64

Basic Science Review: Bioengineering and the diabetic foot ulcer

2012 Volume 5 No 2 Wound Healing Southern Africa
Introduction
Currently, the World Health Organization estimates that worldwide,
more than 346 million people have diabetes.
1
Diabetic foot ulcers
(DFUs) occur in approximately 15% of all patients with diabetes.
2,3

DFUs precede 84% of lower-leg amputations and are the leading
cause of nontraumatic lower limb amputation in developed countries.
Associated costs with diabetes in the USA amount to hundreds of
billions of dollars a year.
4

Bioengineered skin substitutes (BSS) have been used in the last
few decades as a therapeutic tool with which to treat DFUs. It is
designed to replace and interact with the extracellular matrix
(ECM). Presumably, this upregulates growth factors and some
cytokines, thus encouraging wound healing.
5,6
Although the main
pathophysiological deciency in diabetic wound healing relates
to decreased vasculogenesis, current BSS designs lack a direct
vasculogenic stimulatory component.
Natural biomaterials (collagen) are considered to be more
biocompatible with the hosts ECM. Synthetic biomaterials lack
cellular recognition signals, making dynamic reciprocity between
ECM and cells more difcult.
7
Collagen, although mechanically
weak, can be strengthened by altering its cross-linking.
7,8
The
ultimate matrix should be one that promotes intrinsic regeneration
by encouraging cellular incorporation, cellular and extracellular
cross-communication and targeting of cellular abnormalities that
relate to diabetic disease.
9

Pathophysiology
DFUs occur as a result of neuropathy, vasculopathy, excessive pres-
sure and wound infection that is associated with diabetes. Patients
with diabetes have macrovascular disease as well as microvascular
disease, including a reduction of capillary size, thickening of the
basement membrane and arteriolar hyalinosis. This results in
reduced vessel permeability, altered migration of leucocytes and
decreased autoregulation of the vessels.
10,11
Macrophages release
fewer cytokines, particularly the vascular endothelial growth factor
(VEGF).
12
Excessive activation of matrix metalloproteases (MMPs),
such as MMP9, and reduced concentrations of tissue inhibitor of
metalloproteinase-2 can cause excessive degradation of the ECM
and growth factors.
11,13
In addition, these MMPs may generate
antiangiogenic factors.
14,15
These changes collectively result in the
decreased vascularity and angiogenesis that are characteristic of
DFUs.
Normally, hypoxia causes cells to release hypoxia-inducible factor-1
(HIF-1), which in turn results in the release of VEGF by macrophages,
broblasts and keratinocytes.
16,17
VEGF stimulates endothelial
progenitor cells (EPCs) to enter the circulation from the bone marrow
(Figure 1).
1,17,18
The EPCs are attracted to the site of injury by stromal
cell-derived factor-1 (SDF-1 ) to initiate neovasculogenesis. In
diabetes, as in other ischaemia reperfusion pathologies,
19
reactive
oxygen species (ROS) are generated and affect HIF-1 stability.
Phosphorylation in the bone marrow is impaired,
18
limiting EPC
mobilisation from the bone marrow into the circulation. In addition,
decreased SDF-1 limits EPCs being directed to the wound, thus
decreasing angiogenesis and wound healing.
Tissue hypoxia causes the release of HIF-1 , which stimulates the
release of VEGF by fibroblasts, keratinocytes and macrophages.
VEGF activates phosphorylatrion of the endothelial isoform of nitric
oxide synthase in the bone marrow, resulting in increased nitric
Bioengineering and the diabetic foot ulcer
Widgerow AD, MBBCh, FCS(Plast), MMed, FACS
Clinical Professor Surgery (Plastic), Director Laboratory for Tissue Engineering and Regenerative Medicine Aesthetic and Plastic Surgery Institute, University of California, Irvine
Correspondence to: Alan Widgerow, e-mail: awidgerow@adarscience.com
Keywords: bioengineering, diabetic foot ulcer
Abstract
Diabetic disease is increasing exponentially on a global scale. Diabetic foot ulcers (DFUs) are the leading cause of nontraumatic lower limb
amputations. The pathophysiological events need to be considered when designing new interventions. Bioengineered skin substitutes (BSS)
are accepted in the therapeutic armamentarium for DFU treatment. However, newer designs are likely to offer more targeted approaches to
the disease process. This relates to the stimulation of vasculogenesis in particular. This can be achieved by using interactive scaffolds that
stimulate endothelial progenitor cells to increase vascular endothelial growth factor production and reverse some of the damage that is caused
by glycation end-products that are characteristic of diabetes.
Medpharm Wound Healing Southern Africa 2012;5(2):64-67
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Basic Science Review: Bioengineering and the diabetic foot ulcer
2012 Volume 5 No 2 Wound Healing Southern Africa
oxide which stimulates the release of EPCs into the circulation. The
chemokine, SDF-1 , then guides the EPCs to the wounded area,
stimulating vasculogenesis. In diabetes, when ROS are generated,
they affect HIF-1 stability. Phosphorylation in the bone marrow is
impaired and limits EPC mobilisation from the bone marrow into the
circulation. In addition, decreased SDF-1 limits the directioning of
EPCs to wounds, thus decreasing angiogenesis and wound healing.
The intention of bioengineered skin is to change the nature of the
degradative ECM, with decreased MMPs and increased availability of
growth factors, particularly VEGF. However, it is important to recognise
that BSS do not appear to be incorporated into the wound site for any
protracted period of time, and specialised cellular inclusions in BSS
do not appear to survive for very long.
10
Additionally, no disease-
specic targeted approach has been adopted with current BSS.
Current BSS products and standard of care
The three approved BSS products in the USA for use in DFUs are
Dermagraft

(Advanced BioHealing, California, USA), Apligraf

(Organogenesis, Massachusetts, USA) and more recently, Oasis

(Cook Biotech, Indiana, USA).
15,20,21
Dermagraft

includes neonatal
broblasts from human foreskin cultured on a polyglactin scaffold.
It is contraindicated in infected ulcers and used for DFUs of greater
than six weeks duration and with full thickness in depth, but without
tendon, muscle, joint or bone exposure. It must remain stored at
-70C until ready for use.
20
Apligraf

is derived from broblasts

that are cultured in a collagen matrix and used for full-thickness
neuropathic DFUs of greater than three weeks duration, that are
resistant to standard therapy (also without tendon, muscle, capsule or
bone exposure). It is also contraindicated in the case of infection and
its shelf life is 10 days. It is stored at a temperature from 21-30C.
20

Oasis

is porcine-derived small intestinal submucosa that contains

glycosaminoglycans, proteoglycans and bioactive growth factors,
such as broblast growth factor-2 (FGF-2), transforming growth
factor-beta 1 and VEGF.
15,21
The US Food and Drug Administration
has issued a black-box warning about becaplermin [recombinant
human platelet-derived growth factor-BB (PDGF-BB)] because it has
carcinogenic potential. This has limited its use.
20
BSS is not used in isolation to treat diabetic foot ulceration. As with
all wound healing regimens, wound bed preparation is essential.
This may involve restoration of vascular supply, removal of pressure,
control of infection (including biolm) and debridement of the
wound in DFUs. The aim of wound bed preparation is to convert the
molecular and cellular environment of a chronic wound to that of
an acute wound,
1,22,23
and to prepare the appropriate environment
for BSS transplantation.
23
Peripheral ischaemia is one of the
pathological characteristics of DFU and a critical contributing factor
that affects BSS transplantation. Usually, surgical revascularisation
and decompression are carried out to improve ischaemia.
24
Even
with such attempts to achieve healing, a large number of DFUs
progress to a nonhealing status. BSS may then be indicated in an
effort to change this healing trajectory.
Looking at new possibilities
The primary goal in healing diabetic wounds is to increase vascularity.
Inefcient angiogenesis prolongs ulceration and increases the
probability of amputation.
1,11,20
Current therapies do not adequately
target vasculogenesis. Possible interventions should be directed at
the sequence of pathophysiological events that occur in diabetic
patients:
Diabetes is characterised by the formation of advanced glycation
end-products (AGE). These result from the increased methylglyoxal
that is formed in association with hyperglycaemia.
25
Methylglyoxal
detaches the endothelial cells from the basement membrane
so that they become free-oating and senescent, resulting in
eNOS: endothelial isoform of nitric oxide synthase, EPCs: endothelial progenitor cells, HIF: hypoxia-inducible factor, HIF-1 : hypoxia-inducible factor-1 , ROS: reactive oxygen species, SDF-1 : stromal cell-derived
factor-1 , VEGF: vascular endothelial growth factor
Figure 1: Vasculogenesis pathway and its limitation in diabetic patients
Tissue hypoxia (releases HIF)
Hyperglycaemia impairs
HIF-1 stability via ROS
HIF causes VEGF release by
macrophages, broblasts
and keratinocytes in
response to hypoxia
Phosphorylation
activation by eNOS in the
bone marrow
Increased nitric oxide
Mobilises EPCs in the
bone marrow to enter
the circulation
SDF-1 guides EPCs to the site
of the injury (impaired in diabetes)
Vasculogenesis
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Basic Science Review: Bioengineering and the diabetic foot ulcer
2012 Volume 5 No 2 Wound Healing Southern Africa
retinopathy and generalised microangiopathy.
27
Pharmacological
scavenging of methylglyoxal can prevent endothelial cell
detachment and maintain angiogenesis. Thiamine, benfotiamine
and pyridoxamine decrease protein glycation by methylglyoxal.
25
Much of the tissue damage and limited neovascularisation that
accompanies the DFU is initiated by ROS generation.
27,28
As a
protective mechanism, normal EPCs express high levels of the
antioxidant enzyme, manganese superoxide dismutase, which
scavenges mitochondrial ROS and is decreased in diabetes.
27

HIF stability is affected by hyperglycaemia, thus the HIF response
to hypoxia (increased EPCs and VEGF) is diminished.
29,30
Hydroxylase inhibitors, dimethyloxalylglycine and the iron chelator
and antioxidant deferoxamine, have been demonstrated to
stabilise and activate HIF-1.31 This counters the suppression of
VEGF-A and SDF-1 expression.
17

SDF-1 homes mobilised EPCs to the wound. This expression
appears to be decreased in diabetes.
30
The extrinsic addition
of SDF-1 , combined with hyperbaric oxygen, was shown to
substantially promote angiogenesis and the deposition of collagen
in the granulation tissue of the diabetic wound.
30
Lipid-derived molecules have been identied as important
mediators in inammation, wound healing and angiogenesis.
32
The
administration of exogenous lipid molecules to wounds in diabetic
animals was reported to rescue healing and angiogenesis. It was
suggested that it enhances VEGF release, vasculature formation
and the migration of endothelial cells.
32
Additionally, matricellular
proteins, such as osteopontin and syndecans, may improve
cellular and extracellular communication, promoting growth factor
stimulation, neovascularisation and improved granulation.
33-35
Controlled inammation and restored immunity. The initial
acute inammatory phase involves the secretion of cytokines,
chemokines and growth factors from immune cells in normal
wound healing. In diabetes, hyperglycaemia disrupts the activity
of these essential inammatory mediators in wound healing.
36
Thus, the impairment of the natural wound healing process
in diabetes may be attributed to alterations in the interaction
between cytokines and neuropeptides.
36,37
These neuropeptides
include neuropeptide Y and substance P, and the cytokines,
interleukin-6, interleukin-8, tumour necrosis factor-, PDGF, FGF,
VEGF and TGF-.
37
It appears that the chronic inammation that
accompanies DFUs suppresses the focused acute inammatory
response to injury that is needed for normal wound healing
and which results in impaired leukocyte function and aberrant
expression and activity of inammatory chemokines, cytokines
and growth factors, all required for wound healing.
37
Discussion
The intention of current DFU therapy with BSS is to replace the
degraded and destructive milieu of the ECM by introducing a new
ground substance matrix with cellular components aimed at starting
a new healing trajectory. Pure cellular incorporation into BSS may
not contribute very much to the healing process. The introduction of
growth factors does very little, especially if the destructive wound
milieu is not corrected rst. In addition to this, chronic wound uid
has been shown to be particularly corrosive and may contribute
directly to the pathology that is seen in many chronic wounds.
38
Thus more goal-directed, disease-directed BSS are needed with
mechanical and structural design nuances that are tailored to
the wound and disease background. The logic is that BSS should
promote intrinsic regeneration of growth factors, cellular proliferation
and vasculogenesis, rather than extrinsically adding specialised
components that have questionable incorporation or effect on the
underlying molecular processes.
From a structural design standpoint, three aspects are important
in design construct: mechanotension and inherent resistance of
the matrix, porosity within the scaffold bres, and hydration within
the functional scaffold.
9,39
With the establishment of the basic
structural components of BSS, consideration should be given to
possible additive components that can influence the background
disease process. The main goal in diabetes, as described above, is
to re-establish vasculogenesis and to avoid infection. To that end,
the sequence of pathophysiological events has been determined.
This provides an opportunity to incorporate substituents that can
influence this sequence. ROS scavengers,
16,17,27,31
methylglyoxal
inhibitors,
25,26,40
EPC and VEGF stimulators,
32,33,35
and even
neuropeptides,
37
have the capacity to restimulate bone marrow
production of EPCs, redirect them to the area of injury and promote
neovascularisation. Coupled with this, newer antibacterial and
anti-inflammatory advances, e.g. nanocrystalline silver, can be
incorporated to complete the picture.
41
Conclusion
DFUs are a major drain on the economy. They cause tremendous
morbidity and mortality and are likely to increase in occurrence in
the future. Molecular biological advances have allowed identication
of the critical components behind the background pathophysiological
events that surround the evolution and progression of DFUs. The
major background impairment is that of vasculogenesis, brought
about as a direct result of hyperglycaemia, AGE and methylglyoxal
generation and its direct effect on EPC production, and homing to
the wound site. It is time to adopt a specic target-focused approach
with a well-structured matrix that incorporates strategic elements
to counter the specic disease process. In this manner, intrinsic
healing with balanced growth factors and cellular proliferation
is encouraged, rather than current crude attempts to add varying
quantities of specialised cellular and growth factor components to
the wound interface.
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