxynucleotides; CX3CL1, chemokine C-X3-C motif ligand-1;
CX3CR1, chemokine C-X3-C motif receptor-1; HSP, heat shock protein; ICAM, intercellular
adhesion molecule; IL-1ra, IL-1 receptor antagonist; Nrf, nuclear factor-E2-related factor; green
arrows, positive regulation: activation; red arrows, negative regulation: inhibition.

modulated by natural resistanceeassociated macrophage protein-1 (Nramp1) (solute carrier family 11

member-1), which is also a modulator of inflammatory toll-like receptor (TLR)-7 signaling. Secretory
leukocyte protein inhibitor-1 down-regulation leads
to prolonged wound healing as shown in an Nramp1
e/e macrophage model. In wild-type Nramp1 1/1
mice, secretory leukocyte protein inhibitor-1 levels
were significantly higher and cutaneous wound
healing was significantly faster than in Nramp1
knockout mice.7
The regulation of genes encoding growth factors
and cytokines or chemokines is also of great interest.
Beyer et al8 summarized the specific role of NF-E2related factor (Nrf)2 in the regulation of wound
healing. The gene is the target of keratinocyte growth
factor (KGF), and the protein is an important transcription factor inducing detoxifying enzymes and
antioxidant proteins. Nrf2 messenger RNA was significantly up-regulated in a murine full-thickness
excisional wound model, while other members of
this transcription factor family either remained unaffected (Nrf1) or were even down-regulated (Nrf3).
Using transgenic mice expressing a dominant-negative Nrf2 variant, Beyer et al8 were able to show that
Nrf2 plays a crucial role in inflammatory wound

pathogenesis but not in re-epithelialization. Again,

this finding shows that wound closure is a highly
regulated process in terms of time-dependent expression profiles of cytokines/chemokines and
growth factors. These factors not only account for
enhancing inflammation but also set well-defined
stop signals to block the inflammatory cascade when
appropriate during the wound-healing process. This
is important as a prolonged inflammatory reaction is
the main reason for impaired wound healing.
Therefore, therapeutic strategies aimed at reducing
inflammation at the appropriate time point could
have a significant impact. Mori et al9 found that
knocking down connexin-43 (gap junction component that mediates cell migration and proliferation)
by antisense oligodeoxynucleotides (asODN) resulted in faster wound closure and a reduced inflammatory reaction, as also reflected by reduced levels
of CC chemokine ligand (CCL)-2 and tumor necrosis
factor. Correspondingly, histologic analysis showed
reduced macrophage and leukocyte infiltration. A
few years earlier, Coutinho et al10 had already shown
the dynamic regulation of connexin expression during wound repair. Therapeutic strategies based on
these observations are being developed; for instance, a single topical application of connexin-43

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antisense gel resulted in a transient down-regulation

of connexin-43 protein levels and subsequently
accelerated wound closure.11
One of the major breakthroughs in our understanding of wound healing during the past years was
the discovery that antimicrobial peptides have an
impact on both wound healing and on different
cellular and subcellular functions. For example,
cathelicidin, more specifically LL-37, a C-terminal
fragment of human cathelicidin (human cathelicidin
antimicrobial protein-18), is up-regulated by common growth factors, such as insulin-like growth
factor-1 or TGF-a.12 These peptides function as
regulatory factors by inhibiting cytokine release
and triggering respiratory burst (generation of reactive oxygen species [ROS]) in the early phase of
wound healing.13 The antagonist role of LL-37 may
be relevant for keeping the level of inflammation at
the wound site under control.14,15 Moreover, LL-37
acts as a potent antimicrobial peptide, thereby also
limiting inflammatory processes.14,15 In contrast,
other regulatory peptides such as the human b-defensins even trigger the release of proinflammatory
cytokines and chemokines.16 Besides, LL-37 has
been shown to transactivate the epidermal growth
factor (EGF) receptor and thus to stimulate
keratinocyte proliferation during the subsequent
proliferative phase.17,18 The important in vivo activity of LL-37 in wound healing has been studied in
ob/ob (obese, ob-gene encodes leptin) mice.19
Adenoviral transfer of LL-37 to ob/ob mice with
excisional wounds was shown to result in significantly improved wound healing. But how can LL-37
be therapeutically modified in a clinical setting?
Many questions still need to be answered before
adenoviral transfer is incorporated into routine clinical practice. On the other hand, LL-37 expression
can be modulated by the application of short-chain
fatty acids such as butyrate (not in all cell types
involved).20 This approach is relatively easy and may
be suitable for daily practice.
A well-known problem in delayed wound healing
is oxidative stress induced by reactive oxygen species (ROS).21,22 Peroxiredoxin-6 has been identified
as one of the factors protecting endothelial cells and
keratinocytes from ROSeinduced damage to membranes, DNA, and proteins.23 The peroxiredoxin
family comprises 6 members, which reduce hydrogen peroxide and other organic peroxides by means
of redox-active cysteine. Kumin et al23 have shown
in vitro that knocking down peroxiredoxin-6 in
cultured endothelial cells increases their susceptibility to oxidative stress. In addition, peroxiredoxin-6
deficient mice showed massive hemorrhage in granulation tissueedependent on the amount of

oxidative stressewhich counteracted proper wound

healing.
Cell membrane damage leads to a release of a vast
variety of intracellular proteins from the cytoplasmic
compartment into the wound bed. One highly conserved family of intracellular proteins contributing to
the early inflammatory phase after cutaneous injury
is the heat shock protein (HSP) family. Exogenously
administered HSP70 and HSP90 (normally located in
the cytoplasm) as well as HSP gp96 (located in
endoplasmic reticulum) produced enhanced wound
healing in a full-thickness skin wound mouse
model.24 One mechanism involved is HSP70-mediated activation of phagocytosis by macrophages,
which are essential for removing cell debris from
the wound to allow 3-dimensional tissue reconstruction and remodeling.
Chen et al25 reported another factor, which is
temporarily overexpressed in the early stage of
wound healing. Tissue factor (TF) (thromboplastin
or factor III) plays an important role in the coagulation cascade. In a murine diabetic model, TF may be
one of the cross-links between the early inflammatory phase and the subsequent proliferative phase.
Chen et al25 showed that TF was up-regulated as
early as 1 hour after injury and that high TF levels led
to improved wound vascularization. Moreover, a
relative deficiency of TF in diabetic mice compared
with nondiabetic controls resulted in longer woundhealing times. These results underline that a perfect
spatial and chronological regulation of proteinases
and their inhibitors is needed for normal wound
closure.

PROLIFERATIVE PHASE
The proliferative phase includes: (1) neoangiogenesis (Fig 2, A), (2) formation of granulation tissue
and ECM (Fig 2, B), and (3) re-epithelialization
(Fig 2, C ).
Neoangiogenesis
Neoangiogenesis, one of the major processes in
wound healing, is absolutely necessary for proper
wound healing (Fig 2, A). The switch of macrophages from producing proinflammatory cytokines
to secreting VEGF is controlled by TLR cross-talk
with adenosine A2A receptors. Myeloid differentiation primary response gene (MyD)-88, a cytoplasmic
adaptor protein for TLR signaling, is responsible for
this switch. Wounds of myeloid differentiation primary response protein-88 e/e mice heal significantly slower than those of wild-type mice. In
different knockout models, it has been proven that
IL-1 receptoreassociated kinase-4 (IRAK-4) and tumor necrosis factor-receptoreassociated factor-6

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Fig 2. Continued

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Fig 2. Proliferative phase. A, Neoangiogenesis is induced by activation of mammalian target of

rapamycin (mTOR), sonic hedgehog (Shh), neuropeptide Y (NPY ), and toll-like receptors
(TLRs) (cross-talk with adenosine A2A receptors, myeloid differentiation primary response
protein-88 [MyD88]: important intracellular TLR adaptor in wound healing), leading to
production of angiogenic factors, such as vascular endothelial growth factor (VEGF ),
cysteine-rich angiogenic inducer-61 (Cyr61), and interleukin (IL)-b1. Thus, de novo vessel
formation is induced. B, Proliferation and collagen production of fibroblasts is activated by
increase in activin/follistatin ratio, matrilin-2, and activation of sphingosine signaling pathway
(sphingosine-1 kinase [Sp1-K]). Secreted protein acidic and rich in cysteine (SPARC ) (part of
extracellular matrix [ECM]) controls excessive fibroblast proliferation and facilitates production
of collagen-1. IL-4-induced wound-healing macrophages contribute to fibroblast proliferation
and collagen production. C, Re-epithelialization is triggered via epidermal growth factor (EGF )
(its receptor is transactivated by LL-37), activation of mTOR pathway by keratin-17 production
and hepatocyte growth factor (HGF ) (acts via c-Met receptor on keratinocytes). Leptin is
important for proliferation/migration of keratinocytes, which interact with ECM through
galectins in their podosomes. AGE, Advanced glycation end product; c-Met, mesenchymal
epithelial transition factor = hepatocyte growth factor receptor (HGF-R); EGF-R, epidermal
growth factor receptor; Fhl2, four and a half LIM domains-2; IGF, insulin-like growth factor;
MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF, nuclear factor;
PAI, plasminogen activator inhibitor; PI3K, phosphatidyl inositol-3 kinase; RAGE, receptor of
advanced glycation end products; RELM, resistin-like molecule; Sp1-P, sphingosine-1-phosphate; TGF, transforming growth factor; TH2-cells, T helper cell type 2; USF, upstream
transcription factor; YM, chitinase-like protein; Y2-receptor, neuropeptide Y receptor-2; green
arrows, positive regulation: activation; red arrows, negative regulation: inhibition.

(TRAF-6) participate in this complex process of

macrophage phenotype change, which is essential
for vascular proliferation in wounds.26
In addition to VEGF, which has been studied in
great detail, other barely known angiogenic factors
need to be investigated. One is cysteine-rich angiogenic inducer-61 (Cyr61), a heparin-binding, ECMassociated protein, which is known to contribute to
angiogenesis by modulating cell proliferation, migration, and adhesion of endothelial cells and fibroblasts. Chen27 was the first to show that Cyr61 is
significantly up-regulated in dermal fibroblasts

during granulation tissue formation and that Cyr61

levels return to basal levels thereafter. Remarkably,
Cyr61 regulates genes encoding for major proteins
that are involved in different aspects of cutaneous
wound healing, such as angiogenesis and lymphogenesis (VEGF-A and -C), inflammation (IL-1b), ECM
remodeling (matrix metalloproteinase [MMP]; tissue
inhibitor of MMP [TIMP]), and cell-matrix interactions (eg, integrins a3 and a6).
In 2006, Asai28 reported that the sonic hedgehog
(Shh) gene is a genetic factor contributing to de novo
vessel formation in wounds. Shh is known to be

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Fig 3. Remodeling phase. Matrix metalloproteinases (MMPs) are positively regulated by

mammalian target of rapamycin (mTOR), inhibited by a1-antichymotrypsin (a1-ACT ) and by
tissue inhibitors of MMPs (TIMPs). Rather unspecific stimuli, such as bacterial colonization and
pH value modifications, alter MMP activity. Syndecan-4 (on fibroblasts) is induced upon
cutaneous injury and interacts with integrins and growth factor receptors. These processes
contribute to extracellular matrix remodeling (especially MMP1-3), chronic wound pathogenesis (MMP8/9), and keratinocyte migration (especially MMP28 = epilysin). RE, Response
element; green arrows, positive regulation: activation; red arrows, negative regulation:
inhibition.

important in epithelial-mesenchymal interactions. In

a patched-1/lactose operon Z (LacZ) mouse model,
Asai28 topically applied the Shh gene DNA in form of
a human Shh plasmid. Patched-1 is a receptor for
hedgehog signaling that is activated by Shh binding.
LacZ encodes for b-galactosidase. The LacZ reporter
gene is particularly useful for studies of the cisregulatory element for tissue-specific expression in
transgenic mice because of the ease of the enzyme
assay and the visualization on sections. In the model
of Asai,28 topical Shh gene therapy increased the
angiogenic cytokine production of dermal fibroblasts, leading to an increase in endothelial cell
proliferation, migration, and vessel tube formation.
In 2003, Ekstrand et al29 reported on the role of
neuropeptide Y (NPY), a well-known neurotransmitter, in wound healing. NPY acts via interaction
with NPY-receptor-2 and initiates the formation of
treelike vessel structures. In a mouse corneal micropocket model and in a chick chorioallantoic membrane model using NPY-receptor-2-deficient mice,
NPY failed to induce angiogenesis and wound healing was drastically reduced in these mice.29
Furthermore, hypoxia is a major factor contributing to delayed healing of chronic cutaneous

wounds. A key target may be mammalian target of

rapamycin (mTOR) (synonym of FK506 binding
protein-12/rapamycin-associated protein kinase-1)
as it has a significant regulatory impact on hypoxiainduced angiogenesis.30 In normoxic conditions,
mTOR does not seem to play a central role in
angiogenesis. Hypoxia-induced activation of proliferation and angiogenesis by mTOR is a new molecular link between exogenous stimuli and cellular
response. This link may be clinically relevant as
mTOR inhibitors (eg, rapamycin, everolimus) are
known to lead to delayed wound healing in the early
postoperative phase after solid organ transplantation. Another argument for the importance of mTOR
in wound healing is the observation that blocking or
even knocking down signaling pathways interacting
with mTOR complex-1esuch as extracellular regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase, and phosphatidyl inositol-3 kinase
(PI3K)delay, accelerate or even prevent wound
closure.31 mTOR integrates signals from these
pathways,30 and a fragile balance of positive
wound-healing signals (eg, p38 mitogen-activated
protein kinase and ERK1/2) and negative ones (eg,
PI3K pathway) is needed for normal healing.31

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Sufficient vascularization is absolutely necessary

for the delivery of nutrients and oxygen to the
wound site during the subsequent energy-consuming proliferative phase.
Fibroblast proliferation and collagen
production
In the proliferative phase (Fig 2, B), nutrients and
oxygen are limiting factors because of the tremendous metabolic activity. Just as in neoangiogenesis,
macrophages are also being rediscovered in their
interaction with fibroblasts. A special subtype of
wound healing macrophages is induced mainly by
IL-4-mediated T helper cell type 2 (TH2) responses.32
This macrophage subtype is characterized by the
expression of membrane-bound molecules that enable interaction with the ECM, for example chitinaselike protein (YM1) and resistin-like molecule
(RELM)-a. In addition, the expression of the antiinflammatory IL-27 receptor a is induced via IL-4,
marking the shift from inflammation to the stimulation of fibroblast proliferation and collagen production, for example via insulin-like growth factor-1.
Wound healing macrophages also contribute to collagen production as arginase activity in this subtype
is up-regulated, allowing macrophages to convert
arginine to ornithine, which is an important precursor in collagen production.
A number of other relevant factors important for
fibroblast proliferation in cutaneous wound healing
have been investigated in the past years. In 2002,
Bradshaw et al33 reported on the involvement of an
ECM protein, secreted protein acidic and rich in
cysteine (SPARC) (synonym of osteonectin or BM40) in cutaneous wound healing. The authors compared wound healing in SPARC e/e knockout mice
and wild-type mice. SPARC-null mice showed
quicker wound healing and higher proliferation rates
of fibroblasts but only half as much collagen I
content as wild-type mice, as documented by hydroxyproline levels. The increased contractibility of
collagen fibers produced by SPARC-null dermal
fibroblasts may be partially explained by the decreased collagen I content.33
Little is known about matrilin-2, a protein involved
in fibroblast proliferation and ECM interaction.
Matrilin-2 is a member of the von Willebrand factor
A domain-containing protein family and a component of extracellular filament networks. It interacts
with ECM components, fibroblasts, and keratinocytes. DNp63/bone morphogenic protein-7/Smad
signaling (DNp63, N-terminal isoform of p53; Smad,
Sma and mothers against decapentaplegic homolog)
has been shown to regulate matrilin-2 transcription in
wound healing, elucidating the promising potential

of this pathway.34 As matrilin-2 is known to contribute to wound healing and as skin injury leads to
transient DNp63-up-regulation, further study of this
cascade appears promising.35 After completion of
wound healing processes through involvement of
matrilin-2, DNp63 is down-regulated to basal levels,
again showing the fragile complexity of cutaneous
wound healing.
Another important, time-dependent mechanism
involves fibroblast growth factor (FGF) binding protein. FGF itself is a prominent stimulus for fibroblast
proliferation and differentiation. This protein is also
up-regulated during the initial phase after skin injury
and quickly decreases to baseline levels as shown in
a severe combined immunodeficiency (SCID) xenograft mouse model with human upper eyelid skin.36
In turn, however, excessive proliferation and collagen production rates of fibroblasts may lead to
hypertrophic scarring or keloid formation.
Hypertrophic scars are marked by excessive collagen
production, leading to scar elevation and hardening.
An enzyme needed to form a stable triple helical
collagen molecule by hydroxylating procollagen
proline residues is termed prolyl-4-hydroxylase.
Kim et al37 demonstrated that inhibition of this
enzyme by the topically applied inhibitor FG-1648
reduced scar hypertrophy in a rabbit ear hypertrophic scar model. Moreover, wound re-epithelialization and granulation remained unimpaired.
The potential of lysophospholipids (eg, sphingomyelin, phosphatidylcholine) in modulating fibroblast functions is far from being fully understood.38
The protein four and a half LIM-domains (Fhl)-239ea
downstream effector of sphingosine-1-phosphate
signalingeis translocated to the nucleus in cutaneous
wound repair. (LIM domains are named after their
initial discovery in the proteins Lin-11, Isl-1; and
Mec-3; LIM is a protein structural domain composed
of two contiguous zinc finger domains.) Fhl-deficient mice show impaired wound healing as a result
of reduced collagen contractibility and cell migration
to the wound. Furthermore, there is molecular evidence for these observations as p130Cas (a protein
important for cell migration; Cas, Crk associated
substrate) was down-regulated in Fhl e/e mice.39
This new signaling cascade may be an attractive
target for the relatively new class of sphingosine1-phosphate receptor inhibitors and activators.
Many other factors also control fibroblast activity,
for instance TGF-b and activins (TGF-b-superfamily
members). Activins are known as important proteins
in wound repair, and the expression of two activinbinding proteinsefollistatin and follistatin-related
proteinehas been investigated. As a consequence
of wounding, activins are up-regulated, whereas

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follistatin and follistatin-related protein levels remain

relatively constant or even decrease. As more free
activin is available by increasing the ratio of activin
and follistatin-related protein, activin is able to activate TGF-b-mediated signaling, which is important
for wound repair.40 Of course, receptors not only act
on a single cell type but rather mediate cross talk
between different cell types in wound healing.
Ghahary and Ghaffari41 studied the important cross
talk between keratinocytes and fibroblasts with a
special focus on the regulation of MMPs and TGFb1 signaling. They showed that wound-edge keratinocytes produce significantly higher amounts of
TGF-b1, which is a potent paracrine stimulus for
fibroblast and macrophage proliferation as well as
migration. This cross talk between keratinocytes and
fibroblasts in different skin layers is very important,
particularly for the initiation of the next step:
re-epithelialization.

Re-epithelialization
Re-epithelialization (Fig 2, C ) aims at covering the
wound surface with a layer of epithelium and is
based on the differentiation, proliferation, and
migration of epidermal keratinocytes. After the
wound bed has been properly established with
proliferating fibroblasts, a new collagen matrix, and
new vessels, the process of re-epithelialization can
start. Keratinocytes are activated and migrate into the
wound site from the edges. Even highly conserved
pathways such as the Wnt (combined from wingless
Wg and Int) pathway are involved in this process.42
New mechanisms in the complex process of reepithelialization have been discovered during the
past few years. The role of a Ca21-dependent
e-(g-glutamyl)lysin cross-linking enzyme termed
transglutaminase-1 was investigated in a neonatal
mouse skin model.43 This enzyme colocalizes with
involucrin, which is essential for the composition of
the cornified envelope of the stratum corneum.
Inada et al43 showed that transglutaminase-1 e/e
mouse skin grafted on athymic mice showed delayed
wound healing and uncontrolled keratin 6a messenger RNA expression, possibly to compensate for
transglutaminase-1 deficiency.
Another previously unknown mechanism in reepithelialization was found in a mouse model with a
targeted deletion of the signaling domain of a6b4
integrin (laminin-332; formerly known as laminin-5).
a6b4 Integrin is essential for proper EGF-mediated
ERK and Jun-N terminal protein kinase signaling.
The crucial finding was that a6b4 integrin is required
for the nuclear translocation of mitogen-activated
protein kinases (MAPK) and NF-kB, which, in turn,

regulates keratinocyte proliferation and migration in

wound healing.44
KGF is also known for its potential in wound
healing. Lin et al45 used Sprague-Dawley rats in
which wound healing was impaired by sepsis as a
result of cecal ligation after punch biopsy. By means
of electroporation transfection techniques using
plasmids containing KGF-1 DNA, they showed significantly decreased wound healing times. This
model even provided evidence that KGF may be
therapeutically useful in the future.
Epithelial injury stimulates specific signaling cascades, which may be potential therapeutic targets in
future. Providence46 was the first to show a specific
keratinocyte response to epithelial monolayer
wounding in a cell culture model. Plasminogen activator inhibitor-1 is a serine protease inhibitor essential
for barrier proteolysis and cell-to-matrix adhesion.
Plasminogen activator inhibitor (PAI)-1 messenger
RNA levels are elevated after epithelial injury.
Upstream transcription factor-1 (USF-1, a helix-loophelix transcription factor)which binds to an E-Box
motif in the plasminogen activator inhibitor-1 proximal gene promoteris induced by tissue injury in
vitro. These data implicate upstream transcription
factor-1 as a transcriptional regulator of genes involved in wound repair. Even though specific modification of this signal transduction pathway is still far
from being routine, it is tempting to speculate on novel
drugs interacting with upstream transcription factor-1.
mTOR (FK506 binding protein-12/rapamycin-associated protein kinase; RAFT1, rapamycin and
FKBP12 target-1) is a highly conserved protein
kinase and a component of two different protein
complexes: mTOR complex-1 regulates proliferation, DNA synthesis, transcription, and translation,
whereas mTOR complex-2 is predominantly involved in the control of cell size and actin skeleton
ultrastructure.30 In 2006, a novel link between keratin 17 (an intermediate filament)47 and mTOR was
shown by Kim et al.48 Keratin 17, which is upregulated in wounded stratified epithelia, stimulates
the Akt/mTOR (Akt is protein kinase B) pathway by
binding to the adaptor protein 14-3-3s. As a consequence, proliferation and protein synthesis of keratinocytes increase, which is absolutely necessary for
cutaneous wound healing.48 The differential regulation of the PI3K/Akt pathway upstream of mTOR30
seems to be involved in wound-healing gene expression patterns.49 In addition, PI3K/Akt signaling
is extremely important in preventing keratinocytes
from going into apoptosis.50
The galectins, ie, b-galactoside binding lectins,
are carbohydrate-binding proteins that also play a
key role in wound healing. Cao51 reported that

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specific galectins (3 and 7, but not 1) play a role in

epithelial wound healing of the cornea of galectin-3
e/e mice. Surprisingly, exogenous galectin-3 administration did not lead to improved wound healing
in galectin-3 e/e mice, whereas galectin-7 showed
potential. However, in galectin-3 1/1 mice, galectin-3 was of additional benefit when administered
exogenously. The emerging role of galectin-7 in
epidermal wound healing was studied in a mouse
model with galectin-7 e/e mice.52 Although the skin
structure remained unaltered in galectin-7 e/e mice,
re-epithelialization proved to be significantly slower
than in wild-type controls. As galectin-7 is located in
keratinocyte podosomes, the authors speculated on
a mechanism of impaired cell/ECM interaction
through reduced galectin-7 expression.52 The galectin family may also be a promising wound treatment
target, particularly as the topical application of
galectin-3 or -7 should not be clinically difficult.
Chronic wounds not only occur more frequently
in patients with diabetes mellitus but these wounds
are also more difficult to treat. Diabetes plays a
pivotal role in chronic wound pathogenesis.53 In the
past years, a molecular link between advanced
glycation end products (AGEs) and microvascular
as well as macrovascular complications in diabetes
has been established. Glycated proteins (AGEs) act
through receptors of AGEs (RAGEs).54 The pathways
activated by AGEs (RAGEs) include NF-kB-mediated
inflammation, ERK- and PI3K/Akt-signaling. A review of these effects has been published recently,54
and the link between carbohydrates and epithelial
repair has been studied in great detail.55 The fact that
AGEs also modify dermal fibroblast proliferation56
underlines the clinical observation of extremely
difficult wound care in patients with diabetes.
These pathways are integrated via the highly conserved mTOR30 and represent important signaling
cascades in wound healing and pathogenesis.
Changes induced by diabetes also comprise altered
migration, proliferation, and differentiation patterns
of keratinocytes in patients with chronic ulcers.57
A deficiency of leptin seems to be co-causative for
diabetes and obesity. Adipocytes secrete leptin,
which is the product of the obese (ob) gene and an
important regulatory feedback signal for energy
homeostasis. The impact of leptin on wound healing
was proven by Frank et al,58 who reported improved
wound healing in leptin-deficient mice after topical
and systemic leptin administration. Wild-type mice
also showed improved wound healing after leptin
administration. By studying the direct impact of
leptin, Frank et al58 were able to show that the
classic model of ob/ob-mice for impaired re-epithelialization can not be explained simply by the mild

diabetic phenotype but rather by the lack of a direct

mitogenic effect of leptin on hyperproliferative
wound-edge keratinocytes via leptin receptor subtype obese receptor b (ObRb). People with diabetes
and obesity may benefit from future leptin treatment
strategies in wound therapy.
A cytokine with multiple roles in acute and
chronic wound healing is hepatocyte growth factor
(HGF), which interacts with c-Met (mesenchymal
epithelial transition factor is HGF receptor).59
HGF/scatter factor is a paracrine cellular growth,
motility and a morphogenic factor. HGF/scatter factor is secreted by mesenchymal cells, targeting and
acting primarily on epithelial cells and endothelial
cells. The question of whether HGF is differently
expressed in acute compared with chronic wounds
has been controversially discussed in recent years. In
2007, Conway et al60 found that HGF was significantly higher in chronic ulcers than in acute wounds.
This finding confirmed the results by Nayeri et al,61
who studied HGF in chronic wounds. In addition,
Conway et al60 showed that HGF expression differs
spatially in chronic wounds, being up-regulated at
the wound edge and down-regulated in the surrounding normal-appearing skin with levels decreasing with increasing distance from the wound. In
contrast, in acute wounds, HGF expression was
down-regulated at the wound edge and up-regulated with increasing distance from the wound.
Furthermore, Conway et al60 showed that c-Met
(mesenchymal epithelial transition factor, an HGF
receptor) is nearly undetectable in wound edge
keratinocytes of chronic wounds, whereas, in acute
wounds, c-Met expression is significantly higher than
in normal-appearing skin. These results are in accordance with animal models using c-Met-deficient
keratinocytes, which are unable to contribute to the
re-epithelialization process, indicating that c-Met is
essential for physiologic wound healing.59
In 2003, Heilborn et al62 found the antimicrobial
peptide LL-37 to be lacking in chronic wound
epithelia. The administration of LL-37 antibodies
led to reduced Ki67 levels in the epithelium, reflecting a reduced proliferation rate and a subsequent
deceleration of wound healing. LL-37 peaked after
48 hours postinjury and returned to baseline levels
until wound closure. As bacterial colonization is a
major issue in chronic ulcers, the effects of antimicrobial peptides, such as LL-37, are interesting. One
of the recently discovered pathways by which LL-37
acts is kallikrein-mediated proteolysis.63 This is of
great importance as the balance of proteolytic activity controls innate immune responses at epithelial
surfaces. Against this background, LL-37 may be one
of the most promising targets for future treatment

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regimens in chronic wound healing. It could be of

major relevance that antimicrobial peptides affect
proliferation and migration of keratinocytes as well
as their cytokine and chemokine production.16

REMODELING PHASE
The tissue remodeling phase (Fig 3) starts as early
as a few days after injury and lasts up to 2 years. In
this phase, a variety of proteinases contribute to
coordinated wound healing, which are regulated by
time-dependent and spatial modification of expression patterns. Apart from this regulation, almost all
proteinases are altered in their activity and conformation by the wound milieu itself, for instance by pH
changes caused by wound healing64 as compared
with physiologic conditions.65
An important group of proteinases are MMPs
which are known to be precisely regulated by the
pH value.64,66 They play a central role in wound
healing as they degrade certain constitutes of provisional wound tissue, such as collagen I, III, IV, and
VII.67 Once the provisional wound tissue has been
removed, the presence of TIMPs is crucial, as otherwise the continuing degradation would counteract
tissue formation and subsequently wound closure.
Wound healing is influenced by both the ratio of
certain MMPs and TIMPs (eg, MMP1/TIMP ratio in
diabetic foot ulcers)68,69 and the ratio of MMPs to
each other (eg, high levels of MMP1 are needed for
wound healing, whereas excessively high levels of
MMP8 and MMP9 impair wound healing).70 Potent
MMP inhibitors and synthetic MMPs are available.
MMP2 (gelatinase 2) has turned out to be important for angiogenesis, inflammation, and fibrosis in
wound healing. Jansen et al71 were able to identify
the essential role of the enhancer element response
element (RE)-1 in skin injuryeinduced MMP2 expression in a MMP2/LacZ reporter mouse model.
RE-1 is known to regulate most of the constitutive
MMP2 promoter activity. Jansen et al71 showed that
RE-1 is an important cis-regulatory element in dermal
wound healing. Modulation of MMP2 expression
may be a way to reorganize cell-cell interactions in
skin wounds. Injuries are known to regulate MMP
expression by distinct pathways, for example, MMP2
by the enhancer element RE-1.71
A connection between MMPs and mTOR has been
investigated.72 Although mTOR up-regulates MMP1
and MMP3 expression after ultraviolet Beinitiated
DNA damage, it does not affect IL-1b-mediated
MMP1 and MMP3 production by fibroblasts.
Ultraviolet B radiation was suggested as a therapeutic option to activate MMPs in wound healing.
However, in chronic wounds, excessively high levels
of certain MMPs such as MMP8 and MMP9 exist, and

factors such as a1-antichymotrypsin appear capable

of regulating MMP9 function in skin wound healing.70,73 Again, the link between inflammation (IL1b) and tissue remodeling (MMPs) becomes evident
and shows that wound healing phases are not strictly
chronological but rather interwoven. A newly found
member of the MMP family is MMP28, also known as
epilysin. In 2008, Illman et al74 reviewed the role of
epilysin, which is known to be overexpressed in
basal keratinocytes after cutaneous injury. Epilysin
not only plays an important role in wound cell
migration but also in tumor cell invasion.
In recent years, a few other biomarkers for
chronic wound pathogenesis have been characterized. Low serum levels of Factor XIII, for instance,
are known to be one factor contributing to delayed
chronic venous ulcer healing. FXIIIa can bind to ECM
proteins and thereby contribute to wound healing.
Gemmati et al75,76 studied a gene polymorphism in
the FXIIIa subunit V34L in 91 patients with chronic
venous ulcer versus 195 healthy control subjects.
FXIII V34L carriers are known to exhibit reduced
a2-antiplasmin incorporation into fibrin. Gemmati
et al75,76 speculated on a mechanism involving plasmin activation of pro-MMPs by direct plasminantiplasmin interaction. The major observation was
significantly reduced ulcer size in patients with
increasing numbers of polymorphic FXIII L34 alleles,
which was independent of total FXIII activity. A
higher number of FXIII L34 alleles seemed correlated
to a decrease in the higher fibrinolytic activity seen in
patients with chronic venous ulcers.75,76
Cell-ECM interactions are of major relevance for
wound healing. Integrins are cell surface receptors
that interact with the ECM and mediate various
intracellular signals. Factors such as integrins need
to interact with cell membraneebound receptors for
efficient cell-ECM interactions. One newly studied
co-receptor interacting with b1-integrins and growth
factor tyrosine kinase receptors is syndecan-4. This
heparan sulfate proteoglycan crosses the cell membrane and is up-regulated in response to cutaneous
injury. Echtermeyer et al77 demonstrated its importance by using mice that were heterozygous or
homozygous for mutated syndecan-4 genes. Both
types exhibited delayed wound healing as compared
with wild-type controls but were otherwise
indistinguishable.

NEW AND FUTURE THERAPEUTIC

APPROACHES
For the evaluation of different therapeutic approaches, a reliable scoring system is indispensable.
The wound bed score78 and the ulcerated leg severity assessment score79 have been established to

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evaluate the stages and progress of wound healing in

clinical practice. Not only clinical scoring systems are
used for wound evaluation; more sophisticated
methods are available. Optical wound measurement
is in vogue and promising for certain questions
arising in ulcer treatment plans.80,81
One recently introduced, promising technique is
the application of biodegradable polymers that
release certain growth factors such as FGF-2 in a
pH-dependent manner.82 pH dependency is very
important as pH values change during wound repair,
and enzyme activity may fluctuate as pH changes.1
To make things even more complex in this special
case, basic FGF is triggered by histamine (a mediator
also released upon cutaneous injury), leading to
accelerated wound closure.83
In addition, pH changes affect bacterial colonization84,85 and function, such as causing structural
changes of the enterotoxin C2 of staphylococci.86
In addition, raised pH values in chronic wounds
facilitate infections by Candida albicans.87 A major
fact highlighting the importance of pH on cells is that
even protein expression patterns and transcriptional
processes are altered by extracellular protons, for
instance musculoaponeurotic fibrosarcoma oncogene homolog G-2 (an important transcription factor) expression may be induced by lower pH values
(higher proton concentration).88 Surprisingly, specific pH-dependent signaling pathways exist even
though pH seems to be a rather unspecific stimulus at
the first glance. Therefore, modifications in wound
pH could be promising, simple, and inexpensive
strategies in future chronic wound treatments.89
However, further research is indispensable to elucidate the impact of wound pH on the healing progress
and on available treatment options.
Antimicrobial peptides also play a pivotal role in
preventing bacterial colonization.15 They may therefore be a promising target for future therapeutics in
wound healing.18,20 It could be of major relevance
that antimicrobial peptides also affect proliferation
and migration of keratinocytes as well as their
cytokine and chemokine production.16 Therefore,
influencing the inflammatory and the re-epithelialization phase by stimulating these peptides, for
instance via vitamin D, seems to be feasible.
Another promising target in wound healing is
mTOR. For instance, increased levels of mTOR and
p70S6 kinase (a downstream target of mTOR)30 are
present in keloids.90 Therefore, the local inhibition
of mTOR by inhibitors such as rapamycin (sirolimus)
or everolimus may be promising in the prevention of
hypertrophic scarring and keloids. On the other
hand, systemic inhibition of mTOR (eg, in posttransplantation immunosuppression) leads to impaired

wound healing, which necessitates the assessment of

mTOR activity before treatment. Another problem is
that these inhibitors are not yet available in a topical
formulation.
The endoplasmic chaperone protein calreticulin
induces proliferation of keratinocytes, fibroblasts,
and endothelial cells in vitro affecting all stages of
wound healing.91 Nanney et al91 showed enhanced
macrophage recruitment to the wound site (inflammatory phase), improved granulation (proliferative
phase), and re-epithelialization in a porcine excisional skin wound model after topical administration
of calreticulin. They emphasized the potential of
calreticulin in wound treatment with the advantage
that it can be applied topically.
A novel therapeutic approach is the use of ex vivo
gene transfer by allogenic keratinocyte cell suspensions. For example, allogenic keratinocytes transfected with human EGF by pCEP/h EGF (plasmid
chromosomal expression platform/human epidermal growth factor) plasmids were transplanted in a
full-thickness porcine wound model, leading to
higher rates of re-epithelialization.92 This strategy
may be applicable for a variety of different genes
important in wound healing, but there are still too
many unknown factors for using gene transfer in a
clinical setting.
In addition, some approaches rely on the use of
intact stem cells instead of modifying the cells
present in the wound. Of course, this strategy can
not be specifically assigned to any of the phases
discussed above. As fetal wound healing differs from
adult wound healing in many aspects, gene expression in foreskin and fetal skin cells was compared.93
The overall TGF-b expression (predominantly TGFb1) in fetal cells was 6-fold up-regulated. In addition,
bone morphogenic protein (BMP)-2 levels were
about 4-fold higher in fetal cells. However, growth
differentiation factor (GDF)-10 (synonym of bone
morphogenic protein-3B) was down-regulated 12fold in fetal cells. The authors concluded that fetal
cells offer advantages for cell-based wound therapy
compared with foreskin cells.93
A newly discovered field in wound repair is the
network between the nervous system and the immune system. Numerous important mediators exist
in this cross talk, which include neuropeptides and
cytokines released from nerve fibers, immune cells,
and cutaneous cells. A link between wound healing
and the nervous system is clinically apparent as
peripheral neuropathy is reported in 30% to 50% of
patients with diabetes, presenting the most common
and sensitive predictor for foot ulceration.94 One
important player in this context is substance P. In
addition to its role in pain perception, substance P

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acts as an injury-inducible factor early in the wound

healing process and induces CD291 stromal-like cell
mobilization.95 Remarkably, mobilization of such
cells also occurs in uninjured mice, rats, and rabbits
if substance P is intravenously injected.95 Both substance P injection and transfusion of autologously
derived substance Pemobilized CD291 cells from
uninjured rabbits accelerated wound healing in an
alkali burn model.95 Hong et al95 showed that
epithelial engraftment of transfused cells into injured
tissue occurred during wound healing. Moreover,
they showed that substance P can stimulate transmigration, cell proliferation, activation of the extracellular signalerelated kinases (Erk)-1 and -2, and
nuclear translocation of b-catenin in vitro. This
pioneer work highlighted a new function of substance P as a systemically acting messenger of injury
and as a mobilizer of CD291 stromal-like cells in
wound healing.

CONCLUSIONS
The variety of molecular and cellular targets and
signaling cascades in wound healing may start
speculations on future treatment strategies based
on these results. However, additional attention
should be drawn to widely used techniques, even
though evidence-based data are still missing that
may underline the promising results in daily clinical
routine. Prominent examples are negative pressure
wound therapy96,97 or hyperbaric oxygen therapy.98,99 Regulating the activity of cells involved in
wound healing seems to be a hot topic in future
wound therapy as many new and interesting factors
are being discovered and studied in cutaneous
wound healing. A promising new strategy for
chronic wound treatment is tissue engineering for
skin substitutes. However, this procedure involves
the problem that wound healing gene expression
patterns are different in fetal and adult cells.93
Nevertheless, fetal cells seem to be promising as a
few specific gene expression patterns are known to
be essential for scarless wound healing.93,100 These
findings may influence research on bone-marrowederived stem cells for skin repair101 as little is
known about their specific wound gene expression
patterns. New studies also focus on the role of hair
follicle-bound epithelial stem cells in cutaneous
wound healing.102 These cells may give rise to the
development of new follicles, glands, and epidermal
regeneration.
Even though most strategies still have to be
transferred from bench to bedside, many of them
are promising, and the future will show which
strategies will be implemented into clinical routine.

The authors would like to thank Walter Burgdorf for the

precise correction of the manuscript and Yvonne Egle for
the thorough proofreading. The editorial assistance of
Monika Schoell is gratefully acknowledged.
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