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Basic Science Review: Bioengineering and the diabetic foot ulcer

2012 Volume 5 No 2Wound 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 deficiency 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 host’s ECM. Synthetic biomaterials lack

cellular recognition signals, making dynamic reciprocity between

ECM and cells more difficult.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

PathophysiologyDFUs 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,

fibroblasts 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 α (SDF-1 α) 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 α limits EPCs being directed to the wound, thus

decreasing angiogenesis and wound healing.

Tissue hypoxia causes the release of HIF-1 α, 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: [email protected]

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.

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oxide which stimulates the release of EPCs into the circulation. The

chemokine, SDF-1 α, 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 α limits the directioning of

EPCs to wounds, thus decreasing angiogenesis and wound healing.

The intention of bioengineered skin is to change the nature of thedegradative 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-

specific 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

fibroblasts 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

-70°C until ready for use.20 Apligraf® is derived from fibroblasts

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 21-30°C.20

Oasis® is porcine-derived small intestinal submucosa that contains

glycosaminoglycans, proteoglycans and bioactive growth factors,

such as fibroblast 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 biofilm) 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.

Inefficient 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-floating and senescent, resulting in

eNOS: endothelial isoform of nitric oxide synthase, EPCs: endothelial progenitor cells, HIF: hypoxia-inducible factor, HIF-1 α: hypoxia-inducible factor-1 α , ROS: reactive oxygen species, SDF-1 α: stromal cell-derived

factor-1 α, VEGF: vascular endothelial growth factor

Figure 1: Vasculogenesis pathway and its limitation in diabetic patients

Tissue hypoxia (releases HIF)

Hyperglycaemia impairs

HIF-1 α stability via ROS

Phosphorylation

activation by eNOS in the

bone marrow

SDF-1 α guides EPCs to the site

of the injury (impaired in diabetes)

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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 aprotective 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 α homes mobilised EPCs to the wound. This expression

appears to be decreased in diabetes.30

The extrinsic additionof SDF-1 α, 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 identified as important

mediators in inflammation, 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 inflammation and restored immunity. The initial

acute inflammatory 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 inflammatory 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-α, PDGF, FGF,

VEGF and TGF-β.37 It appears that the chronic inflammation that

accompanies DFUs suppresses the focused acute inflammatory

response to injury that is needed for normal wound healing

and which results in impaired leukocyte function and aberrant

expression and activity of inflammatory 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 maynot contribute very much to the healing process. The introduction of

growth factors does very little, especially if the destructive wound

milieu is not corrected first. In addition to this, chronic wound fluid

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 fibres, 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 identification

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 specific target-focused approach

with a well-structured matrix that incorporates strategic elementsto counter the specific 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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