The International Journal of Lower Extremity

Wounds
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Altered Molecular Mechanisms of Diabetic Foot Ulcers

Robert Blakytny and Edward B. Jude
International Journal of Lower Extremity Wounds 2009 8: 95
DOI: 10.1177/1534734609337151

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Altered Molecular Mechanisms
of Diabetic Foot Ulcers
Robert Blakytny, MA, DPhil, and Edward B. Jude, MD, MRCP
The continuously increasing worldwide prevalence of
diabetes will be accompanied by a greater incidence of
diabetic foot ulcer, a complication in which many of the
morphological processes involved in normal wound
healing are disrupted. The highly complex and inte-
grated process of wound healing is regulated by a large
array of molecular factors. These often have overlap-
ping functions, ensuring a certain degree of tolerance
through redundancy. In diabetes, changes to the expres-
sion of a large number of molecular factors have been
observed, overwhelming this inbuilt redundancy. This
results in delayed healing or incomplete healing as in
ulceration. Understanding the relationship between
T
he worldwide pandemic of obesity, including in
children, is being accompanied by an increasing
prevalence of type 2 diabetes in adults, together
with its appearance in children.1-3 Furthermore, type
1 diabetes is also becoming more common.4
Consequently, the occurrence of diabetic foot ulcer
(DFU) will become ever more common. This debilitat-
ing condition greatly affects the quality of life of
patients, as well as impacting on their carers, resulting
in greater levels of amputation in patients, in turn
leading to increased mortality.5-9 The management of
DFU and its consequences puts a great strain on both
health and social services, with huge related costs for
treatment, as well as causing the loss of general eco-
nomical productivity.7,10,11
Normal wound healing involves an overlapping
series of events, with clot formation, inflammation,
From the Institute of Orthopaedic Research and Biomechanics,
University of Ulm, Germany (RB) and Diabetes Centre,
Tameside Hospital NHS Foundation Trust, Ashton-under-Lyne,
United Kingdom (EBJ).
Conflict of interest: none.
Address correspondence to: Edward B. Jude, MD, Tameside
General Hospital, Fountain Street, Ashton-under-Lyne, Lancs
OL6 9RW, UK; e-mail: edward.jude@tgh.nhs.uk.
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The International Journal of
Lower Extremity Wounds
Volume 8 Number 2
June 2009 95-104
© 2009 Sage Publications
10.1177/1534734609337151
http://ijlew.sagepub.com
hosted at
http://online.sagepub.com
altered levels of molecular factors and the inhibited
healing process in such ulcers will permit the develop-
ment of targeted treatments aimed to greatly improve
the quality of life of patients, at the same time helping
to reduce the huge costs associated with treating this
diabetic condition and its long-term consequences.
This short review examines how changes in the expres-
sion of molecular factors are related to altered mor-
phology in diabetic foot ulceration and very briefly
considers treatment strategies at molecular level.
Keywords:  angiogenesis; chronic wound; diabetes;
extracellular matrix; growth factors; nerve regeneration
formation of granulation tissue and a new blood
supply (angiogenesis), re-epithelialization leading to
wound closure, nerve regeneration, and laying down
of extracellular matrix (ECM) as scar tissue that will
eventually be remodeled (see Figure 1).12 All these
morphological events are compromised in DFU,
hindering the normal healing process. Altered expres-
sion of many molecular factors has been found in
DFU, including growth factors and their receptors,
neuropeptides, and proteolytic enzymes as presented
in tabular form in Table 1. Most of these will be detri-
mental to healing although some, such as the increase
in granulocyte macrophage–colony-stimulating factor
(GM-CSF) and the interleukin-8 (IL-8) receptor
CXCR1,13 might be compensatory. Many of these
molecular factors often have multiple and overlap-
ping functions in normal wound healing, resulting in
redundancy and creating a complex set of interac-
tions.14,15 However, the degree of changes in diabetic
wound healing can become overwhelming, leading
to DFU formation. Understanding how altered
expression of such molecular factors lead to modi-
fied cellular function and thus ulcer formation and
maintenance will provide an insight into potential
points for intervention, not just in terms of which
factors but also the relevant timing for optimum
95
96   The International Journal of Lower Extremity Wounds / Vol. 8, No. 2, June 2009
Inflammation Angiogenesis
Redox ECM
Wound Healing
Balance Formation
Nerve Re-epithelialisation
Regeneration
Figure 1.  Morphological contributions to wound healing that
are altered in diabetic foot ulcer (DFU).
effect. It is also important for completeness to state
that although altered levels of tumor necrosis factor-α
(TNF-α) have been associated with the Charcot foot,
there is a paucity of literature relating to this factor
with respect to DFU. Although it could be argued
that there may be a change involved with DFU, this
would be based on an assumption and hence TNF-α
is not included in this review.
This review considers how the morphological
changes associated with DFU could arise through
the altered expression of molecular factors reported
in the literature for DFU. We will also briefly con-
sider treatment strategies at the molecular level. We
searched PubMed, cross-referencing the subjects
discussed to diabetes and ulceration and followed up
on quoted references in the English language. There
is a vast quantity of literature related to this subject
in languages other than English; it has not been pos-
sible to consider all databases-related literature.
Inflammation
Extension of the inflammatory phase is associated
with DFU, with altered numbers of several inflam-
matory cell types reported (Figure 2).16-18 Up-regulated
and continued presence of such activated inflamma-
tory cells will delay the switch from inflammation to
proliferation of skin-forming cells. High levels of
transforming growth factor-β3 (TGF-β3) within the
diabetic epidermis, particularly at the ulcer edge
could inhibit the elevation of TGF-β1 found in nor-
mal wound healing, resulting in the lack of TGF-β1
produced in the basal layer and in fibroblasts
observed in DFU.19 As TGF-β1 can downregulate
macrophage activity,20 its diminished levels in DFU
will result in increased activity of these cells. This
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Table 1.  Altered Expression of Molecular Factors
in Diabetic Foot Ulcer
Chronic Wound
in Diabetic Patient
Growth factors and receptors
  IGF-1 ↓
  IGF-2 ↑
  TGF-β1 ↓
  TGF-β2 ↑
  TGF-β3 ↑
  TGF-βR1 and R2 ↓
  PDGF ↓
  PDGF-R ↓
  EGF ↑/↓ (cell type)
  GM-CSF ↑
  MCP-1 ↑
  IL-8 ↓
  CXCR1 ↑
  IL-10 ↓
  IL-10R ↓
  IL-15 ↓
  NGF ↓
  trkA ↓
  NT-3 ↓
  trkC ↓
Enzyme activity
  NOS ↑
  Neutrophil elastase ↑
  Cathepsin G ↑
  MMPs ↑
  TIMP concentration ↓
  NEP ↑
  Arginase ↑
Other factors
  HIF-1α ↓
  Substance P ↓
  CGRP ↓
  K2 ↓
  K6 ↓
  K10 ↓
  LM-3A32 ↓
  GSH ↓
  Cysteine ↓
Abbreviations: IGF, insulin-like growth factor; TGF, transform-
ing growth factor; PDGF, platelet-derived growth factor; EGF,
epidermal growth factor; GM-CSF: granulocyte macrophage–
colony-stimulating factor; MCP-1, macrophage chemoattrac-
tant protein-1; IL, interleukin; NGF, nerve growth factor; trk,
tyrosine kinase receptor; NT-3, neurotrophin-3; NOS, nitric
oxide synthase; MMP, matrix metalloproteinase; TIMP, tissue
inhibitor of metalloproteinase; NEP, neutral endopeptidase;
HIF, hypoxia-inducible factor; CGRP, calcitonin gene–related
peptide; K, keratin; LM: laminin; GSH: reduced glutathione.
will be exacerbated by elevated presence of mac-
rophage chemoattractant protein-1 (MCP-1) in DFU,13
which would attract greater numbers of macrophages.

↑TGF-β3
↓TGF-β1
↓Activated T-cell Apoptosis ↑ Macrophage activity
↓ IL-10 Extended Inflammation
↑ Macrophage & Leukocyte Infiltration
↑ MCP-1
Figure 2.  Extended inflammatory phase.
Additionally MCP-1 would lead to increased leuko-
cyte infiltration of the wound.21 Greater activity of
inflammatory cells in addition to high levels of glu-
cose at the ulcer will generate more reactive oxygen
species (ROS),22,23 the many cell disrupting actions
of which are discussed below. The anti-inflammatory
cytokine IL-10 is down regulated in DFU,13 thus being
less effective in stimulating an end to the inflamma-
tory phase.
Angiogenesis
Wound healing requires a good blood supply to deliver
sufficient oxygen and nutrients as well as allowing
infiltration of the wound site by cells and molecular
factors required to stimulate the healing process.24
The extension of blood vessels into the wound occurs
through proliferation and chemotaxis of endothelial
cells from preexisting capillaries is termed angiogen-
esis. Disruption of this process occurs in DFU.25,26
Changes to many molecular factors associated with
DFU could contribute to inhibition of angiogenesis
(Figure 3), resulting in anaerobic conditions and a
poor supply of nutrients.
High glucose and related greater glycation, poten-
tially play a direct role in inhibiting angiogenesis.27
Clot formation and sealing of damaged blood vessels
on wounding to stop blood loss results in initially
hypoxic conditions. Such conditions induce upregu-
lation of the expression of the transcription factor
hypoxia-inducible factor-1α (HIF-1α). This combines
with constitutively expressed HIF-1β to form the
HIF complex. The complex acts in the nucleus to
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Molecular Function in Diabetic Ulcers / Blakytny, Jude   97
↓ TGF-β1 ↑ iNOS
↑ NO
↓ HIF-1α
↓ Substance P ↓ Angiogenesis ↑ Glycation
↓ CGRP
↑ ROS
↓ IL-8 ↑ MMPs
↓ IGF-1 ↑ Neutrophil Elastase
↓ PDGF ↑ Cathepsin G
↓ EGF
↓ NGF
Figure 3.  Altered angiogenesis.
stimulate expression of various molecular factors,
including ones that induce angiogenesis, such as vas-
cular endothelial growth factor (VEGF).28,29 Increased
local osmotic conditions due to high glucose will
diminish expression of HIF-1α as seen for fibro-
blasts in DFU.30 This lack of HIF-1α response in
DFU will thus contribute to the poor blood supply.
Although the free radical nitric oxide (NO) at lower
concentrations can stimulate angiogenesis, exces-
sive amounts become inhibitory.31 Increased expres-
sion of 2 isoforms of the enzyme that synthesize NO,
nitric oxide synthase (NOS), have been found in
DFU (endothelial constitutive and inducible, ie,
ecNOS and iNOS),32 which may lead to greater NO
in the wound, with elevated plasma NO associated
with DFU.33 The growth factor TGF-β1 downregu-
lates expression of iNOS,34 such that lack of upregu-
lation of this factor in DFU19 will at least in part
contribute to the elevated appearance of iNOS. In
addition to TGF-β1, a number of other growth factors
that are able to enhance angiogenesis through stimu-
lation of endothelial cell proliferation/differentiation
and/or migration, including platelet-derived growth
factor (PDGF), insulin-like growth factor-1 (IGF-1),
epidermal growth factor (EGF), nerve growth factor
(NGF), and IL-8 similarly display diminished expres-
sion in DFU,13,35-42 thus contributing to the poor
blood supply (Figure 3). Decreased levels of the
angiogenesis-stimulating neuropeptides, substance P,
and calcitonin gene–related peptide (CGRP), could also
contribute to diminished angiogenesis though poor
innervation of the DFU41 (as discussed in a following
or later section). For blood vessels to penetrate the
98   The International Journal of Lower Extremity Wounds / Vol. 8, No. 2, June 2009
↓ PDGF ↓ TGFβ-1 & IGF-1
↓ Fibronectin ↓ Collagen ↓ GAG
↑ Neutrophil
Elastase
↓ Extracellular Matrix
Cathepsin G
↓ IGF-1 & IL-10 ↑ MMPs ↓ TIMP
↑ EGF & MCP-1 ↓ TGFβ-1 ↓ PDGF
Figure 4.  Extracellular matrix (ECM).
wound site, sufficient ECM needs to be present to act
as a scaffold and physically support extension of the
blood capillaries. The ulcer environment is highly
proteolytic, which will be detrimental to matrix regen-
eration and subsequently angiogenesis.
Formation of Extracellular Matrix
Rapid formation and deposition of ECM, in particu-
lar by fibroblasts, is required for efficient cellular
binding at the wound. The ECM provides a scaffold
to which cells involved in the healing process can
attach too. It also lends physical support for the regen-
eration and expansion of the blood vessels in angio-
genesis. Additionally, it allows chemotaxis of epithelial
cells, especially of the basal layer of the epidermis,
along the wound bed, thus sealing the wound through
re-epithelialization. Eventually, the size of the wound
is diminished by contraction of the ECM through the
activity of mesenchymal cells.
Poor ECM formation is associated with DFU and
can arise through both diminished synthesis and an
increased rate of removal through elevated activity of
proteolytic enzymes (Figure 4). The growth factors
TGF-β1, IGF-I, and PDGF all stimulate the synthesis
of ECM components, including collagen and gly-
cosaminoglycan (GAG) by fibroblasts.43-47 All these fac-
tors are downregulated in DFU,13,19,38,39 leading to poor
ECM formation. Compromised fibroblast function in
DFU has been reported, such cells displaying reduced
proliferative ability and response to growth factors,
together with altered morphology.48,49 Interestingly,
treatment of DFU with in vitro expanded autologous
fibroblasts induced healing.50
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The DFU environment has long been known to
be highly proteolytic, with fluid from such wounds
able to degrade fibronectin.51 This fluid displayed
increased activity of both neutrophil elastase and
cathepsin G,52 with elevated levels of the elastase
enzyme indicated in DFU,18 causing breakdown of
ECM (Figure 4). The matrix metalloproteinases
& (MMPs) are a family of zinc-dependent endopepti-
dases that are necessary for maintaining ECM integ-
rity, as well as removing damaged ECM and later
remodeling during wound healing.53,54 They also play
a role in angiogenesis, both through and indepen-
dent of their direct action on ECM.55 The MMPs
display varying substrate specificities, often with
some degree of overlap, including of different col-
lagen types, together with gelatin, elastin, fibronec-
tin, and proteoglycans. Thus, as a group, the MMPs
can degrade the structural components of the ECM.
As excessive activity of MMPs would be detrimental
to maintaining ECM, their activity is normally
tightly regulated by various factors, amongst these
being their inhibitors, the tissue inhibitors of metal-
loproteinases (TIMPs).56 Increased expression and/
or activity in DFU have been shown of various
MMPs, together with the down regulation of
TIMPs,52,57-60 which will result in excessive removal
of ECM, inhibiting cellular function and angiogen-
esis. Several growth factors able to regulate the
expression of MMPs and/or TIMPs, including TGF-β1,
IGF-1, PDGF, IL-10, EGF, and MCP-1, display
changed expression in DFU.13,19,38,39,42,61-65 Such
growth factor alteration would contribute to elevated
MMP activity and downregulation of TIMPs in DFU
(Figure 4).
As well as inducing loss of the ECM, the highly
proteolytic environment of the DFU is able to degrade
growth factors,52 possibly contributing to the low
levels of such molecules in the DFU. This may in
part explain why early successful studies with recom-
binant growth factors for the treatment of acute
wounds in diabetic animals often did not translate to
trials in patients with DFU where the wound envi-
ronment is much harsher.66,67
Re-epithelialization
Closing of the wound by re-epithelialization presents
a barrier to the external environment, keeping out
foreign bodies that lead to infection, as well as pre-
venting the wound drying out. In DFU, this process
is not completed, leaving an open wound. Restoration

↑ MMPs
↓ K2
↓ K6
↓ TIMP Cell Activity ↓ K10 ↓ LM-3A32
Inhibited Re-Epithelialisation
↓ IL-8 ↓ TGF-β1 ↓ NGF ↓ Substance P
↓ IL-10 ↓ IGF-1 ↓ NT-3 ↓ CGRP
↓ IL-15 ↓ PDGF
↑ TGF-β3
Figure 5.  Inhibited re-epithelialization.
of the epithelium involves keratinocyte prolifera-
tion, differentiation and migration. These cellular
activities are regulated by a large array of molecu-
lar factors. Growth factors, including cytokines
and nerve growth factors, along with neuropep-
tides (TGFβ1, IGF-1, PDGF, IL-8, IL-10, IL-15,
NGF, neurotrophin-3 [NT-3], substance P, and
CGRP) that stimulate epithelial cell activity are
downregulated in DFU (Figure 5).13,15,19,39,41,42,63,68-71
In contrast, TGFβ3, which inhibits the migration of
dermal cells, is upregulated in DFU.19,72 As well as
inhibiting MMPs, TIMPs also stimulate growth of
epidermal cells.56 However, when a TIMP molecule
combines with an MMP molecule it loses this stim-
ulatory effect. Thus, low levels of TIMP in DFU,
exacerbated by greater amounts of MMPs,52,57-60 will
contribute to inhibited re-epithelialization. The
expression of the cytoskeletal keratins K2, K6, and
K10 by keratinocytes that are associated with the
differentiation of these cells is diminished in DFU.73,74
This indicates that the differentiation of keratino-
cytes, which is inhibited in DFU, is necessary for
migration of these cells and subsequent wound clo-
sure. Reduction of the expression of LM-3A32 by
keratinocytes was observed in DFU.74 LM-3A32 is
the uncleaved precursor of the α3 chain of various
laminin isoforms, including laminin-5. This laminin
isoform regulates binding of epithelial cells to the
basement membrane together with the motility of
these cells via integrins. Disruption of the LAMA3
gene results in reduced keratinocyte survival and
differentiation,75 such that loss of the protein itself
would inhibit re-epithelialization.
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Molecular Function in Diabetic Ulcers / Blakytny, Jude   99
↓ IGF-1
↓ PDGF
↓ NGF ↑ NEP
↓ Neuroinflammatory
↓ SP Signalling
↓ Nerves &
↓ CGRP
Cellular
Stimulation
↓ PDGF
↓ TGF-β1
↓ NT-3
Figure 6.  Inhibited nerve regeneration.
Nerve Regeneration
Sensory nerves within the skin are in contact with
dermal and epidermal cells, and can regulate local
blood flow and stimulate wound healing through
their release of neuropeptides such as substance P
and CGRP that display a range of healing-associated
properties (Figure 6).71 Loss of nerve function in
diabetes and thus sensitivity to injury contributes to
DFU formation. Diminished nerve content was
found in DFU, with specific markers indicating a
lack of their regeneration in the epidermis whereas
in the dermis reformation occurred but without
achieving maturity.41,76 Consequently, staining for
substance P and CGRP-related nerves is generally
lacking in DFU in both these skin layers,41 contrib-
uting to poor angiogenesis and re-epithelialization
(Figures 3 and 5). Various growth factors know to
have neurotrohpic properties, including the neu-
rotrophins NGF and NT-3 together with TGF-β1,
IGF-1, and PDGF,77,78 are all downregulated in
DFU,13,19,38,39 and may all contribute to poor nerve
regeneration and function, as well as reduced pro-
duction of neuropeptides.
Even low levels of neuropeptides that can still be
produced will be susceptible to removal by elevated
activity of neutral endopeptidase (NEP) in DFU, a
neuropeptide-specific peptidase, potentially directly
through high glucose levels.79,80 As CGRP inhibits
degradation of substance P by NEP,81 diminished
expression of CGRP in DFU41 will leave substance P
even more vulnerable to increased NEP activity in
DFU.
100   The International Journal of Lower Extremity Wounds / Vol. 8, No. 2, June 2009

↑ iNOS ↑ Glucose ↑ Glycation Anti-Protease

↑ NO

Viral Vectors NOS Inhibition

↓ L-Arginine ↑ ROS ↓ GSH & Cysteine

DFU Treatment

↑ Arginase ↑ Direct damage: ↑ MMPs

DNA
Protein Platelet Grafts
Lipid

Growth Factors
Figure 7.  Formation of reactive oxygen species (ROS) and
consequences.
Figure 8.  Approaches to diabetic foot ulcer (DFU) treatment
at the molecular level.
Reactive Oxygen Species
Many recombinant growth factors have been inves-
Increased oxidative stress in diabetes, including
tigated.66,67,88 Although earlier studies of these fac-
through the formation of ROS, plays a major role in
tors in diabetic animal models with acute wounds
diabetic pathogenesis, including DFU formation
often resulted in accelerated healing, an improve-
(Figure 7).22,23 Elevated levels of such free radicals
ment in DFU in subsequent trials with patients did
will lead to poor wound healing.82 These molecules
not automatically follow. The highly complex nature
damage proteins, lipids, and DNA (Figure 7), caus-
of DFU formation and the changes in expression of
ing disruption to cellular function and even leading
many growth factors may have contributed to this
to cell death. ROS can affect platelet aggregation
diminished effectiveness of growth factor treatment.
and the release of growth factor, as well as inducing
The response elicited by growth factors still expressed
increased expression of MMPs that will degrade
will further be decreased by the diminished presence
ECM (Figures 4 and 7). Free radicals possess the
of various receptors. Amongst these are TGF-βR1
property to directly degrade ECM components.83,84
and TGF-βR2, PDGF-R, IL-10R, tyrosine kinase
ROS can arise directly from high glucose and
receptor A (trkA: for NGF), and trkC (for NT-3);
increased advanced glycation end products (AGEs)22
(Table 1).13,19,41 Only PDGF has been given approval
associated with DFU. Increased levels and thus activi-
for the treatment of human DFU, with recombinant
ties of both iNOS and arginase in DFU32 will increase
protein being expensive and large amounts neces-
their competition for their mutual substrate the amino
sary. As the wound fluid of DFU is highly proteolytic,
acid l-arginine. This results in NOS synthesizing ROS,
even being able to degrade growth factors, this
including superoxide.85 The increased NO produced by
might contribute to the need for such large amounts
elevated NOS activity can react with superoxide to gen-
of recombinant growth factor. Using a protease
erate the even more toxic free radicals OH• and NO2,
inhibitor in combination may be one way to improve
the free radicle spot’.86 Apart from generating greater
the chance of successful intervention with a growth
amounts of ROS in diabetes, the ability to remove them
factor. To provide more sustained levels viral expres-
is compromised in DFU. The universal reducing agent
sion vectors are being developed that contain the
reduced glutathione (GSH) together with the reducing
sequence for a given growth factor that will be syn-
amino acid cysteine are diminished in DFU.87 In addi-
thesized in vivo at the DFU.89
tion to its direct action, GSH acts as a cosubstrate for
The fact that many growth factors are altered in
the reducing enzyme glutathione reductase.
DFU means a multiple treatment approach may be
warranted. Indeed, treatment with platelets and
Treatment material derived from this cell, which produces mul-
tiple factors involved in wound healing, has been
Various approaches at the molecular level have been promising.88 The timing of the appearance of molec-
developed for the treatment of DFU (Figure 8). ular factors in wound healing is also crucial. To

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 Molecular Function in Diabetic Ulcers / Blakytny, Jude   101
change the level at the appropriate time a response
mechanism to the local environment of the wound is
necessary, which will include cellular activity. Artificial
skin grafts that contain cells provide a long-term source
of molecular factors, with the cells themselves able to
respond to the changing environment within the
wound. The graft itself provides a scaffold onto which
cells from the patients themselves can bind when
ECM is lacking in the DFU. Many such products have
now been developed.66,88 Mesenchymal cells are of
interest as they have the potential to develop into
different cell types. Such cells would differentiate to
the appropriate cell type during the time course of
healing, and consequently produce relevant molecular
factors as needed. The value of these cells in DFU is
being studied in current projects.90
Discussion
It is clear that despite the degree of overlap in the
function of molecular factors involved in wound
healing, the many changes associated with DFU will
eventually overwhelm the inbuilt redundancy of the
normal healing process. When considering molecu-
lar changes in DFU, these should not be seen in
isolation but rather be considered how they fit into
the greater picture. This will allow a better under-
standing of the whole process, and for targeted and
comprehensive treatment. With the various morpho-
logical processes of wound healing often being inter-
linked through the action of the same molecular
factors, with many of these being changed, interven-
tion to stimulate healing in DFU will most likely
need a multiple approach. With a continually increas-
ing prevalence of both forms of diabetes1,4 and sub-
sequent increased incidence of DFU, a coordinated
approach to the treatment of DFU will improve the
quality of life for patients. Additionally, this will help
to make substantial savings to the astronomical
costs faced by health and social services associated
with treating DFU and the long-term consequences
of this chronic condition.
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