Colin Davies - Measurements in Wound Healing - Science and Practice-Springer-Verlag London (2013)

Measurements in Wound Healing
Raj Mani • Marco Romanelli • Vijay Shukla

Editors
Measurements
in Wound Healing
Science and Practice
Editors
Dr. Raj Mani, D.Sc., FACA Dr. Marco Romanelli, M.D., Ph.D.
Department of Clinical Measurements Wound Healing Research Unit
Wound Healing and Vascular Laboratory Department of Dermatology
Southampton University Hospital University of Pisa
NHS Foundation Trust Pisa
Southampton Italy
UK
Dr. Vijay Shukla, M.B.B.S., M. Ch. (Wales)
Shanghai Jiao Tong University Department of General Surgery
School of Medicine Institute of Medical Sciences
Shanghai Banaras Hindu University
China Varanasi
Benares
India
ISBN 978-1-4471-2986-8 ISBN 978-1-4471-2987-5 (eBook)

DOI 10.1007/978-1-4471-2987-5
Springer London Heidelberg New York Dordrecht
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This book is dedicated to care.
We dedicate it to our own families and
those of our corresponding authors for their
collective patience that permit us (authors)
to devote time to such scholarly pursuits as
writing chapters after long days in hospitals
and laboratories.
It is also dedicated to the patients with
chronic wounds as well as our colleagues every-
where who support the care such patients.
Raj Mani, D.Sc., FACA
Marco Romanelli, M.D., Ph.D.
Vijay Shukla, M.B.B.S., M. Ch. (Wales)
Foreword
For an obstinate ulcer, sweet wine and a lot of patience should be enough
Hippocrates (460–370 BC)
The complex clinical management of chronic wounds has struggled to move from
being an art to being true science. The lack of evidence-based medicine to guide us
in the treatment of complex chronic wounds is apparent to most working in this
challenging area. If we search, for example, for evidence to guide us as to which
dressing to choose for a neuropathic foot ulcer, it is soon clear that we have not
progressed far from the statement of Hippocrates thousands of years ago: there are
no randomised controlled trials to confirm the efficacy or superiority of any particu-
lar dressing.
One of the results of advances in medical technology is increased longevity that
is apparent in most western countries: associated with this is an increased preva-
lence of chronic diseases and consequently chronic wounds, especially the common
venous and diabetic foot ulcer. In my area of diabetes, there is almost an epidemic
of type 2 diabetes across the world, including likely >250 million people with dia-
betes in Asia, and the prevalence in the USA predicted to increase from 10% today
to over 30% in 2050. Thus we can expect significant increases in chronic wounds in
the next few decades.
There is therefore an urgent need to provide an evidence-based approach to the
management of chronic wounds: an essential prerequisite is to have accurate mea-
surements in the science of wound healing. The information provided in the second
edition of this important volume is therefore most welcome and timely. The editors,
each of whom is a leader in the field, have amassed a team of true experts to provide
the basis of the science of measurement in wound healing. The first section covers
the common chronic wounds from venous leg ulcers and diabetic foot disease to
pressure ulcers and also includes a welcome contribution on the epidemiology of
wounds. The next sections include papers on the histopathology, biomarkers, pres-
sure and vascular measurement and papers on burns and scarring. Finally, the all
important topics of research in this area, randomised controlled trials and modeling,
are discussed.
vii
viii Foreword
By far the most important aspect of this area of medicine is the need for team
working. The editors have clearly succeeded in meeting this need as they have
brought together authors from numerous disciplines from basic science to clinical
medicine. Thus this volume is an important step in progressing the science of wound
healing, but, as Charcot said [1] “sera continué” or, “to be continued”.
Andrew J.M. Boulton, M.D., D.Sc. (Hon), FRCP
Prof of Medicine, University of Manchester
Consultant Physician, Manchester Royal Infirmary, Manchester, UK
Visiting Professor, University of Miami, Miami, FL, USA
Vice-President and Director of Postgraduate Education for the European
Association for the Study of Diabetes
Reference
1. Charcot JM. Sur quelques arthropathies qui paraissent dépendre d’une lésion du cerveau ou de
la moelle épinière. Arch Physio Norm Pathol. 1868;1:161–78.
Preface
When is an idea formed? At the moment it is actively discussed or somewhere in the

back of beyond? When did the planning for this book begin?
In 1998, I was honoured to be invited to the first meeting on Wound Management
in Benares, India that was hosted by Vijay Shukla. I was hugely excited at the prospect
of returning to northern India to meet Vijay whose papers I had read and who was
setting up a multidisciplinary clinic in a city that was on my ‘to visit list’. The day
arrived. When the flight landed at this small provincial airport with pretty pink and red
bougainvillaea and marigolds growing in a tidy lawn in front of a small, ancient ter-
minal building, I was met by a young resident from the hospital who escorted me to
the hotel and politely enquired when ‘we’ would be ready to meet the Dean. I was
puzzled and asked who else might be involved to be told ‘Oh sorry, I thought you were
aware that Professor Hunt (he meant Tom Hunt or ‘TK’ from San Francisco) arrived
yesterday’. ‘Dr. Shukla would like both of you to meet the Dean.’ The news that I was
going to get to meet TK instantaneously cured me of all travel fatigue I was experienc-
ing; instead I became nervous and apprehensive. Tom Hunt was (and continues to be)
a most knowledgeable person in matters relating to hypoxia and wound healing.
I had been measuring extremely low transcutaneous oxygen pressures on peri-
wound skin some of which healed – this was surprising and, at that time, inexplica-
ble. The prospect of discussing this (with TK) was daunting. But often in life
apparently unrelated events occur and effect changes. First of all, in one of his papers,
TK had observed ‘to measure a difference, there must be a difference’. Second of all,
I was working on a book focused on measurements in wound healing.1 Third of all,
a colleague editor/author and now friend, Vincent Falanga, had introduced me to a
young, bright and enthusiastic dermatologist and scientist called Marco Romanelli
who was contributing a chapter to this book. Once I had overcome anxiety nervosa
and survived the first day in TK’s company, I plucked up the courage to invite him to
write a foreword to our book. To my surprise and delight, he agreed. Since those 3
days in Benares I have enjoyed many meetings with TK and have come to admire
1
Chronic Wound Healing – Basic Science and Clinical Measurements by Mani R, Falanga V,
Shearman CP and Sandeman DD. Harcourt Brace, London 1999.
ix
x Preface
one of the finest minds in wound healing. The book was published and received very
favourably based on feedback received from a variety of sources.
During the following years, Marco developed his innate interests in wound
assessments and began to make an impact. Vijay’s ideas progressed well and he
duly received national and international recognition for his contributions to wound
management. As a decade passed, Vijay and Marco began to encourage me to write
a sequel to the first book on measurements in wound healing. I cogitated about the
changing scene. The concepts and the need for measurements in both clinical and
research sectors were accepted and there were changes on the horizon, some could
be game changing for a wound healer. My inward ruminations were largely based
on determining the audience for the new book.
In Europe as in the USA, nurses play a frontline role in clinical wound healing.
We have witnessed the development of tissue viability nurses (TVN) (initiated in
the UK) who manage clinical problems very well indeed; any worries expressed by
TVNs seem to stem from the frustrations of workload and so on. TVNs interface
very well with such other professionals as surgeons and podiatrists who are also
essential to wound healing. Also during this decade, the diabetic foot problem has
grown in immensely in prevalence and in the nature of its complications; the num-
ber of amputations is not significantly reducing either. Multidisciplinary team work
is essential to manage the diabetic foot. The success of the Copenhagen Wound
Centre developed by Finn Gottrup and colleagues is worth emulating. In other soci-
eties, wound healing is the domain of the surgeon working in close collaboration
with diabetologists, podiatrists, dermatologists, tissue viability nurses and the vas-
cular family (surgeons and angiologists). Research into wound healing had flourished
during the decade evidenced by the growth of journals, research reports and learned
societies. A new book would need to be valuable to the specialist as well as those on
track to developing a special interest in wound healing.
During a visit to India in the autumn of 2009, Vijay provoked discussion on the
current state of measurements concluding ‘You have to write the book’. I responded
positively but conditionally. The three of us (Vijay, Marco and I) had to work on it.
They were up for it. This book is the result. Its purpose is to revisit the measure-
ments in wound healing this time giving emphasis to its significance.
Measurements imply a vast range of techniques used to diagnose, to learn the
pathophysiology of wounds, to quantify morphological changes and to learn more
about complications that make wound healing challenging. And then there are mea-
surements with promise. We decided to present this book in sections dealing with
the established, the less established and those with promise for the future. In this
way, the established value of measurements to identify pathology and guide man-
agement, the specificities on measurements waiting to gain widespread clinical
acceptance and finally the futuristic measurements are covered. We trust the balance
is just there or thereabouts.
Regarding its value to researchers: wound healing research through the 1980s was
devoted to vascular physiology, angiogenesis and microcirculation. Through the
1990s it was mainly focussed on growth factors and their potential to improve wound
healing based on the premise that there is an imbalance between the factors that pro-
mote synthesis and degradation of the extracellular matrix. The chapter on research
Preface xi
in this book (ably led by Finn Gottrup) is focused on randomised controlled trials
(RCT) that has become a mantra to wound researchers. The main purpose of the RCT
is to compare a new or untested technique/device/treatment/ with a comparable
equivalent. Ideally, a control should be equal in all respects (except of course in terms
of effectiveness). It is widely considered as the best way of deriving a very high level
of evidence of effectiveness. Our readers should bear in mind that robust experimen-
tal design with clear exclusion and inclusion criteria, a clear definition of outcomes
also permit good, reliable evidence to be derived. When the best design requires huge
sample sizes, collaborate rather than negotiate to limit the aims of the study. It is only
when workers across the globe regularly submit large numbers of requests to fund
studies in wound healing will governmental agencies begin to recognise our efforts.
The chapter on models in wound healing is small with a focus on animal models
for the reason that there is an urgent need for us to think about the translational value
of our research and innovative work. Findings from bench studies may lead to clini-
cal research of the same theme or vice versa; translation of the concept requires that
the ways and means of using the concept in wound management. We are hopeful
that this book will help researchers in their quest for translation.
This book has chapters that indicate what you ‘must do’ and the need for further
research as in the chapter by Anba Soopramanian. The chapters on burns and scars
reinforce the need for more measurements in these areas while pointing to suc-
cesses. The chapters on histopathology of wounds and on wound fluids are impor-
tant to track The possibility of imaging oxygen available to tissues with a clear
indication of hypoxia is the future; the progress is reported in a chapter. Atypical
wounds are discussed with confidence and clarity. Certain aspects have been
excluded and must be followed up with respective specialities, for example the
problem of self-inflicted chronic wounds in children discussed by Paul Russell.2
So when does an idea form? From where does original thought arise? And clearly
where does it lead to? It brings to mind a stanza in Sanskrit that I learnt rather a long
time ago.
Aakasath pathitham thoyam
Yatha gacchathi sagaram
Sarva Deva namaskara
Keshavam prathigacchathi.
Literally translated these lines describe how rain water forms streams that flow
into rivers thence to the sea. It is used allegorically to excite wound healers and
readers of this book; use the experience and evidence in this book constructively but
critically. Think, measure carefully, and you will make a difference to the practice
of wound healing.
Raj Mani, D.Sc., FACA
Marco Romanelli, M.D., Ph.D.
Vijay Shukla, M.B.B.S., M. Ch. (Wales)
2
Russell P. Self injurious behaviour to the lower extremity among children with atypical
development – a diagnostic and treatment algorithm. Int J Low Extrem Wounds 2006;5(1):10–7.
Contents
1 The Importance of Vascular Investigation and Intervention

in Leg Ulcer Management ................................................................... 1
Colin Davies and Keith Poskitt
2 Diabetic Foot Disease and Wound Healing ........................................ 17
Agbor Ndip and Edward B. Jude
3 Atypical Ulcers ...................................................................................... 39
Jennifer Tang and Robert S. Kirsner
4 Nutrition and Wound Healing ............................................................. 63
Giuseppe Benati and Maria S. Bertone
5 Measurement of Wound Healing and Tissue Repair ........................ 73
Visweswar Bhattacharya, Nilesh K. Agarwal,
and Siddhartha Bhattacharya
6 Pressure Ulcers in Neurologically Compromised Patients ............... 93
Anbananden Soopramanien and Shivram Singh
7 Complications of Wound Healing ....................................................... 109
Somprakas Basu and Vijay Shukla
8 Epidemiology of Wounds ..................................................................... 145
David J. Margolis
9 Histopathology of Wounds ................................................................... 155
Mariya Miteva and Paolo Romanelli
10 Measuring Pressure in the Diabetic Foot ........................................... 175
Thanh Dinh, Aristidis Veves, and Francesco Tecilazich
11 Skin and Vascular Assessments ........................................................... 193
Marco Romanelli, Valentina Dini, and Raj Mani
xiii
xiv Contents
12 Wound Tissue Oximetry: A Cornerstone in Wound Care ................ 225

Jaideep Banerjee and Chandan K. Sen
13 Measurement of Biomarkers for Impaired Healing
in Fluids and Tissues ............................................................................ 243
Gregory S. Schultz and Daniel J. Gibson
14 Measurements in Burns ....................................................................... 259
Tom McKinnell and Sarah A. Pape
15 Managing Scars: Measurements to Improve
Scar Management ................................................................................ 291
Luc Téot, Claude Roques, Sami Otman, Antonio Brancati,
and Rainer Mittermayr
16 The Use of Biophysical Technologies in Chronic
Wound Management ............................................................................ 313
Uwe Wollina, Birgit Heinig, and Luther Kloth
17 Research Studies in Wound Healing: The Role
of Outcomes/Endpoints for the Evidence in RCTs ........................... 355
Finn Gottrup, Jan Apelqvist, and Patricia Price
18 Models in Wound Healing.................................................................... 369
Ming Yuan Miao, Ting Xie, Shuliang Lu, and Raj Mani
Appendix ........................................................................................................ 385
Index ............................................................................................................... 387
Contributors
Nilesh. K. Agrawal, M.S., M.Ch. Department of Plastic Surgery, Institute of

Medical Sciences, Banaras Hindu University, Naria, Varanasi, Uttar Pradesh, India
Jan Apelqvist, M.D., Ph.D. Department of Endocrinology, Malmö University
Hospital, Malmö, Sweden
Jaideep Banerjee Department of Surgery, The Ohio State University
Wexner Medical Center, Columbus, OH, USA
Somprakas Basu, M.B.B.S., M.S. Department of General Surgery,
Institute of Medical Sciences, Banaras Hindu University, Varanasi,
Uttar Pradesh, India
Giuseppe Benati, M.D. Clinical Nutrition Service, Geriatric Department,
Morgagni Pierantoni Hospital, Forlì, Italy
Maria S. Bertone, CD Department of Dermatology, Wound Healing Unit Re-
search, University of Pisa, Pisa, Italy
Siddhartha Bhattacharya Department of Plastic Surgery, Institute of Medical
Sciences, Banaras Hindu University, Naria, Varanasi, Uttar Pradesh, India
Visweswar Bhattacharya, M.S., MCh, Ph.D., FICS Department of Plastic
Surgery, Institute of Medical Sciences, Banaras Hindu University, Naria, Varanasi,
Uttar Pradesh, India
Antonio Brancati, M.D. Wound Healing Medico-Surgical Unit Burns Unit,
Montpellier University Hospital (MUH), Montpellier, France
Colin Davies, B.Sc. (Hons), M.Sc. Leg Ulcer Service,
Cheltenham General Hospital, Cheltenham, Gloucestershire, UK
Thanh Dinh, D.P.M. Surgical Department, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, MA, USA
Valentina Dini, M.D., Ph.D. Wound Healing Research Unit, Department
of Dermatology, University of Pisa, Pisa, Italy
xv
xvi Contributors
Daniel J. Gibson, M.S. Department of Biochemistry and Molecular Biology,

Institute for Wound Research, University of Florida, Gainsville, FL, USA
Finn Gottrup, M.D., DMSci Department of Dermatology, Copenhagen Wound
Healing Center, Bispebjerg University Hospital, Copenhagen, Denmark
Birgit Heinig, Dipl-Med Department of Wound Healing, Academic Teaching
Hospital Dresden-Friedrichstadt, Dresden, Germany
Edward B. Jude, M.D., MRCP Department of Medicine, Consultant
Diabetologist and Honorary Reader in Medicine, Tameside Hospital NHS
Foundation Trust, Ashton-under-Lyne, UK
University of Manchester, Manchester, UK
Robert S. Kirsner, M.D., Ph.D. Department of Dermatology and Cutaneous
Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
Luther Kloth, PT, M.S., FAPTA, CWS, FACCWS Department of Physical
Therapy, College of Health Sciences, Marquette University, Milwaukee, WI, USA
Shuliang Lu, M.D., Ph.D. Burns Centre and Wound Healing Department,
Ruijin and 9th Peoples’ Hospitals, Shanghai, China
Raj Mani, D.Sc., FACA Department of Clinical Measurements,
Wound Healing and Vascular Laboratory, Southampton University Hospital NHS Trust,
Southampton, UK
David J. Margolis, M.D., Ph.D. Departments of Biostatistics and Epidemiology
and Dermatology, University of Pennsylvania, Philadelphia, PA, USA
Tom McKinnell, MB, ChB, M.Sc., MRCS (Ed) Department of Plastic Surgery,
James Cook University Hospital, Middlesbrough, UK
Ming Yuan Miao, M.D. Burns Centre and Wound Healing Department,
Wound Healing Research Group, Southampton University Hospital NHS Trust,
Southampton, UK
Mariya Miteva, M.D. Department of Dermatology and Cutaneous Surgery,
University of Miami Miller School of Medicine, Miami, FL, USA
Rainer Mittermayr, M.D. Department of Wound Healing, Ludwig Boltzmann
Institute for Experimental and Clinical Traumatology,
Vienna, Austria
Agbor Ndip, M.D., Ph.D. Department of Diabetes and Medicine,
Manchester Royal Infirmary, University of Manchester, Manchester, UK
Sami Otman, M.D. Wound Healing Medico-Surgical Unit Burns Unit,
Montpellier University Hospital (MUH), Montpellier, France
Contributors xvii
Sarah A. Pape, MB, ChB, FRCSEd(Plast), MClinEd Department of Plastic

Surgery, Royal Victoria Infirmary, Newcastle upon Tyne, UK
Keith Poskitt, M.D., FRCS Vascular Department, Cheltenham General Hospital,
Cheltenham, Gloucestershire, UK
Patricia Price, Ph.D., CHPsychol School of Healthcare Studies,
Cardiff University, Cardiff, UK
Macro Romanelli, M.D., Ph.D. Wound Healing Research Unit,
Department of Dermatology, University of Pisa, Pisa, Italy
Paolo Romanelli, M.D. Department of Dermatology and Cutaneous Surgery,
Claude Roques, M.D. Département of Physical Therapy,
Pediatric Rehabilitation Centre, CSRE Lamalou le Haut,
Lamalou les Bains, France
Gregory S. Schultz, Ph.D. Department of Obstetrics and Gynecology, Institute
for Wound Research, University of Florida, Gainsville, FL, USA
Chandan K. Sen, Ph.D. Department of Surgery, The Ohio State University
Wexner Medical Center, Columbus, OH, USA
Department of Surgery, Davis Heart and Lung Research Institute, Ohio State
University Medical Center, Columbus, OH, USA
Vijay Shukla, M.B.B.S., M. Ch. (Wales) Department of General
Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi,
Benares, India
Shivram Singh, FRCS Department of Plastic Surgery, Birmingham Hospital
NHS Trust, Birmingham, UK
Anbananden Soopramanien, M.D., Ph.D., FRCP The Glenside Hospital
for Neuro-Rehabilitation, Salisbury, Wiltshire, UK
Centre of Postgraduate Medical Research and Education,
University of Bournemouth, Bournemouth, Dorset, UK
Jennifer Tang, B.S. Department of Dermatology, University of Miami Miller School
of Medicine, Miami, FL, USA
Francesco Tecilazich Surgical Department, Beth Israel Deaconess Medical
Center, Boston, MA, USA
Luc Téot, M.D., Ph.D. Wound Healing Unit, Department of Surgery,
Montpellier University Hospital, Montpellier, Hérault, France
Aristidis Veves, M.D., D.Sc. Surgical Department, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA, USA
xviii Contributors
Uwe Wollina, M.D. Department of Dermatology and Allergology,

Academic Teaching Hospital Dresden-Friedrichstadt, Dresden, Germany
Ting Xie, M.D., Ph.D. Burns Centre and Wound Healing Department,
Chapter 1
The Importance of Vascular Investigation
and Intervention in Leg Ulcer Management
Colin Davies and Keith Poskitt
Keywords Leg ulcer • Photoplethysmography • Vascular • Venous • Foam

sclerotherapy • Doppler • Pinch skin graft • Duplex
Introduction
Chronic ulceration of the leg is a common condition which is time consuming to

treat and reduces health related quality of life [1, 2]. The financial burden has been
previously reported and it has been estimated that the cost to the UK National
Health Service (NHS) of treating venous leg ulcers alone consumes around 2% of
the health service budget [3].
Historically, leg ulcers have been managed in the community and have tradition-
ally fallen within the domain of the District Nurse with little consensus as to the
most appropriate means of assessing and managing leg ulceration [4, 5]. A survey
in Leeds found that district nurses’ knowledge of the assessment and management
of leg ulcers was often inadequate [6]. Another survey reported that 51% of nurses
made a diagnosis of the cause of the ulcer based on visual assessment alone [7].
The vast majority of chronic leg ulceration will have a vascular aetiology, with
chronic venous insufficiency (Fig. 1.1) the primary cause in around 70% and a major
C. Davies, B.Sc.(Hons), M.Sc.

Leg Ulcer Service, Cheltenham General Hospital,
Sandford Road, Cheltenham, Gloucestershire GL53 7AN, UK
e-mail: colin.davies@glos.nhs.uk
K. Poskitt, M.D., FRCS (*)
Vascular Department, Cheltenham General Hospital,
Sandford Road, Cheltenham, Gloucestershire GL53 7AN, UK
e-mail: keith.poskitt@glos.nhs.uk
R. Mani et al. (eds.), Measurements in Wound Healing, 1

DOI 10.1007/978-1-4471-2987-5_1, © Springer-Verlag London 2012
2 C. Davies and K. Poskitt
Fig. 1.1 Venous ulcer
Fig. 1.2 Differential Other causes*

diagnoses of leg ulcers. Arterial 5%
* Malignancy (1–2%), 10%
diabetes, rheumatoid arthritis,
vasculitis, haematological
disorders, trauma
Mixed arterial &

venous 15%
Venous
70%
contributory factor in a further 15% [8, 9]. However, despite a wealth of research into
lower extremity wounds, the precise mechanism of skin breakdown as a result of
chronic venous insufficiency is poorly understood. Peripheral vascular disease
accounts for around 10% of leg ulcers, the principal cause being occlusive arterial
disease and a further 5% of causes attributed to other vascular and non-vascular aeti-
ologies such as vasculitis, malignancy, diabetes and rheumatoid arthritis (Fig. 1.2).
Assessment
It is essential to differentiate between arterial and venous ulcers not only to get a
precise diagnosis but also because the mainstay of treatment of venous ulcers –
compression therapy [10] may lead to skin necrosis, and worse, if applied to an
1 The Importance of Vascular Investigation and Intervention in Leg Ulcer Management 3
ischaemic leg [11]. In the past, assessment of the arterial supply to the leg was made
by palpating the pulses on the foot. This assessment is a poor predictor [12] making
it essential to use objective tests. The use of Doppler ultrasound to measure the
ankle brachial pressure index (ABPI) and in the assessment of arterial waveforms is
now recognised as essential to select appropriate treatment pathways [13, 14].
Developments in ultrasound imaging over the past quarter of a century have had
a dramatic impact on non-invasive investigations of the body’s vasculature. Duplex
ultrasound imaging is useful in the assessment of venous reflux in patients with
chronic leg ulceration [15] and is now the most commonly performed procedure
used for investigating and diagnosing chronic venous insufficiency in the leg. Both
ABPI assessment and venous duplex ultrasound offer validated methods of identify-
ing abnormalities in blood vessels and can be achieved in a ‘one-stop’ assessment
clinic [16].
In some patients, it will be important to establish the relative importance of
superficial and deep venous reflux in order to predict the effect of treating any
superficial reflux. Objective measurement of the haemodynamics of venous return
may be made using ambulatory venous pressure measurement (AVP). This inva-
sive technique is done using a small needle into one of the veins on dorsum of foot
and connecting the needle through a transducer to a blood pressure measurement
machine. Digital photoplethysmography (PPG) is a sensitive, non-invasive test that
can be performed by non-medical staff using cheap, portable equipment and pro-
vides functional information equivalent to the gold-standard AVP measurement
[17, 18]. The importance accurate assessments for a reliable diagnosis to ensure
selection of the most effective treatment pathway for the patient and cannot be
overstated.
Doppler Ultrasound Examination
The use of handheld Doppler ultrasound as a diagnostic tool is now mandatory

when assessing patients with leg ulcers. Objective measurement of the ABPI using
a hand held Doppler probe is important and easily repeated [19–23] (Fig. 1.3). In a
continuous wave Doppler system, a sound wave is continuously transmitted and
received with two (usually adjacent C shaped) transducers. The transmitted and
reflected beams begin to overlap a short distance from the surface of the probe, and the
overlap extends until the beams attenuate [24, 25].
The Doppler phenomenon states that the frequency of an incident light or sound
wave is altered when it is incident on a moving object (in this case a red blood cell
or pulsating blood vessels). The change in Doppler frequency due to the relative
motion between the observer and the object is known as the Doppler shift [26, 27].
The Doppler shift (fD) can be calculated with the following formula:
f D = 2f tvCos q / c
Fig. 1.3 Hand-held Doppler ultrasound assessment of arteries
where v is the speed of the moving target, ft is the frequency of the emitted pulse (i.e.
the frequency of the transducer), q is the angle between the direction of emitted
sound wave and the direction of the moving target, and c is the average speed of
sound within the tissue [27, 67].
In moving blood, it is the red blood cells that reflect the sound waves that result
in the Doppler shifted echo. This reflection, or ‘backscattering’ as it is often referred
to in the texts [24, 26, 67] is detected by the transducer. Ultrasonic frequencies in
the range of 5–10 MHz are commonly used for measurements of systolic blood
pressures in the limbs. These frequencies, the range of tissue velocities encountered
in the body (around 0–5 m/s), and the velocity of sound in blood, all fall within an
audible range [26].
In blood vessels, the Doppler shift is dependent on the speed of blood flow, the
angle between the transducer and the vessel, and the operating frequency of the
Doppler transducer [27, 67]. Thus, interpretation of the audible response can be
susceptible to user error if the angle between the transducer beam and the moving
target (i.e. blood flow) is too great. When the probe is directed precisely along the
direction of flow, the Doppler shift is maximum since the angle Q is zero and
cosine Q is 1. If the transducer is held at 90° to the blood vessel there will be no
component of velocity along the beam (cosine Q is zero) and therefore no detect-
able Doppler shift.
Table 1.1 Interpretation of ankle brachial pressure indices

ABPI What it means
>1.25 May indicate incompressible arteries (atherosclerosis)
0.85–1.25 Normal range of values: no significant arterial insufficiency
0.5–0.85 Moderate arterial insufficiency: ulcer considered to be mixed aetiology in
the presence of clinical signs associated with chronic venous insufficiency
<0.5 Severe arterial insufficiency
Validity and Reliability of Doppler
ABPI may be accurately derived using a handheld Doppler probe accurately. This is
essentially a comparison between the systolic blood pressure in the arm and in the
lower leg and is illustrated in the following formula:
ABPI l = Pl
Pa ABPI1 = ABPI for a leg
Pl = Highest pressure obtained from the ankle vessels for that leg
Pa = Highest brachial pressure of the two arms
In a normal subject the pressure at the ankle is often slightly higher than at the
brachial fossa as there is reflection of the pulse pressure from the vascular bed of the
feet. The shape of the arterial pressure pulse wave changes from the ascending aorta
to the periphery; the systolic pressure increases while diastolic pressure falls at
peripheral sites. A range of 0.85–1.25 may be considered to be consistent with the
presence of normal peripheral arterial supply (see Table 1.1).
Discrepancies in the assessment of patients with leg ulcers have previously been
reported [12] with many community nurses relying on evaluation of pedal pulses or
ABPI as a diagnosis in itself. However, much of the evidence supporting palpation
of pedal pulses in order to detect arterial disease has originated from studies per-
formed in a vascular unit by a trained observer as opposed to routine assessment by
nurses. This underlines the importance of education and training in order to main-
tain good reliability and reproducibility between and among users.
There is generally poor agreement between manual palpation of foot pulses and
ABPI. Two large studies have shown that 37% and 15% of limbs respectively with
an ABPI < 0.9 had palpable foot pulses, with the consequent risk of applying com-
pression to people with arterial disease [12, 30]. Both studies concluded that mea-
surement of arterial circulation using a hand-held Doppler should be considered
mandatory in the assessment of leg ulcers.
However, there appears to be disagreement as to what should be regarded a nor-
mal ABPI. The above two studies considered an ABPI of <0.9 to indicate the pres-
ence of peripheral arterial disease, whilst another study suggested that an ABPI of
<0.8 was significant [29]. Other researchers have compromised by referring to <0.85
as an indicator of arterial insufficiency [8, 16, 28]. From a simple arithmetic perspec-
tive, 0.85 may be rounded off to 0.9 significant to the first place of decimals.
Other potential limitations to the validity of Doppler ultrasound can be derived

from environmental factors and user error. Fluctuations in room temperature can
affect measurement of ABPI as an environment that is too hot or too cold can cause
vasodilation and vasoconstriction respectively [31] though this will affect both bra-
chial and foot pressure values. Two practical limitations that affect the measurement
of ABPI are size of patients’ legs and incompressibility of arteries. This is can be
bothersome and UK nurses who are constrained by practice guidelines, seek support
from trained staff in vascular laboratories.
User error can be related to the positioning of the ultrasound probe. Measurement
of the Doppler shift is dependent on the angle of insonation to the direction of target
movements, i.e. transducer to blood vessel. The probe should be held at an angle of
45–60° to detect optimum Doppler signals. In practice, it is possible to steady the
probe using a free finger once the best signal (corresponding to the highest Doppler
frequency shift) thereby minimising artifacts due to probe movement.
Duplex Ultrasound
Duplex ultrasonography has become the investigation of choice in patients present-

ing with varicose veins and is now widely used in the assessment of patients with
leg ulcers. It combines real-time B-mode ultrasound with pulsed-wave Doppler and
colour flow duplex imaging enabling more precise anatomical and haemodynamic
assessment in order to localise and specify the source of the venous problem and to
provide a map to help select best treatment and evaluate outcomes [32] (Fig. 1.4).
Venous return to the heart from the legs is achieved by the pumping of the leg
muscles and is helped by a series of bicuspid non-return valves inside the veins in
order to prevent back flow of venous blood into the lower extremities. Gravitational
forces significantly affect venous return and if a physiological deterioration in the
mechanics of venous return develops such as valve incompetence or muscle atro-
phy, then venous pooling of blood will ensue thus giving rise to chronic venous
hypertension. Venous reflux is considered to be the retrograde flow of blood lasting
for more than 1 s [33]. Using duplex imaging, the duration of venous reflux can be
determined using distal calf augmentation and spectral Doppler analysis as dis-
cussed in greater detail in a later chapter in this book (Chap. 13). The same technol-
ogy can be used to visualise the arterial network in the leg to identify and classify
the severity of arterial disease.
Digital Photoplethysmography
Digital photoplethysmography (d-PPG) is a non-invasive test, which measures the

change in skin blood volume using a small light-emitting diode that is placed on the
surface of the skin just above the ankle. After a period of rest the subject exercises
Fig. 1.4 Duplex ultrasound scan performed in the vascular laboratory and screenshots from
duplex showing changes in flow direction – ante grade (blue) and retrograde (red)
%
PPG
4
0 10 20 30 40s
Quantitative Parameters:
Venous refilling time: To = 38s
%
PPG
4
0 10 20 30 40s
Quantitative Parameters:
Venous refilling time: To = 8s
Fig. 1.5 Photoplethysmography (shown with below knee tourniquet). The graphs display venous
refilling time in seconds
the calf pump by moving the foot up and down at the ankle (Fig. 1.5). The d-PPG
probe measures the reduction in skin blood content that is a result of the pumping
action of the calf muscles.
This is a relatively cheap but sensitive test that can be performed by non-medical
staff and measures venous haemodynamics in the leg. It has been shown to be par-
ticularly useful in predicting the influence of superficial surgery on venous function
in chronically ulcerated limbs when combined with inflatable blood pressure cuffs
placed on the leg above and below the knee [34]. When the cuff is inflated to
80 mmHg, this controls superficial reflux and the effect of the cuff on the PPG mea-
surement gives further information about the severity and nature of the venous prob-
lem. This is useful when assessing the patient’s suitability for further intervention
such as surgery or foam sclerotherapy.
Leg Ulcer Diagnosis and Further Intervention
The majority of leg ulcers have an underlying venous or arterial abnormality with a
large proportion of these having a potentially correctable problem (Figs. 1.1 and
1.5). The importance of making an accurate diagnosis in order that the appropriate
treatment can be carried out has created the need for a structured and coordinated
Fig. 1.6 Venous reflux and Superficial and

indications for further total deep
intervention [40] 15%
Superficial and Isolated

segmental deep superficial reflux
24% 61%
approach. Despite many areas in the UK still lacking a dedicated specialist leg ulcer
service, it is now widely acknowledged that this is the ‘gold standard’ when devel-
oped alongside a ‘one-stop’ vascular assessment clinic [8, 16, 35].
In Gloucestershire, a vascular led, specialist nurse-run service was intro-
duced in 1995 in order to provide clearly defined protocols for treating patients
with leg ulcers and to provide an evidence-based approach to leg ulcer manage-
ment [36, 37]. Prior to this, the provision of leg ulcer management was less than
ideal [16].
Improvement in outcomes for these patients were clearly seen after just 12 months
[16, 36]. Similar improvements have been demonstrated in Sheffield [38], Trafford
[39] and Riverside (Charing Cross) [40] following the development of a similar
model. Studies from Gloucestershire have shown that a significant proportion of
patients with chronic venous insufficiency could be suitable for further intervention
if they present with isolated superficial venous reflux or superficial and segmental
deep reflux [1] (Fig. 1.6).
The Role of Surgery
Stripping of the long saphenous vein (LSV) has been widely recognised as essential
to minimising recurrence of venous leg ulcers [1, 41, 42]. The traditional surgical
approach is to ligate and divide the saphenous trunk and all proximal tributaries,
followed by stab avulsions of any lower limb varices. Such practice has become an
established intervention for patients with reflux identified in the long and short
saphenous trunks [37].
Previous studies have suggested that surgery may improve healing of venous leg
ulcers [43]. More recently the ESCHAR randomised controlled trial compared the
effects of surgery and compression therapy with compression therapy alone in the
healing and recurrence of venous ulcers. It concluded that following ulcer healing,
superficial venous surgery significantly reduced ulcer recurrence at 1 and 3 years for
patients who did not present with total deep venous incompetence [1, 42].
Foam Sclerotherapy
Injection sclerotherapy has been an established treatment for varicose veins for over
100 years and there is evidence to suggest its use goes back much further. Foam scle-
rotherapy (FS) has developed from earlier methods and uses a standard sclerosant
such as sodium tetradecyl sulphate or polidocanol. This is mixed with air or carbon
dioxide in various ratios to create a microfoam, which is then injected into the vein
(Fig. 1.7). The treatment is ultrasound guided allowing safe positioning of the cannula
or needle into the vein and careful monitoring of the foam’s dispersion [44, 49].
At present, the evidence for ultrasound guided foam sclerotherapy shows that it
is a clinically effective intervention for the treatment of incompetent superficial
veins. A Cochrane review comparing surgery to FS concluded that FS has greater
benefits than surgery in the short term but surgery has greater benefits in the longer
term. Sclerotherapy was better than surgery in terms of treatment success, compli-
cation rate and cost at 1 year, but surgery was better after 5 years [45, 46]. Cabrera
and co-workers published a clinical series of 500 lower limbs treated by FS and
reported long saphenous vein occlusion rates of 81% at 3 years although the authors
acknowledge that there is still a significant chance of recanalisation of the treated
vessels, resulting in recurrence of superficial venous incompetence [47, 48].
There are very few reported complications, but those that have been reported
include localised inflammation and haematoma formation, pigmentation, throm-
bophlebitis, nerve damage [50] and, more rarely, leakage of foam into the deep
venous system resulting in deep venous thrombosis (DVT) [51]. The literature
reports an incidence of post procedural DVT ranging from 1% to 6%. Clinical audit
data of FS in 7,027 patients reported DVT in less than 1% (36/7,027) patients [53].
In one case series of 243 patients treated at the University Hospital in Malmo,
Sweden, the reported incidence of DVT was 2% with one patient suffering a
Pulmonary Embolism [52]. Unpublished work from the Cheltenham unit reports
seven cases of DVT and one pulmonary embolism from a case series of 856.
Fig. 1.7 Foam sclerotherapy

Visual disturbances have also been recognised as a potential side effect, particu-
larly in patients who have a history of migraine although these symptoms tend to
resolve spontaneously after 2 h and no long-term or permanent visual impairment
has been reported [53].
In terms of cost, FS can be performed without hospitalisation or anaesthesia, allow-
ing the patient a rapid return to normal daily activity. Furthermore, it is cheap in com-
parison to almost all of the other methods of treatment available today. Two reports
both from the UK compared FS and surgery in terms of cost. In both trials, general
anaesthesia was used for surgery and local anaesthesia for FS. Overall procedure cost
was significantly less for FS at £672.97 compared to £1120.64 for surgery [54]. The
authors acknowledged that the lower costs were achieved through:
• Shorter treatment time (therefore lower staff costs)
• Lower anaesthetic costs (no need for a general anaesthetic)
• Reduced recovery time (no ward or theatre used)
• Reduced capital and overhead expenditure.
Undoubtedly, this is more favourable for patients and for healthcare providers
alike. However, further studies are required to evaluate its long term efficacy.
Endovenous Ablation
The two commonly used endovenous ablation techniques are Radiofrequency and
endovenous laser ablation (EVLA). Radio frequency ablation (RFA) offers another
minimally invasive alternative to surgery and uses radiofrequency to heat and seal
the refluxing segment. When the RFA catheter is in the diseased vein, radiofre-
quency energy is delivered to a heating element to heat and contract the collagen
within the vein walls. This subsequently leads to the shrinkage and eventual col-
lapse of the vessel.
EVLA works by means of thermal destruction of venous tissues. Laser energy is
delivered to the incompetent segment inside the vein through a laser fibre that has
been passed through a sheath to the desired location. As with FS, both RFA and
EVLA are performed under ultrasound guidance. Current evidence suggests EVLA
and RFA are as safe and effective as surgery, particularly in the treatment of saphen-
ous veins [53].
Bruising and pain are frequently reported side effects of EVLA. Nerve injury,
skin burns, deep vein thrombosis and pulmonary embolism occur more rarely [54].
Whilst long-term data are not yet available, particularly in relation to ulcer recur-
rence, such minimally invasive methods can provide useful treatment options in the
elderly leg ulcer population.
Importantly, the use of endovascular techniques frees up valuable theatre time for
other surgical procedures and allows patients to be treated in an outpatient clinic with
quicker recovery times and lower costs. Cost benefits may also be shared by the
patient and the wider economy with a speedier return to work and normal activities.
Compression Therapy after Surgery and Sclerotherapy
Graduated compression stockings or bandages are recommended after surgery to

reduce bruising and thrombophlebitis [55]. Following foam sclerotherapy, the use
of compression assists in the occlusion of the superficial vein lumen. However, the
compression value produced by bandages is very much dependant on the practitio-
ner applying them [55, 56]. In the past, there has been little consensus as to the
optimum compression following such procedures but it is agreed that some form of
compression is required after varicose vein treatments.
One randomised controlled trial found that there was no benefit in wearing com-
pression for more than 1 week, but found that the wearing of compression bandages for
the first week did significantly reduce pain, bleeding and the extent of bruising [57]. In
another randomised study, no significant differences between compression dressings
with regard to the degree of postoperative pain experienced, and concluded that the
choice of compression dressings used for varicose vein surgery should depend primar-
ily on the personal preference of surgeons as well as financial considerations [58, 67].
Pinch Skin Grafting for Venous Leg Ulcers
Skin grafting has been shown to expedite ulcer re-epithelialisation and improve
healing rates [58–60]. Pinch skin grafting provides partial coverage of the ulcer
area and increases the total length of skin margin from which epithelium can grow
(Fig. 1.8). This technique is simple, can be performed under local anaesthesia in
the outpatient or primary care setting and avoids the problems associated with the
full-thickness or split skin grafting, such as general anaesthesia, poor healing of
the donor site, immobilisation and lifting of the skin graft by exudate. Pinch skin
Fig. 1.8 Pinch skin grafting

for venous leg ulcer
grafting has been shown to improve healing rates in large venous ulcers [61]. It is
a practical and cost-effective procedure [62] and has been used successfully in the
treatment of chronic leg ulcers of various aetiologies [62–66].
However, despite being widely used in Europe, pinch skin grafting is infrequently
seen in the UK.
Conclusions
The management of patients with chronic leg ulceration has continued to evolve
with dramatic improvements developing in recent years. A comprehensive assess-
ment of the underlying aetiology is the key to achieving optimum patient outcomes
with research demonstrating that the best results are achieved when patients are
assessed and managed by specialist leg ulcer teams in dedicated clinics providing
direct access to vascular surgeons and close links with primary care teams.
Whilst strong evidence supports the use of multilayer graduated compression in
the treatment of venous ulceration, minimising the recurrence of chronic venous leg
ulcers remains a challenge and many patients are plagued with multiple episodes of
ulceration throughout life. If investigated appropriately and corrective procedures
performed, the recurrence of leg ulcers may be reduced by half. As the vast majority
of leg ulcers will have a vascular aetiology, it is vital that patients with this disabling
condition are offered appropriate investigations to allow an accurate diagnosis
allowing subsequent treatment pathways to be optimised and the possibility of
potentially curative intervention explored.
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Chapter 2
Diabetic Foot Disease and Wound Healing
Agbor Ndip and Edward B. Jude
Keywords Diabetic foot ulcers • Neuropathy • Infection • Wound healing •

Amputation
Introduction
Foot ulcers are one of the most common complications of diabetes affecting up to
25% of patients during their lifetime [1] and frequently resulting in hospitalization
[2–4]. Approximately one quarter of all hospitalised days for persons with diabetes
are related to foot complications [5–7]. Diabetic foot ulcers are also associated with
significant morbidity and mortality [8]; 80% of all diabetes-related lower extremity
amputation being preceded by a foot ulcer [9]. Prevention strategies as well as
proper management must be a mainstay in the reducing to reduce the burden of foot
ulcers. Effective management of diabetic foot ulcers rests on the ability to perform
accurate identification, measurement and classification of ulcer characteristics, to
decide on therapeutic strategies, and recognise the need for referral to specialist
teams. Furthermore, accurate recognition and monitoring of ulcer characteristics
A. Ndip, M.D., Ph.D.

Department of Diabetes and Medicine,
Manchester Royal Infirmary, University of Manchester, Manchester, UK
E.B. Jude, M.D., MRCP (*)
Department of Medicine,
Ashton under Lyne and University of Manchester, Manchester, UK
University of Manchester,
Ashton-under-Lyne, UK
e-mail: edward.jude@tgh.nhs.uk

18 A. Ndip and E.B. Jude
facilitates benchmarking therapeutic response which makes it essential that ulcer

characteristics are well defined. This chapter will focus on summarising key clinical
measurements that help define ulcer characteristics in people with diabetes, with a
view to guide therapeutic choices.
Initial Assessment of Diabetic Foot Ulcers
At the first encounter with a diabetic patient with a chronic ulcer, the question must
be asked: is this a neuropathic or a neuro-ischaemic ulcer? This key question gains
prominence from the fact that it guides management and provides an initial progno-
sis. Although good clinical acumen often provides useful cues as to the aetiology of
diabetic foot disease, it is mandatory to perform an objective assessment of vascular
and neuropathy status. Table 2.1 summarises key features that distinguish neuro-
pathic from neuro-ischaemic ulcers.
Neuropathy
As far as diabetes neuropathic complications go, loss of protective sensation (LoPS)

is the more significant predictor of foot ulceration. Other manifestations of neuropa-
thy such as painful neuropathy and autonomic neuropathy may co-exist with LoPs.
It must be recognized that diabetic neuropathy is a diagnosis of exclusion and care
must be taken to rule out other causes of neuropathy.
A complete physical examination should include a systematic and meticulous
testing for the different sensory modalities. Assessments are best performed with
the patient supine and with their eyes closed to eliminate visual bias. The following
are usually included in the assessment for sensory neuropathy (Table 2.2).
The 10-g Monofilament (10-g MF)
The standard 10-g Semmes-Weinstein monofilament is designed to buckle at a force

of 10-g when applied perpendicularly to the skin surface briskly (1 s) such that the
nylon filament bows. It is the most widely used device in clinical practice in the
assessment of diabetic neuropathy [11]. It is also a good predictor of foot ulcer risk
[12, 13]. Other types of 10-g monofilament (10-g MF) exist today. However, not all
10-g MFs supplied by different companies buckle at this force and clinician’s should
identify reliable suppliers. The technique for using the 10-g monofilament is illus-
trated in Fig. 2.1. Briefly, buckling is first tested on the hand or forearm to demon-
strate the procedure to the patient. With the patient relaxed, supine and eyes closed,
2 Diabetic Foot Disease and Wound Healing 19
Table 2.1 Differences between neuropathic and neuro-ischaemic diabetic foot disease
Characteristic Neuropathic Ischaemic/neuroischaemic
Demographic/history Loss of sensation, numbness Usually history of vascular disease,
smoking, claudication
Harbingers Deformity, callus, dry skin, Discoloration
distended dorsal foot veins (if
Charcot present)
Mode of occurrence Fortuitous, patient may be Usually sudden in onset with
unaware of its onset discoloration and pain
Caused by Trauma: usually footwear Predominantly occurs spontane-
related, but may include sharp ously but may occur following
objects, burns from hot objects trauma
e.g. radiators, hot beaches
(‘holiday foot’)
Foot temperature Normal (or warm if A–V shunt) Usually cold
Foot pulses Palpable, usually bounding May or may not be palpable.
pulse Usually feeble pulse if present
ABPIs Normal or elevated Reduced (<0.9). On rare occasions,
may be elevated if incompressible
arteries
Duplex ultrasound Usually normal – triphasic Biphasic or monophasic
waveforms
Edges of ulcer Raised: Callused due to Surrounding skin atrophic. May be
pressure, may be undermined red in the absence of infection
(dependent rubor)
Site of ulcer Mostly pressure/friction areas, Edges of the foot, apices of toes,
plantar aspects of the foot, back of the heel. But may be
inter-digital areas located anywhere on the foot
Appearance of ulcer Indented borders Same as neuropathic, but the
presence of gangrene is pathogno-
monic of ischaemic ulceration
Pain during Mitigated by loss of sensation. Neuroischaemic usually moderate
debridement Usually very little pain (mild to pain but purely ischaemic
moderate neuropathy) or no ulceration is exquisitely tender to
pain (severe neuropathy) touch
Patient behavioural Disconnected. May walk into Usually very much aware of ulcer
response to ulcer pain office with a ‘large’ plantar and presents with a limp
ulcer without a limp
Adapted from Ndip and Jude [10]
the free end of the 10 g MF is approached to touch the skin. Gentle pressure is then
applied (avoiding sliding movements on the skin) until the MF starts to bow and this
is maintained for 1 second. The patient should start feeling the MF as it starts to bow
and responses are recorded as ‘yes’ or ‘no’. The International Working Group on the
Diabetic Foot (IWGDF) recommends that for each site tested, the procedure is
repeated three times [14]. A normal response (‘yes’) is recorded if the patient is able
to correctly detect two out of three applications.
Table 2.2 The modified neuropathy disability score (NDS)

Normal response
Present after Maximum
Modality tested Yes No reinforcement score
Pinprick 0 1 – 1
Monofilaments 0 1 – 1
Vibration 128 Hz 0 1 – 1
Ankle reflex 0 2 1 2
Total score per foot 5
Note that the overall NDS reported for a given patient is the total score derived from the sum of the
individual score for each foot; maximum = 10 for both feet
Fig. 2.1 Technique for

assessing neuropathy using
the 10-g monofilament
Repetitive use of the MF causes them to lose their strength and therefore not
provide the requisite buckling force of 10-g. It is generally recommended that the
same MF should not be used for more than ten measurements per day [15].
The Modified Neuropathy Disability Score (NDS)
A composite score known as the neuropathy disability score has been proposed to
grade the severity of neuropathy based on the testing of four modalities. The sensory
modalities include: heat/cold, vibration, pinprick, and the ankle reflex. An NDS score
3–5 is considered to be consistent with the presence of mild neuropathy; 6–8 moder-
ate neuropathy and 9–10 severe neuropathy respectively [16] (Table 2.2).
Heat/Cold Discrimination
When used in the evaluation of NDS, this modality is assessed using a neurotherm
which is a rod with one end being plastic (feels ‘normal’) and the other end made
of metal (feels ‘cold’). With the patient supine and rested, the free end of the rod
is touched to the dorsum of the hallux. Scores 0 = normal; 1 = abnormal (not able
to distinguish hot from cold ends). Alternatively, the base of a tuning fork is
immersed alternately in a beaker of ice/warm water for testing cold/hot sensation
respectively. This latter method is less practical and hence rarely used in routine
practice.
Pinprick
A neurotip is a suitably designed pin with a sharp and a blunt end. It is applied on
the dorsum of the foot proximal to the toenail. Inability to correctly identify the
sharp and blunt ends when applied to the skin indicates LoPS (score = 1).
Tuning Fork
The standard 128 Hz tuning fork is used to assess vibration sensation. It is typically
applied to the apex of the big toe (hallux).
Deep Tendon Reflex
Deep tendon reflex can either be present (normal, score = 0), present after rein-
forcement (Jendrassik maneuver) (abnormal, score = 1) or absent (abnormal,
score = 2). It is assessed using a suitable tendon hammer and when used to evalu-
ate the neuropathy disability score, the ankle reflex is the preferred site for
testing.
Vibration Perception Threshold (VPT)
This is assessed using a neurothesiometer (powered by a rechargeable battery) or a

biothesiometer (needs current source) (Fig. 2.2). A VPT > 25 V is generally consid-
ered as evidence of clinically significant sensory neuropathy (score = 1) and a risk
factor for foot ulceration [15–17].
Fig. 2.2 Technique for assessing neuropathy using the biothesiometer
Peripheral Arterial Disease
Femoral, popliteal, dorsalis pedis and posterior tibial pulses should be palpated com-
paratively on both lower limbs and their strength noted [15]. Absent or weak pulses
indicate occlusion, stenosis or calcification at a level proximal to the abnormal finding.
Although for research purposes (and where no other tests are available) the absence of
two or more of the four foot pulses is commonly taken to represent PAD [18], this
does not obviate the need for further assessment if only one foot pulse is abnormal. In
fact, the presence of palpable foot pulses does not necessarily exclude PAD as collat-
eral blood flow may be enough to maintain a palpable pulse but not enough to heal a
distal ulcer especially in patients with infrapopliteal disease [19]. However, a system-
atic review of published data showed that in asymptomatic patients, a foot pulse
abnormality is a useful tool with which to diagnose PAD (likelihood ratio, 3.10; 95%
CI, 1.40–6.60); conversely, the presence of normal pulses decreases the likelihood of
moderate to severe disease (likelihood ratio 4.70; 95% CI, 2.20–9.90) [20].
Ankle-Brachial Pressure Index (ABPI) Measurement
The ABPI is the ratio of the systolic blood pressure taken at the ankle (posterior
tibial artery) to that taken at the ipsilateral brachial artery. It is a reproducible and
reasonably accurate, non-invasive method for the detection of PAD and the determi-
nation of its severity. The tools required to record an ABPI include a 5–10 MHz
Doppler probe and a blood pressure cuff. An ABPI < 0.9 is generally taken as a cut-
off to diagnose PAD. The complete diagnostic criteria for PAD based on the ABPI
are as follows [21].
• Normal if 0.91–1.30
• Mild obstruction if 0.70–0.90
• Moderate obstruction if 0.40–0.69
• Severe obstruction if <0.40
• Poorly compressible if >1.30
In the diabetic population, an ABPI value greater than 1.3 suggests poorly com-
pressible arteries at the ankle level due to the presence of medial arterial calcification
(MAC). In fact, MAC can be present in up to a third of people with diabetes.
Furthermore, ABPI will not detect obstructions in branches of the popliteal artery
nor in segments distal to the ankle itself. Therefore, the diagnosis of PAD by ABPI
alone in people with diabetes is less reliable.
Toe Blood Pressure and Toe-Brachial Pressure Index (TBI)
The digital arteries are usually spared in diabetic patients with MAC. In addition,
great toe blood pressure readings are a reflection of overall occlusions in all seg-
ments. In patients with possible critical limb ischaemia, further NIVAs may help in
the clinical decision for performing re-vascularisation. A toe pressure of less than
40 mmHg or a toe waveform <4 mm may predict impaired wound healing. It has
been reported that toe pressures less than 30 mmHg substantially increases the risk
of amputation and a toe brachial index (TBI) of less than 0.10 is associated with
higher mortality [22, 23]. This has led to the widespread use of toe pressure mea-
surements in individuals with suspected critical limb ischaemia (CLI). However, it
remains uncertain whether routine assessment of toe pressures has any impact on
clinical outcome in individuals with CLI.
Transcutaneous Oxygen Tension (TcPO2)
The transcutaneous oxygen tension (TcPO2) is essentially a research tool that is

rarely used in day-to-day wound management. It is time consuming and not ideally
suitable in primary care settings. TcPO2 is an indicator of skin perfusion. In diabetic
foot disease, it is mainly used as a predictor of wound healing in individuals with
neuro-ischaemic ulcers in whom revascularisation is either not possible or is not
indicated. A TcPO2 less than 20 mmHg is associated with nearly 40-fold increase in
the risk of early wound healing failure [24].
Doppler Ultrasound Waveforms
The detection of abnormal waveforms on Doppler ultrasound probing of peripheral

arteries is a learned skill. The waveform can either be monophasic (normal), biphasic
(abnormal) or triphasic (abnormal). Any abnormal waveform recorded warrants

careful vascular assessment.
Other non-invasive vascular assessments include plethysmography and segmen-
tal blood pressures. Although foot pulses, the TBI, and qualitative waveform analy-
ses are highly sensitive screening methods, the presence of peripheral neuropathy
has been shown to be associated with a reduced sensitivity and poor specificity of
foot pulses, a reduction in sensitivity of the ABPI, and a reduction in the specificity
of the TBI and qualitative waveform analysis [25].
Wound Assessment
Wound measurement is integral to the successful management of diabetic foot

ulcers [26]. Wound assessments may include subjective assessment of wound
colour, odour, slough/exudates and the degree of surrounding inflammation (red-
ness, swelling), but also include more objective measurement of wound size. The
subjective assessments of colour, odour and slough/exudates may be important in
determining the presence and extent of wound infection, but also guide manage-
ment strategies including the choice of wound dressing and the use of empirical
first line antibiotic. Similarly, the amount of callus formation around an ulcer is
directly related to the wound pressure, and thus provides a subjective visual
assessment of the adequacy of offloading. More objective assessment such as
biomechanical evaluation and gait/pressure analysis are not routinely used in
everyday clinical practice but may be useful in evaluating recalcitrant neuro-
pathic ulcers.
Wound probing using a suitably designed probe is an important test to assess the
depth of wound infection and can provide invaluable information on the presence of
diabetic foot osteomyelitis.
However, wound size is an important measurement integral to foot care, widely
used to assess progress in healing. In fact, regular assessment of wound size predicts
healing and provides information used to guide treatment decisions [26, 27]. Current
methods used for wound measurement in clinical practice are based on ruler tech-
niques or wound tracing.
Ruler Technique
This technique assumes that the wound is approximately rectangular. The wound is
measured after adequate debridement and removal of slough and just prior to apply-
ing the wound dressing. The longest axis (length) and the greatest width perpen-
dicular to this axis are measured, and the product of these two variables gives the
area of the wound.
The Elliptical Wound Measurement Method
It is based on the assumption that wound shape can be modelled as an ellipse. The
(visually) longest axis of the wound (length) and that of the orthogonal axis (width)
must be measured as described for the ruler technique. The area is then calculated
using the formula: Area (mm2) = Length (mm) × Width (mm) × 0.25 × p.
Wound Tracing (Visitrak)
Two types of wound tracing methods are available: the hand (acetate) planimetry and
the digital planimetry (Visitrak™, Smith & Nephew, Hull UK). Recently, a modification
of elliptical method has been described – the digital elliptical method (DEM) – which
uses optical photographic wound imaging techniques and specific software to create a
three-dimensional wound image. This image is then fed unto computer software to
provide an accurate, reproducible estimate of wound size [28].
Management of Diabetic Foot Disease
Management of Diabetic Painful Neuropathy
General Measures
Management of diabetic peripheral sensorimotor neuropathy should aim to:

• prevent disease progression and thus forestalling further nerve damage,
• providing symptomatic relief including adequate pain management,
• preventing neuropathic complications including psychosocial aspects.
There is strong evidence linking hyperglycaemia to the development and pro-
gression of diabetic neuropathy [29–31]. Also, sudden changes in blood glucose
control has been associated with the development of neuropathic pain [32]. This can
occur after starting insulin (insulin neuritis) or following a successful pancreas
transplant [33]. For these reasons, we underline the need for optimum as well as
stable glycaemic control in the management of neuropathy and neuropathic pain.
Pharmacological Therapy for Managing Diabetic Sensory Neuropathy
In general, the treatment of DN must be tailored to individual patients, taking into

consideration the specific clinical circumstance and associated co-morbidities.
A patient with neuropathic pain and co-existing depression or anxiety may benefit
from a neuropathic agent that is either anti-depressive or anxiolytic. Side effects are
common and care must be taken when prescribing certain agents to elderly people
(anticholinergic effect), heavy machinery workers (sedative effect) or those with
renal impairment (toxicity, dose modification). Two broad categories of treatment
can be considered: (i), those that target the underlying pathogenetic mechanism of
DN and (ii) those that provide symptomatic/pain relief.
Therapeutic Modalities Based on the Pathogenesis of DN
Almost every pathway involved in the pathogenesis of DN has been explored in an

effort to find novel therapeutic approaches. The rationale for this approach is that
since glycaemic control for most patients is not expected to be optimal, these thera-
pies might be effective even in individuals with poor glycaemic control through
their action on specific neuronal pathways. However, while some have shown initial
promise, the overall results have been varied and mixed. These agents are not
licensed for use in DN in the UK or US.
• protein kinase C (PKC) inhibitors,
• anti-oxidants e.g. g-linolenic acid (GLA)
• vasodilators e.g. ACE inhibitors,
• aldose reductase inhibitors (ARIs), and
• C-peptide.
• Nerve growth factors e.g. vascular endothelial growth factor (VEGF).
Therapeutic Modalities for Symptom (Pain) Relief
It should be emphasized that guidelines for the treatment of diabetic peripheral

neuropathic pain (DPNP) may be different and country-specific.
Tricyclic Antidepressants (TCAs)
Tricyclics have been used for the treatment of neuropathic pain for approximately
three decades. In general, tricyclics have been replaced as first line agents in the UK
and also in the US as a result of the higher frequency of adverse events as compared
to other agents. However, when tolerated, they are efficacious in treatment of DPNP.
They have different potencies and are either noradrenalin reuptake inhibitors (NRIs
e.g. nortriptylline and desipramine), or both noradrenalin and serotonin reuptake
inhibitors (NSRIs e.g. chlomipramine) [34]. TCAs have a good dose–response rela-
tionship with relief of neuropathic pain occurring before their anti-depressive effects
[35]. Adverse side effects are common and dose-related. Hence dose titration is
essential starting from the smallest dose and increasing up until desired pain relief or
development of adverse effects. Their affinity for muscarinic receptors is responsible

for the typical atropine-like side effects of dry mouth, blurred vision, constipation,
urine retention, recumbent tachycardia and memory impairment, which is especially
relevant in the elderly, where delirium can result even from ‘therapeutic’ doses. In a
cost utility analysis of common first line agents, Desipramine (100 mg/day) was
found to be more cost effective than gabapentin or pregabalin for treating painful
diabetic neuropathy; its cost-effectiveness was similar to duloxetine (60 mg/day)
[36]. TCAs are transported in blood protein-bound and metabolised via the CYP450
pathway and thus have the potential of interacting with drugs such as beta-blockers,
levothyroxine and statins [37].
Selective Serotonin-Reuptake Inhibitors (SSRIs)
The effect of SSRIs on neuropathic pain is mediated via beta-2 adrenoceptor activa-
tion resulting in a blocking of the reuptake of the neurotransmitter amines, serotonin
(5-hydroxytryptophane, 5-HT) and/or noradrenalin [38]. Duloxetine is the most
commonly used SSRI in the treatment of DN and has both analgesic and major
antidepressant effects. It is approved by the Food and Drug Administration (FDA)
for the management of DPNP (US) and the recent NICE guidelines, place this drug
as first line agent for DPNP in the UK. Duloxetine and venlaflaxine are mainly
serotonin reuptake inhibitors with minimal noradrenalin reuptake inhibition.
Duloxetine is effective, generally well tolerated even by elderly patients [39], has a
simple dosage and these properties are likely to enhance patient compliance. Nausea
is a common side effect reported by 10% of individuals [40].
Anticonvulsants
Anticonvulsant therapies are now used widely for symptomatic management of

painful neuropathies. Their primary mechanism of action includes calcium channel
blockade, sodium channel blockade, potentiation of gamma-amino butyric (GABA)
activity and antagonism of glutamate at N-methyl-D-aspartate (NMDA) receptors
or a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) [41].
Pregabalin is an a2-d ligand similar to g-amino butyric acid (GABA) whilst gabap-
entin is a GABA analogue. GABA modulates the influx of calcium at presynaptic
voltage-sensitive calcium channels, resulting in a decrease in the release of several
neurotransmitters, including glutamate and norepinephrine. The anticonvulsants
have relatively lesser drug interactions and side effects. The newer ones like pregab-
alin, gabapentin, and lamotrigine have fewer side effects and are now being used in
lieu of carbamazepine. In general, anticonvulsants are efficacious and have a more
favourable side effect profile compared to TCAs. The main adverse effects reported
with pregabalin in studies are dizziness, somnolence, and peripheral edema [42].
Pain and sleep improvements noted with pregabalin are dose related: the greatest
and quickest effects noted in patients treated with 300 mg b.d. [43]. Pregabalin also
has anxiolytic properties and this is of potential benefit in patients with anxiety
states. Pregabalin, lamotrigine and gabapentin have no interaction with CYP450
pathway hence are unlikely to have major drug interaction. Carbamazepine in con-
trast, is metabolised in the liver via CYP450 pathway and can affect the metabolism
of drugs commonly co-prescribed in diabetes such as gliclazide, glimepiride, biso-
prolol, atorvastatin and simvastatin.
Opioids
Opioids are mostly used as adjuncts in the treatment of painful neuropathy. Their
concomitant use with anticonvulsants may achieve better pain relief at lower doses
hence mitigating the occurrence of side effects. The co-administration of prolonged-
release oxycodone in patients on existing gabapentin therapy has been shown to
improve pain relief and to reduce sleep disturbance without exacerbation of opiate-
induced side effects [44]. Similarly, the combination of gabapentin and morphine
has been shown to achieve better analgesia at lower doses of each drug than either
as a single agent [45]. This apparent synergism between anticonvulsants and opiates
is not limited to gabapentin. A recent open-label, prospective, multicenter study
also showed that combining pregabalin with prolonged release oxycodone, was
safer and more effective than either drug alone [46]. Tramadol is a central opioid
analgesic that is occasionally used in the treatment of neuropathic pain although its
efficacy has not been established in randomised studies.
Topical Treatment and Physical Therapy
Topical capsaicin, isosorbide dinitrate spray, lidocaine, and transdermal clonidine

have on occasion been shown to improve neuropathic pain in limited studies.
Management of Neuroischaemic Lesions
General Measures
In addition to the general principles of wound care, optimum glycaemic control is

necessary to enhance wound healing. In addition, other factors that may interfere
with wound healing need to be addressed, such as:
• correction of anaemia;
• treatment of lower limb oedema to improve microcirculation;
• minimizing the likelihood of falls or trauma;
• consideration for smoking cessation;
• adequate nutrition;
• antiplatelet agents;
• Lipid-lowering therapy.
Specific Management
Debridement
When used in the treatment of diabetic foot wounds, the term debridement refers to
the removal of devitalized (dead, damaged, or infected) tissue from an ulcer/gan-
grene until healthy tissue is exposed, hence improving the potential for healing.
Debridement should be done by a trained foot care specialist (usually a podiatrist or
surgeon) and debris and necrotic material removed so that infection is less likely to
occur. There are different types of debridement.
In autolytic debridement, semiocclusive or occlusive dressings (e.g., hydrogels,
hydrocolloids) are used, which maintain a moist environment and enhance the
body’s natural debridement.
The use of maggot debridement therapy (application of sterile larvae of green
bottle fly, Lucilla sericata) on neuropathic ulcers has become widespread. They
digest necrotic tissue (by ingesting slough and debris) and pathogens (bactericidal
secretions), and therefore promote granulation. However, they are not recommended
in ischemic ulcers as they can aggravate or cause severe pain. The mechanism
through which pain is generated is not clearly understood but it is thought to be due
to the relatively low pH (pH 7.2) of the secretions from the maggots, which may
accelerate local tissue damage. Currently, sterile maggots are available in biobags
and this reduces some of the adverse effects related to their use.
There is little information on the utility of newer methods of debridement (e.g.,
ultrasonic Versajet systems, Biogun) in neuroischemic ulceration. The Versajet
Hydrosurgery System (Smith & Nephew, Largo, FL, USA) is a new device intended
for wound debridement. The system is based on the Venturi effect using a high pres-
sure water jet to ‘cut’ away dead tissues that are rapidly sucked up into a container.
It permits cutting over rounded surfaces. The user be it a surgeon or a podiatrist
must be trained before using the equipment.
A Cochrane review recently reported on the role of debridement in the manage-
ment of diabetic foot ulcers [47]. Six randomised controlled trials of debridement
were identified: four assessed hydrogels, with an additional study evaluating larval
therapy against hydrogel and one evaluated surgical debridement. Pooling the three
RCTs which compared hydrogel with gauze or standard care suggested that hydro-
gels are significantly more effective in healing diabetic foot ulcers (Relative Risk
1.84, 95% Confidence Interval (CI) 1.3–2.61). Surgical debridement showed no
significant benefit over standard treatment. One small trial, available in abstract
form only, suggested that larvae resulted in a greater reduction in wound area com-
pared with hydrogel, but this evidence has not been confirmed by publication of full
trial results. In addition, a small study of 13 subjects with MRSA colonised diabetic
foot ulcers showed that application of maggots eradicated MRSA in 12 out of the 13
wounds [48]. Other debridement methods such as enzyme preparations or polysac-
charide beads have not been evaluated in diabetic foot ulcers.
Dressings
Numerous dressings are used in the treatment of diabetic foot ulcers. Owing to lack
of published data, there is no evidence to suggest that a particular type of dressing
is superior to another in diabetic foot ulcers. The choice of any dressing would
therefore depend on its availability as well as on local experience. However, non-
adherent dressings are usually advocated for use on diabetic foot ulcers. Other
desirable properties include ease of removal from the foot and the ability to accom-
modate pressures.
Role of Topical Negative Pressure of Wounds
Vacuum-assisted closure (VAC) using topical negative pressure (−125 mmHg) is a

resurgent therapy in foot ulcer care based on the device developed by Kinetic
Corporation Inc (San Antonio, Texas). [49–51]. It is thought to remove excess
slough, enhance granulation, and accelerate healing through liberation of angio-
genic growth factors from the wound bed [51]. It can also transform a large complex
wound to a simple wound, thus permitting surgical closure or healing by secondary
grafting [52]. There are several makes that offer this technique that is currently
termed Topical Negative Pressure (TNP). Although TNP remains a relatively safe
modality for treatment of diabetic foot wounds, various Cochrane reviews of studies
examining the use of VAC in diabetic foot wounds have concluded that the current
evidence base is only supported by a limited number of under-powered, poorly-
designed studies [53, 54]. !!!
Footwear and Offloading
Offloading is a pivotal part in the treatment plan for diabetic foot ulcers.
Appropriate footwear must be provided to all patients. The main aim is to pro-
tect the foot from high pressure and trauma, while ensuring that blood flow to
the foot is not compromised [55, 56]. Where possible, consultation should be
initiated with an Orthotist or surgical shoe maker. The total contact cast (TCC)
is considered the gold standard to heal diabetic foot ulcers [57, 58] with an
average healing time for foot ulcers of 6–8 weeks. However, its use is limited
by the fact that its application requires a specialized cast technician or physi-
cian, thus consuming significant human resources. Also it is an awkward device
and presents practical challenges to patients that wear them – the physician is
frequently required to explain the risks and benefits before patients are ame-
nable to accepting this type of offloading. Therefore, removal cast walkers
(RCW) have been designed that offer similar advantages to the TCC [ 59, 60]
but are relatively inexpensive. The efficacy of the RCW may be limited by
patient non-adherence, however, this can be overcome by merely wrapping
the RCW with a layer of cohesive tape or plaster bandage – a technique termed
the ‘instant’ total contact cast (iTCC) [61–63]. Examples of a RCW include the
DH Walker, and the AircastÒ, California, USA. Other forms of offloading
include the healing surgical sandals, scotch cast boots, soft/slipper casts, felted
foam dressings and half shoes. Their ability to offload IS variable, but gener-
ally inferior to the TCC and the RCW.
Treatment of Infection
Diagnosis of infection in diabetic foot ulcers in general can be problematic because

local neuroinflammatory signs (pain, redness, swelling, raised local temperature) of
infection may not be present. In addition, systemic signs may only be present in
severe infections (osteomyelitis, septicaemia) although their absence should not
deter the clinician from considering severe infections especially in the face of
delayed wound healing. Pointers of infection in a diabetic foot wound may include
increased slough/exudate, pus, foul smell, pain, warmth, and poor blood glucose
control. Good clinical acumen may be sufficient to diagnose infection of an ulcer
and further investigations may only be warranted to diagnose osteomyelitis and this
should include a plain X-ray. On some occasions, a magnetic resonance imaging
scan and/or labelled leukocyte imaging scan may be required to confirm
osteomyelitis.
Empirical antibiotics are usually started after an appropriate tissue specimen has
been sent to microbiology for stains and cultures, bearing in mind that obligate
anaerobes are most like to be present alongside usual infective organisms. For
empirical therapy, antibiotics with activity predominantly against Gram positive
organisms (staphylococci and streptococci) [64] and broad-spectrum antibiotics
with increased activity against Gram negative organisms and obligate anaerobes
[65] appear equally effective. Ancillary investigations may include liver function
tests, C-reactive protein, full blood counts, urea, and electrolytes. Various guide-
lines have been proposed for the treatment of diabetic foot osteomyelitis, the most
exhaustive being that endorsed by the Infectious Diseases Society of America [66,
67]. Overall, the evidence supporting the use of any specific antimicrobial is thin,
hence local experience and/or guidelines should be consulted where possible.
Table 2.3 lists commonly used antibiotics for diabetic foot infections including their
indications and dosages.
Table 2.3 Commonly used antibiotics in diabetic foot infections

Antibiotic Indications Dose Frequency Route Comments
Co-amoxiclav Cellulitis, mild to 375/625 mg tds po Good first line
moderate soft empirical
tissue infections antibiotic until
sensitivities are
available
1.2 g tds iv Alternative route if
oral intake not
possible
Ciprofloxacin Broad-spectrum 250/500/750 mg bd po/iv Care must be taken
antibiotic for when using the iv
mild-moderate route
infections. Also
used in combina-
tion with
clindamycin for
bone/joint
infections
Flucloxacillin Cellulitis 500/1,000 mg qds po/iv
staphylococcal
soft tissue
infections
Amoxicillin Skin and soft 500 mg tds po
tissue infections
Metronidazole First line 400 mg tds po
anti-anaerobic 500 mg tds iv
agent. Mostly
used when
suspected
infection in
ischaemic ulcers
Clindamycin For joint and bone 150/300/450 mg tds po Excellent
infections, 600 mg iv bioavailability to
particularly bone and joints
Erythromycin staphylococcal 500/1,000 mg bd po
Clarithromycin infections, mild to 250/500 mg bd po
moderate skin and
soft tissue
infections, also
used in cases of
penicillin allergy
bd twice daiuly, tid/tds thrice daily, qds four times a day, po orally
Adjunctive Therapies in the Management of Diabetic Foot Lesions
Platelet Derived Growth Factor (PDGF)
Clinical studies have examined the topical application of exogenous, autologous or

recombinant growth factors and/or cytokines to accelerate wound healing in
chronic wounds. Approved over a decade ago, the human platelet derived growth
factor BB (PDGF-BB), is the only recombinant growth factor, approved for clini-
cal use in diabetic foot ulcer. We found four multicentre RCTs assessing the role of
Lyophilized recombinant human PDGF, beta-beta homodimer (rhPDGF-BB),
purified from genetically engineered yeast in the treatment of chronic non-infected
diabetic foot ulcers and one non-systematic review [68]. All patients in the RCTs
received standard care including adequate debridement. The first RCT was a phase
II company-led (Johnson & Johnson) trial [69] whilst the others were phase III tri-
als [70]. Inclusion criteria were similar in all four studies and comprised: (a)
Chronic (at least 8 weeks’ duration), nonhealing, full-thickness, lower extremity
ulcers resulting from diabetes mellitus; (b) at least one ulcer between 1 and 100 cm2
in area (as determined by simple length × width); (c) ulcers free of infection based
on clinical examination and radiographs; (d) adequate arterial blood supply
assessed by measuring transcutaneous oxygen tensions (TcPO2) of 30 mmHg or
greater on the dorsum of the foot or at the margin of the ulcer if it were not on the
plantar surface. Of the 922 patients treated, 874 (95%) had baseline ulcer areas that
were less than or equal to 10 cm2. The probability of complete wound healing was
significantly higher with rhPDGF-BB gel at a dose of 100 mg/g compared with
placebo gel treatment (p < 0.007). Treatment with rhPDGF-BB gel at a dose of
100 mg/g also significantly decreased the time to complete healing by 30%
(14 weeks versus 20 weeks, p = 0.01). Adverse events and ulcer recurrence rates
were similar in both treatment groups.
Granulocyte Colony Stimulating Factor (G-CSF)
Cruciani et al. performed a meta-analysis to examine the role of G-CSF added to usual
care on rates of infection, cure and wound healing in people with diabetes who with a
foot infection [71, 72]. They found that adding G-CSF did not significantly affect the
likelihood of resolution of infection or wound healing, but was associated with reduced
lower extremity surgical interventions (RR 0.37; 95% CI 0.20–0.68), including ampu-
tation (RR 0.41; 95% CI 0.18–0.95). Additionally, providing G-CSF reduced the
duration of hospital stay (Mean Duration, –1.40 days; 95% CI, –2.27 to −0.53 days),
but did not significantly affect the duration of systemic antibiotic therapy (MD,
–0.27 days; 95% CI, –1.30 to 0.77 days). The authors suggested that clinicians might
consider adding G-CSF to the usual treatment of diabetic foot infections, especially in
patients with a limb-threatening infection, but it is not clear which patients might
benefit
Honey
Although honey has been used since antiquity in the treatment of chronic ulcers,
there is a striking dearth of data from carefully designed RCTs or prospective stud-
ies. Although one study suggested equivalence to polyvidone iodine in Wagner
grade 2 diabetic foot ulcers [73], the design and statistical analysis of this study
failed to use convincing methodologies.
Hyperbaric Oxygen Therapy (HBOT)
Based on the rationale that the common denominator in many wounds is tissue
hypoxia, hyperbaric oxygen therapy (HBOT) has been used for several decades in
the treatment of chronic wounds. Whilst there are several HBOT facilities readily
available in North America, China, Russia and Cuba, it is not widely used in Europe
and Australasia. HBOT can be administered either locally or systemically. Two
independent systematic reviews on HBOT concluded that systemic HBOT may
reduce the incidence of major amputation in people with diabetic foot ulcers [74,
75]. However, most of the studies evaluated were relatively small. Therefore, further
evidence is required from larger, more robust and blinded studies. Nonetheless,
HBOT may be considered in patients with ischaemic ulcers and where revascular-
ization is not possible. The benefit of topically administered HBOT has not been
established.
Conclusion
Accurate, reliable and reproducible measurements have been important corner-

stones in medical practice used in objective assessment and benchmarking success.
In the area of diabetic foot ulceration, accurate determination of neuropathic and
vascular status as well as ulcer characteristics is a prerequisite for assigning risk,
guiding management strategies, monitoring clinical progress and providing evi-
dence based prognosis. Various measurements are possible but few actually influence
clinical decisions. Accurate assessment of clinically relevant neuropathy with loss
of protective sensation involves 10-g monofilament testing, vibration testing and the
composite neuropathy disability score. The presence or absence of foot pulses pro-
vides a reliable indication of vascular status that can be confirmed by determination
of ankle brachial pressure index, and/or where possible, measurement of toe blood
pressure. Different wound characteristics can be assessed but the vast majority
(including slough, callus formation, exudates, redness, swelling) is subjective albeit
in an individual patient, may provide useful insights on clinical progress. Accurate
determination of ulcer size is widely accepted as an indication of wound closure and
thus, of wound healing. Managing diabetic foot disease requires attention to neuro-
pathic pain relief, revascularisation of ischaemic lesions and appropriate wound
care. Although considerable experience has been accrued in the management of foot
disease in diabetes, there remains a significant lack of evidence-based recommenda-
tions and most guidelines are based on consensus panels and expert opinions.
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Chapter 3
Atypical Ulcers
Jennifer Tang and Robert S. Kirsner
Keywords Vasculitis • Pyoderma gangrenosum • Ulcers of infectious etiology

• Vasculopathy • Calciphylaxis • Malignancy • Spider bites • Chemical burns • Post
radiation ulcer • Factitial ulcer • Drug induced ulcer • Epidermolysis bullosa
Introduction of Diagnostic Measures for Atypical Wounds
Wound measurement may be take form of (1) diagnostic measures, (2) measure-
ments to determine wound progress, or (3) measures to determine wound prognosis
and may be achieved through several means. This chapter will focus on wound
diagnosis. With regard to diagnostic measures, depending on clinical suspicion, a
wound swab or tissue biopsy is most commonly pursued either separately or in
conjunction. After a tissue sample is obtained, it may be modified in various ways
including hematoxylin and eosin staining (H&E), immunohistochemical stains,
immunofluorescence, microbiology culture, and polymerase chain reaction (PCR)
in order to assist in diagnosis. These measures should be the forefront approach to
tailoring diagnosis and treatment of an atypical wound.
J. Tang, B.S.
Department of Dermatology,
R.S. Kirsner, M.D., Ph.D. (*)
Department of Dermatology and Cutaneous Surgery,
University of Miami Miller School of Medicine,
1600 N.W. 10th Avenue, RMSB, Room 2023-A,
Miami, FL 33136, USA
e-mail: rkirsner@med.miami.edu

40 J. Tang and R.S. Kirsner
Tissue Biopsy
Various biopsy techniques exist, each with its own advantages and disadvantages.
• Excisional biopsy is excellent for sampling deep inflammatory processes, includ-
ing panniculitis.
• Punch biopsy provides typically a 3–4 mm cylindrical plug in a rapid manner and
good healing without the need for suturing. The only caution is possible sam-
pling error, more common in larger lesions.
• Shave biopsy, appropriate for epidermal processes such as skin cancer, usually
does not provide samples of adequate depth when applied to ulcers and mainly
reserved for protuberant lesions.
• Curettage biopsy is reserved for collecting debridement material for analysis,
specifically for superficial sample collection.
Technique for Wound Swabs
This is a technique where a swab takes a topical sample of a wound. The swab may
then be processed to render a culture or undergo polymerase chain reaction. A
wound swab is advantageous in that it is non-invasive, as it samples the surface of
the wound, and simple to perform. However, its use is restricted to detecting infec-
tious etiologies of atypical ulcers.
Hematoxylin and Eosin (H&E)
Hematoxylin and eosin (H&E) staining is commonplace in dermatology. The biopsy

of the lesion is fixed in formalin, embedded in paraffin, and finally “routinely stained”.
Hematoxylin stains cell nuclei blue and eosin stains cytoplasm red. H&E staining is
useful in visualizing epidermal, dermal, and subcutaneous features of a biopsy. It is a
relatively quick, inexpensive, and practical approach to diagnosing atypical wounds.
Special Stains
Special stains may be applied to biopsy samples when H&E is not sufficient for
diagnosis. Supplemental monoclonal antibodies may assist in identifying
inflammatory infiltrates and tumors. Useful special stains include periodic acid
Schiff (PAS), methanamine silver, Von Kossa, elastin, gram, Brown-Brenn, acid
fast, Fite, Ziehl-Neelsen, Dieterle, Steiner, and Warthin-Starry.
• PAS – fungi, parasites, glycogen, fibrin, cryoglobulinemia
• Methanamine silver (Grocott) – fungi, parasites
3 Atypical Ulcers 41
• Von Kossa – calcium

• Gram, Brown-Brenn – bacteria
• AFB/Fite-Faraco, Ziehl-Neelsen – acid-fast bacilli
• Dieterle/Steiner/Warthin-Starry – spirochetes, fungi
Immunofluorescence
Immunofluorescence testing can be performed in two ways, direct and indirect. In

direct immunofluorescence (DIF), tissue is sampled and sections from biopsies are
incubated with fluorescence labeled antibodies that are directed against imm-
unoglobulin(s), complement factors, or fibrinogen. Excitation of fluorescent label by
ultraviolet light provides the recognition signal of the antibody. In indirect
immunofluorescence, the patient’s serum is incubated with substrate tissue. Rather
than tagging the patient’s skin, the substrate tissue is labeled with fluorescent antibod-
ies. Recognition signal is achieved when patient has circulating antibodies against
antigens on the substrate tissue. A third method, saline split-skin preparation is a
modification of the above two techniques, the placement of tissue (either patients or a
substrate) in a sodium bromide or sodium chloride solution (salt solution) which
causes a separation in the lamina lucida and is reserved mostly for bullous disorders.
Microbiology Culture (Tissue Sampling or Wound Fluid

Sampling)
Microbiology culture is the process of amplifying microbes in media to identify and

quantify the organism causing infection. The aim is to selectively grow the microor-
ganisms that may cause an atypical ulcer. It is a useful tool for deriving a bacterial,
viral, or fungal etiology. The media for growth may be agar for bacteria or cells that
are susceptible to infection for viruses. The proper technique to obtain a sample for
culture remains controversial. There is no consensus of whether tissue biopsy, wound
swabbing, or wound fluid aspiration is the definitive method. Tissue is obtained
optimally. Deep tissue may be obtained by punch biopsy or excision, whereas
superficial tissue may be obtained by curettage. Wound fluid is most commonly col-
lected by a cotton-tipped swab. Needle aspiration may be pursued if fluid is
copious.
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) amplifies limited amounts of DNA million-fold to

allow identification of specific DNA sequences by gel electrophoresis. This method
offers very precise diagnoses in face of trace amounts of DNA or RNA. It is particu-
larly useful when organisms cannot be cultured. Similarly, PCR may be applied to a
tissue biopsy or swab sample. PCR is an aide to diagnosing atypical ulcers because of
the ability to identify various subtypes of an organism. It is able to diagnose viruses,
such as human papilloma virus (HPV) and other herpes viruses. Also, bacterial etiolo-
gies may be detected, such as atypical mycobacteria, as well as parasitic culprits.
Electron Microscopy (EM)
Five major types of electron microscopy exist: transmission electron microscopy

(TEM), scanning electron microscopy (SEM), reflection electron microscopy
(REM), scanning transmission electron microscopy (STEM), and low voltage elec-
tron microscopy (LVEM). Electron microscopy is a diagnostic tool that enables the
examination of tissue at an ultrastructural level. This technique is employed to visu-
alize the structures within epidermis and dermis. Although EM is less utilized than
light microscopy, it remains a valuable tool for diagnosing bullous disorders, pig-
mentary disorders, viral diseases, and genotyping.
Types of Atypical Wounds
Pressure ulcers, venous leg ulcers, diabetic foot ulcers, and arterial ulcers are among
the most common types of chronic wounds. Atypical or unusual causes of wounds
are those that do not fit within these categories, rendering them much less frequently
encountered and less well-studied. Of the estimated 500,000 leg ulcers in the United
States [1], at least 10% may be due to unusual causes [2]. Various etiologies of
atypical wounds [3] include infections, external or traumatic causes, metabolic
disorders, neoplasms, or inflammatory processes. A wound should be evaluated for
an atypical etiology if present on a location unusual of a common chronic wound,
appears different from that of a common chronic wound, has unusual symptoms
such as out of proportion pain or does not respond to conventional therapy.
Etiologies of Atypical Wounds
Inflammatory Causes
Vasculitis
Vasculitis is inflammation and necrosis of the blood vessels and while often idio-
pathic, may also be due to other diseases [4] (Table 3.1). The pathophysiology of
Table 3.1 Potential etiologies Infections

of vasculitis
Bacteria
Staphylococcus spp.
Streptococcus spp.
Mycobacterium tuberculosis
Mycobacterium leprae
Viruses
Hepatitis viruses A, B, and C
Herpes virus
Influenza virus
Human immunodeficiency virus
Fungi
Candida spp.
Parasite
Plasmodium spp.
Schistosomiasis
Medications
Antibiotics: penicillin, streptomycin, sulfonamides
Tamoxifen
Thiazides
Oral contraceptives
Chemicals
Insecticides
Petroleum products
Foods
Milk
Gluten
Connective tissue and other inflammatory diseases
Systemic lupus erythematosus
Dermatomyositis
Sjogren’s syndrome
Rheumatoid arthritis
Behçet’s syndrome
Cryoglobulinemia
Scleroderma
Primary biliary cirrhosis
Malignancies
Lymphoma
Leukemia
Multiple myeloma
vasculitis often is caused by the deposition of circulating immune complexes, com-

posed of antibody-antigen, in blood vessel walls. The clinical manifestations seen
in conjunction with atypical wounds often represents the size of the affected ves-
sel, ranging from reticulated erythema to purpura, necrosis and ulceration depend-
ing on superficial plexuses or larger, deeper vessels respectively. Extracutaneous
Table 3.2 Diagnostic tests To determine etiology

for vasculitis
Complete blood count
Hepatitis profile
Anti-neutrophilic cytoplasmic antibody (ANCA)
Rheumatoid factor (RF)
Anti-nuclear antibody (ANA)
Anti-streptolysin O titer
Cryoglobulin level
Serum protein electrophoresis
Chest radiograph
Purified protein derivative test
Throat culture
Tissue culture
To determine systemic involvement
Urinalysis
Renal function tests
Chest radiograph
Liver function tests
Stool guaiac
Complete blood count
involvement may be seen and may lead to other end organ damage [5]. Tissue
biopsy is most useful if performed early and immunofluorescence also best per-
formed early, may be used to detect the type of immunoglobulin involved. For
example, IgA vasculitis is more commonly associated with renal involvement.
Tissue culture is utilized if suspicion exists for an infectious etiology (Table 3.2).
If the diagnosis is confirmed by histology, it is imperative, as noted above, (1) to
evaluate other organ systems for involvement and (2) to attempt to determine the
etiology. Treatment depends on the extent of the skin disease and whether other
organ systems are involved. This includes agents such as colchicine, dapsone, anti-
histamines, nonsteroidal anti-inflammatory agents are used and if extensive skin or
systemic involvement, systemic steroids and steroid sparing immunomodulatory
agents may be used [6] (Table 3.3). Supportive care consists of leg elevation and
compression.
Pyoderma Gangrenosum
Pyoderma gangrenosum is a misnomer as it does not represent an infectious pro-

cess. Rather it is actually an inflammatory process of unknown etiology presenting
as painful, violaceous skin ulcers with undermined borders [7]. It typically affects
adults with recurring, destructive ulcers, which begin as pustules and resolve with
cribriform scars. Variants include ulcerative, pustular, bullous, vegetative, and per-
istomal types. The mechanism of development is uncertain, although inflammatory
cytokines are involved and pathergy (the development of lesions in areas of trauma)
Table 3.3 Vasculitis Antihistamines

treatment options
Nonsteroidal anti-inflammatory drugs
Anti-inflammatory antibiotics
Topical steroids Mild disease
Dapsone
Colchicine
Potassium iodide
Dapsone
Stanozolol
Immunosuppressant drugs
Systemic steroids
Cyclophosphamide
Methotrexate Extensive or systemic
Azathioprine
Cyclosporin
Mycophenolate mofetil
Tacrolimus
Plasmapheresis
is often considered to be an exaggerated response of these inflammatory cytokines.

In patients with this disorder, even mild trauma to the skin can lead to pustules or
ulcers. The disease tends to have a waxing and waning course. If a diagnosis of
pyoderma gangrenosum is made, it is important to search for underlying diseases as
it coexists with many other conditions [8] (Table 3.4). Currently there is no curative
treatment, but long term remission occurs. For limited or mild disease, topical or
intralesional steroids are indicated [9]. In more severe or widespread disease, anti-
biotics, systemic steroids, and immunosuppressants such as cyclosporine and
infliximab [10] may be used (Table 3.5). To date, only an anti tumor necrosis factor
alpha monoclonal antibody, infliximab has been studied in a randomized fashion
but no comparative studies exist to determine relative efficacy of different treatment
options for pyoderma gangrenosum.
Infectious Causes
Atypical Mycobacterial Infection
Atypical mycobacteria cause cutaneous infection through exogenous inoculation in

genetically predisposed individuals and when predisposing factors such as preceding
trauma, immunosuppression, or chronic disease exist. Mycobacteria marinum is the
most common species to cause skin infection [11] (Table 3.6). Mycobacterial skin
lesions may present as granulomas, small superficial erosions, sinus tracts, or large
ulcers depending on the causative agent. Histology is often not diagnostic for myco-
bacterial infections as they present as granulomas and abscesses that may resemble
Table 3.4 Systemic diseases Inflammatory bowel disease

associated with pyoderma
gangrenosum Ulcerative colitis
Crohn’s disease
Arthritis
Osteoarthritis
Rheumatoid arthritis
Seronegative arthritis
Hematologic abnormalities
Leukemias
Myeloid
Hairy cell
Myelofibrosis
Myeloid metaplasia
Immunoglobulin A monoclonal gammopathy
Polycythemia rubra
Polycythemia vera
Paroxysmal nocturnal hemoglobinuria
Myeloma
Lymphoma
Immunologic abnormalities
Systemic lupus erythematosus
Complement deficiency
Hypogammaglobulinemia
Hyperimmunoglobulin E syndrome
Acquired immunodeficiency syndrome
the ‘typical’ mycobacterial diseases, leprosy and cutaneous tuberculosis. Diagnosis

often relies on tissue culture or newer techniques, such as polymerase chain reaction
and gene rearrangement studies. Interventions include antibiotic therapy, warming of
the area and excision of the cutaneous lesions with or without chemotherapy.
Buruli Ulcer
Buruli ulcer mainly occurs in tropical, developing countries and is caused by M.

ulcerans. Early lesions are nodules, papules, or plaques nodular that progress to the
ulcerative form resulting in loss of skin and soft tissue. If severe, extensive slough-
ing and massive ulceration may occur. Areas affected are the torso, face, or an entire
limb. Although rarely fatal, complications including contractures if joint involve-
ment, amputation if extensive limb involvement, and disfigurement lead to decreased
quality of life [12].
Early diagnosis is crucial and studies show that antibiotics can result in heal-
ing in greater than 90% of patients in less than a year [13] and can prevent late
complications, particularly aminoglycosides [12, 14]. In more severe cases, wide
Table 3.5 Pyoderma gangreno- Topical steroids

sum treatment options
Topical tacrolimus
Nicotine patch Topical treatment
Intralesional steroids
Steroids
Antibiotics
Dapsone
Minocycline
Clofazamine
Immunosuppressants
Cyclosporin
Azathioprine
Tacrolimus
Methotrexate Systemic treatment
Mycophenalate mofetil
Alkylating agents
Chlorambucil
Cyclophosphamide
Immunomodulators
Thalidomide
Infliximab
Etanercept
Intravenous immunoglobulin
Plasmapheresis
Table 3.6 Mycobacteria species that cause skin ulcers

Mycobacterium species Clinical manifestations Treatment options
M. marinum Swimming pool granuloma Antituberculous drugs
M. ulcerans Subcutaneous nodules Surgical excision
Deep ulcers
M. scrofulaceum Cervical lymphadenitis Surgical excision
Fistulas
M. avium-intracellulare Small ulcers with erythematous Surgical excision chemotherapy
borders
M. kansaii Crusted ulcerations Antituberculous drugs
Minocycline
M. chelonei Painful nodules and abscesses Antibiotics: erythromycin,
Surgical wound infection tobramycin, amikacin, or
doxycycline
M. fortuitum Painful nodules and abscesses Antibiotics: ciprofloxacin,
Surgical wound infection sulfamethoxazole, amikacin,
or doxycycline
excision and grafting may be recommended. Treatment of Buruli ulcers is

difficult due to antibiotic resistance mounted by organism’s ability to suppress
the host immune system. Disease progression is correlated to a decrease in the
level of interferon-g, a Th1 cytokine, and concomitant increase in Th2 cytokine

interleukin-10 [12]. Correction of these derangements has been pursued as novel
therapeutic options [12].
Deep Fungal Infections
There are two types of deep fungal infections of the skin, subcutaneous and sys-
temic mycosis. Subcutaneous mycoses develop upon traumatic introduction through
subcutaneous tissue, leading to localized disease, and eventual lymphatic or
hematogenous spread. Systemic mycoses are the result of systemic inoculation of
pathogenic fungi, most frequently the respiratory tract, with subsequent dissemina-
tion through hematogenous or lymphatic routes to other organs, including the skin.
Sporotrichosis
Sporothrix schenckii, a saprophyte, causes a subcutaneous mycosis often associated

with lymphatic spread or lymphangitis due to traumatic implantation into the skin.
S. schenckii exists in the environment and has been found on plants and animals,
including armadillos and cats. Exposure to the environment is a risk factor for infec-
tion. In immunocompromised patients, inhalation of the conidia can lead to pulmo-
nary infection and multi-organ involvement [15]. Sporotrichosis is treated systemically
with potassium iodide, itraconazole, fluconazole, terbinafine, and amphotericin B.
Topical heat may be useful because the organism grows at low temperatures.
Chromoblastomycosis
Chromoblastomycosis, a subcutaneous mycosis, is caused by several pigmented fungi

including Fonsecaea pedrosoi, Fonsecaea compacta, Phialophora verrucosae,
Cladosporium carrionii, and Rhinocladiella aquaspesra [16]. These microorganisms
are most commonly found in tropical and subtropical climates. Chromoblastomycosis
primarily affects the extremities of middle aged men with the onset of a slow-growing
papule that transitions into a verrucous nodule [16]. The lesions may be scaly or may
be ulcerated with serosanguineous crusts. Black dots within the lesions filled with
fungi may be present, representing sites of transepidermal elimination of necrotic tis-
sue. Potential resultant complications include lymphedema, elephantiasis and over
longer term, carcinoma. Diagnosis relies on scrapings from the lesion with potassium
hydroxide 20% or tissue biopsy for culture and histology. Ketoconazole, itraconazole,
terbinafine, amphotericin B, and posaconazole [17] have been used with variable
results. Pulse therapy with Itraconazole or Terbinafine and Itraconazole combination
therapy are currently recommended [18]. Excision, cryosurgery and local heat therapy
of 42°C to 45°C are additional therapeutic modalities. However, chronic lesions are
often resistant to therapy.
Paracoccidioidomycosis
Paracoccidioidomycosis (South American blastomycosis) is systemic myco-

sis caused by Paracoccidioides brasiliensis , a saprophyte of soil and decay-
ing vegetation found in tropical and subtropical climates. Infection begins in
the respiratory tract. Patients develop painful ulcers on the mouth, face, or
the extremities. Regional lymph involvement is also typical [ 19 ] . Diagnosis
can be established by direct mycologic examination, histopathologic or cyto-
pathologic examination, or culture. Treatment options are sulfamethoxazole/
trimethoprim, azoles, such as itraconazole and ketoconazole, and amphoteri-
cin B for severe cases.
Mycetoma
Mycetoma is a chronic infection of the skin and subcutaneous tissues that can be
divided into eumycetomas and actinomycetes caused by fungi and actinomycetes
respectively. As with the majority of fungal infections, it occurs worldwide but
most commonly in tropical and subtropical environments. The most common
agent in Central and South America is the bacteria Nocardia brasiliensis [20],
whereas in the United States it is the fungus Pseudallescheria boydii. A painless
nodule with local edema develops after trauma and may contain purulent dis-
charge and grains. Multiple lesions may interconnect to give rise to characteristic
sinus tracts.
Mainly a clinical diagnosis, additional examinations include visualization of
grains and filaments in discharge, biopsy or tissue culture and ultrasound [21].
Treatment is difficult. Surgical excision with chemotherapy may be effective. Oral
sulfonamides, tetracycline, aminoglycosides, rifampin, ciprofloxacin, amoxicillin-
clavulanate and azoles may be used as well. There is preliminary evidence that sug-
gests ininezolid, imipenem, and the newer triazoles, voriconazole and posaconazole
may be efficacious [22].
Vibrio vulnificus Infection
Vibrio vulnificus is a bacteria commonly found in Atlantic Coast waters. Skin infec-
tion with V. vulnificus occurs when contaminated seawater enters the body through
a break in the epidermal barrier. The bacteria produces proteolytic and elastolytic
enzymes and collagenases that promote tissue invasion. Clinical manifestations
include pustular lesions, lymphangitis, lymphadenitis, cellulitis, myositis and skin
necrosis. More profound infection may lead to hypotension, bullous cellulitis, and
necrotic skin ulcers. Rarely, primary septicemia may occur after ingestion of con-
taminated raw oysters, especially in patients with hepatic cirrhosis, diabetes, or
immunosuppression. Treatment consists of antibiotics, such as doxycycline and cef-
tazidine, and adjuvant wound care [23].
Table 3.7 Conditions leading Age ³50 years

to necrotizing fasciitis [24, 27, 28]
Alcoholism
Chronic leg ulcers
Cirrhosis or chronic liver disease
Chronic Obstructive Pulmonary Disorders
Coronary artery disease
Corticosteroid treatment
Diabetes mellitus
Herpes zoster
HIV
Hypertension
Infection of anogenital regions: anorectal, perianal,
scrotal, ischiorectal, or periurethral abscess
Intravenous drug use
Malignancy
Malnutrition
Obesity
Peripheral vascular disease
Psoriasis
Renal failure
Surgery
Smoking
Trauma: needle puncture, insect bite, fish-fin injury,
burns, lacerations, surgical wound
Necrotizing Fasciitis
Necrotizing fasciitis (NF) is a life-threatening soft tissue infection characterized by

rapidly spreading inflammation and necrosis of the skin, subcutaneous fat, and fas-
cia [24]. Rapid, early intervention is imperative to reduce estimated mortality rates
of over 70% in the past [25], though recent data have shown improvement with
mortality rates of less than 10% [25]. The incidence of necrotizing fasciitis esti-
mated to be 0.40 cases per 100,000 population [26]. Immunocompromised states,
malignancy, obesity, peripheral vascular disease, and trauma may be predisposing
for necrotizing fasciitis in certain patients (Table 3.7).
Three types of necrotizing fasciitis exist. Type 1 is caused by aerobic and anaero-
bic bacteria such as Clostridium and Bacteroides species. Type 2 is due to group A
Streptococcus (S. pyogenes) with or without a coexisting Staphylococcal infection.
Type 3 is caused by a Vibrio infection from a puncture wound by aquatic organisms.
Necrotizing fasciitis most commonly occurs on the extremities. With cephalic or
truncal involvement, mortality is greater due to resistance to treatment. Fournier’s
gangrene is a necrotizing fasciitis when genitalia are involved.
Patients initially present with a poorly demarcated area of redness and edema
with tense and shiny overlying skin resembling cellulitis. A characteristic clue is
the report of severe pain out of proportion to the clinical appearance. Early diag-
nosis is important to rapidly institute treatment and to increase the chance of
survival. Infection and gangrene may extend into the subcutaneous fat and under-
lying fascial planes including muscle. Metastatic abscesses are a rare complica-
tion that may affect viscera and pleural linings. Patients may also have systemic
findings of high fever, chills, constitutional symptoms and even multi-organ
system failure.
Vasculopathies
Cryofibrinogenemia
Cryofibrinogenemia occurs as a primary (idiopathic) disorder or in association with

underlying infection, malignancy, or collagen, thromboembolic, or vascular disease.
The clinical presentation includes painful ulcers with other findings including cuta-
neous livedo reticularis, purpura, ecchymoses, and gangrene on the leg and foot. It is
caused by in vivo occlusion of small blood vessels initiated in the distal extremities
by cryofibrinogen, consisting primarily of fibrin, fibrinogen, and fibronectin, and to
a lesser extent albumin, cold-insoluble globulin, and factor VIII. The complex is
soluble at ambient temperatures (98.6°F, 37°C) but cryoprecipitates at colder tem-
peratures (39.2°F, 4°C). Treatment directed at symptoms or at the underlying disease
[29]. Fibrinolytic agents, stanozolol, streptokinase, and streptodornase have been
used with success.
Cryoglobulinemia
Cryoglobulinemia that causes a vasculopathy is caused by the deposition of

cryoglobulin in medium and small vessel walls [ 30]. Type I or monoclonal
cryoglobulinemia is seen in patients with lymphoproliferative disorders, such
as myeloma and Waldenström’s macroglobulinemia. Type II, or mixed, cryo-
globulinemia consists of both polyclonal and a monoclonal immunoglobulin
and is most commonly associated with infectious or in fl ammatory diseases.
Type III contains polyclonal immunoglobulin and is closely linked to hepatitis
C infection. Both type II and III cryoglobulinemia cause vasculitis as well,
also manifesting as skin ulcers [31 ] . Clinical signs include acral cyanosis,
Raynaud’s phenomenon, livedo reticularis, altered pigmentation of involved
skin, and palpable purpura. Systemic manifestations, such as arthritis, periph-
eral neuropathy, and glomerulonephritis, may also arise. Diagnosis is based
on skin biopsies, detection of cryoprecipitate and analysis of cryoglobulins by
immunoelectrophoresis.
Treatment of cryoglobulinemia is driven by the etiology of disease. If hepatitis C
is the underlying cause of cryoglobunemia, treat with pegylated interferon alpha
and ribavarin. Corticosteroids and immunosuppressive drugs (cyclophosphamide or
mycophenolate mofetil) often induce remission. Plasmapheresis and rituximab are
reserved for severe cases of cryoglobulinemic vasculitis [32].
Antiphospholipid Antibody Syndrome
Antiphospholipid antibody syndrome (APS) is the elevation of different antiphospho-

lipid antibodies leading to venous or arterial thrombosis, recurrent fetal loss, and often
thrombocytopenia. Antiphospholipid antibodies include lupus anticoagulant (LA),
anticardiolipid antibodies (aCL) and anti-b2-glycoprotein-1 (anti-b2-GP1) antibodies
[33, 34]. APS may be a primary disorder or may be secondary to an underlying auto-
immune disease such as systemic lupus erythematosus, malignancy, or infection. The
pathogenic mechanism of antiphospholipid syndrome remains unclear.
The classic presentation of this disease is livedo reticularis; other cutaneous
findings are ulcerations, superficial thrombophlebitis, and infarcts. Any organ sys-
tem may be affected depending on the vasculature involved. A common manifesta-
tion in women is placental insufficiency precipitated by placental vessel thrombosis
and ischemia, leading to miscarriage. Treatment consists of aspirin, warfarin, and
prednisone, but with varying response.
Metabolic Disorders
Calciphylaxis
Calciphylaxis, also called calcific uremic arteriolopathy, is a rare condition charac-

terized by progressive cutaneous necrosis frequently occurring in patients with end-
stage renal disease [35, 36]. The catalyst is usually secondary hyperparathyroidism
[37, 38] that elevates calcium-phosphate product leading to vascular, cutaneous, and
subcutaneous calcification, and ultimately tissue death. The disease course begins
as red, violaceous plaques in a livedo reticularis-like distribution progressing to
gangrenous black plaques that eventually ulcerate and may auto-amputate. Ulcers
due to calciphylaxis are usually bilateral, symmetric, and peripherally surrounded
by vesicles. The prognosis is poor, with an estimated 1-year survival rate of 46%
[39, 40]. Sepsis from superimposed infection of skin wounds and visceral involve-
ment leads to high morbidity and mortality.
Calciphylaxis is typically a clinical diagnosis corroborated by laboratory evidence of
elevated calcium, phosphate, or calcium-phosphate product. Additional findings of ele-
vated intact parathyroid hormone with radiographic and histologic evidence, pipe stem
calcifications and calcification of the intima and media of small and medium sized vessels
beneath the epidermis respectively, also confirm calciphylaxis. Treatment may be medi-
cal or surgical [37, 38, 41, 42], though both are often used concurrently (Table 3.8).
Malignancies
Malignancy is a cause of atypical wounds; conversely a wound may also cause a malig-
nancy. Malignancies such as nonmelanoma skin cancer, lymphomas, and sarcomas
Table 3.8 Calciphylaxis treatment Antibiotics

options
Anticoagulation
Avoidance of systemic steroids
Bisphosphonates
Cinacalcet
Cyclosporine
Medical options
Decreased calcium in dialysate
Hyperbaric oxygen
Low phosphate diet
Phosphate binders
Sodium thiosulfate
Stanozolol
Amputation
Autologous skin graft
Parathyroidectomy
Surgical options
Renal transplantation
Tissue engineered skin
Wound care and debridement
may ulcerate. Up to 2% of chronic wounds, called Marjolin’s ulcers, may develop into
a malignancy, most commonly squamous cell carcinoma, but also basal cell carcinoma
and lymphoma among others [43]. Other entities that may degenerate into Marjolin’s
ulcers include burn wounds, sinus tracts, and chronic osteomyelitis.
Suspicious lesions should be biopsied as early identification is imperative.
Treatment modalities include excision with at least 2 cm margins or, Mohs surgery
[44], Adjuvant therapy with radiation, topical 5-fluorouracil, methotrexate,
L-phenylalanine mustard, lymph node dissection, and amputation of the affected
limb if necessary in some cases [45].
External Causes
Spider Bites
Loxoscelism
The bite of L. reclusae (brown recluse or violin spider) is usually unnoticeable unless
rare late development of pain and systemic symptoms of fever, malaise, headaches,
and arthralgias. However later, a pustule, blister, or large plaque forms at the bite site
that may necrose and form eschar. The classic red, white, and blue sign consists of
peripheral erythema around a clear halo, due to vasoconstriction, that surrounds a
violaceous plaque. Viscerocutaneous loxoscelism, manifested by diarrhea, nausea,
vomiting, petechiae, urticaria, and disseminated intravascular coagulation can occur
in 0.7–27% of patients [46]. Necrosis develops more frequently when bites occur in
areas of greater fat content. When the eschar detaches, an ulcer may develop.
Treatment consists of cooling, elevation (if possible), and analgesics. Additional

therapies include systemic steroids to minimize necrosis and oral dapsone.
Ultimately, the healing of the ulcer is very slow and may take up to 6 months.
Latrodectism
The painless bite of the black widow spider, Latrodectus mutans, is followed by
severe pain, swelling, and tenderness at the puncture site. Systemic symptoms
include headaches and abdominal pain that endure for up to 3 days. Treatment with
local ice, calcium gluconate, and the specific antivenin usually prevents fatality. The
mortality of black widow bites is low, with death mainly occurring in those who are
young, with comorbidities, or elderly.
Chemical Burns
Burns from caustic chemicals often cause progressive damage after the initial
exposure, resulting in difficult to treat ulcers. Factors that increase severity are
higher concentration, longer duration of contact before treatment, and alkaline
chemicals [47]. Typical protocol includes immediate water irrigation for at least
30 min followed by standard burn care. Other treatment options include topical
25% magnesium sulfate for hydrogluoric acid burns, excision of the affected area
for chromic acid burns, and topical polyethylene glycol with alcohol 2:1 for phenol
burns [48].
Radiation Dermatitis
Mild erythema, edema, and pruritus may occur when exposed to ionizing radia-
tion greater than 10 Gy. The course of irritation begins with onset between 2 and
7 days after exposure, peaks within 2 weeks, and eventually subsides. At higher
doses, intense erythema, vesiculation, erosion, and superficial ulceration devel-
ops. Current treatment options are limited to wound excision and hyperbaric
oxygen.
Factitial Dermatitis
Factitial dermitis or ulcers are characteristically linear edged and located in areas
of easy access, especially the extremities, abdomen, and anterior chest, due to
self-imposed injury. Treatment is difficult and should be focused on underlying
psychological disease and limiting accessibility to the wound via an overlying
dressing or cast.
Drug-Induced Causes
Coumadin Necrosis
Coumadin (warfarin) induced necrosis is a rare complication of anticoagulant treat-

ment with an incidence of 1:1,000 and 1:10,000 of patients treated with anticoagu-
lants. Skin necrosis occurs between the 3rd and 10th day after the administration of
a large initial loading dose of the anticoagulant drug. The pathophysiology is not
well understood and relies on postulates involving physiologic coagulation cascades
[49]. Cutaneous manifestations run the gamut of urticarial ecchymoses and purpura
to hemorrhagic necrosis and purple toes. The painful wounds usually progress into
full-thickness skin necrosis within days. Management may be conservative or
involve debridement and grafting. Previously uncomplicated courses of warfarin do
not preclude the risk of skin necrosis with future warfarin administrations. Low-
dose warfarin with heparin may reduce the risk of this reaction.
Extravasation
Extravasation injury typically occurs on the hands from calcium, potassium, bicar-
bonate, hypertonic dextrose, cardiac drugs, chemotherapeutic drugs, cytotoxic
drugs, and antibiotics. Tissue loss may lead to extensive wounds. Mild injury can be
managed conservatively [50]. If injury is extensive, debridement and skin grafting
may be required. Optimal prevention involves appropriate needle gauge, maximal
dilution of the medication, minimal infusion rate, and immediate termination at first
signs of irritation with rapid initiation of treatment. Complications include scarring
and loss of function as these injuries tend to occur on the hands.
Epidermolysis Bullosa
Epidermolysis bullosa (EB) is an inherited disorder that renders a patient highly

susceptible to blister formation after minor mechanical insult to the skin.
Compounded with impaired healing, ulcer formation occurs. EB is a rare bullous
disorder with an incidence of 19 per million live births and prevalence of 8 per one
million population [51]. There are three major types, classified by the affected pro-
tein and its location. Epidermolysis bullosa simplex (EBS) involves basal keratino-
cytes leading to intraepidermal separation. Another subtype is junctional
epidermolysis bullosa (JEB) involving the dermoepidermal junction proteins to
cause disruption in the lamina lucida. The third type is dystrophic epidermolysis
bullosa (DEB) characterized by defective type VII collagen resulting in anchoring
fibril alteration or deficiency below the lamina densa. Each major class is attributed
to unique mutations Table 3.9.
56
Table 3.9 Epidermolysis bullosa subtypes and their gene encoding protein defects
EB type EB subtype Gene encoding protein defect Major clinical manifestations
EB simplex Basal subtypes:
EB simplex localized Keratin 5 or 14 Blisters on palms and soles; extracutaneous intraoral lesions
EB simplex generalized (Dowling Keratin 5 or 14 Vesicles in arcuate array; keratoderma of palms and soles; may
Meara) improve with fever; increased risk of basal cell carcinoma
EB simplex other generalized Keratin 5 or 14 Blisters on any surface, usually sparing palms and soles
(non-Dowling Meara)
EB simplex with mottled Keratin 5
pigmentation
EB simplex with muscular Plectin
dystrophy
EB simplex with pyloric atresia Plectin, a6/b4 integrin
EB simplex Ogna Plectin Susceptible to bruising, hemorrhagic blistering, onychogryphosis
EB simplex autosomal recessive Keratin 5
Suprabasal subtypes:
Lethal acantholytic Desmoplakin
Plakophilin deficiency Plakophilin-1
EB simplex superficialis ?
Junctional EB Junctional EB Herlitz subtype Laminin 332 Excessive granulation tissue, microstomia [52], ankyloglossia [52],
growth retardation, anemia, esophageal strictures, corneal
scarring, increased risk of squamous cell carcinoma
Junctional EB Non-Herlitz subtypes:
Junctional EB, non-Herlitz, Laminin 332, type XVII collagen Generalized blistering, atrophic scarring, nail dystrophy,
generalized anonychia, scarring alopecia [53]
Junction EB, non-Herlitz, localized Type XVII collagen
Junctional EB with pyloric atresia a6/b4 integrin Congenital pyloric atresia and blistering at birth
Junctional EB inversa Laminin 332 Lesions in intertriginous folds, esophagus, vagina
Junctional EB late onset ?
LOC syndrome Laminin 332 (a3 chain) Blisters on face and neck; nail deformities; conjunctival lesions
J. Tang and R.S. Kirsner
Table 3.9 (continued)
EB type EB subtype Gene encoding protein defect Major clinical manifestations
Dystrophic EB Dominant dystrophic EB:
DDEB acral Type VII collagen Hand and feet involvement
DDEB pretibial Type VII collagen Anterior lower leg involvement
3 Atypical Ulcers
DDEB pruriginosa Type VII collagen Blistering associated with severe pruritus
DDEB nails only Type VII collagen
DDEB bullous dermolysis of the Type VII collagen Presents at birth, focal atrophic scarring, disease regresses by
newborn 6–24 months of life
Recessive dystrophic EB:
RDEB severe generalized Type VII collagen Generalized blistering, mutilating scarring, corneal scarring [54],
growth retardation [55], anemia, failure to thrive, esophageal
strictures, mitten deformities, pseudosyndactyly, ankyloglos-
sia, microstomia, chronic renal failure, dilated cardiomyopa-
thy, high risk of cutaneous squamous cell carcinoma,
malignant melanoma (rare)
RDEB generalized other Type VII collagen Less severe presentation of RDEB severe generalized
RDEB inversa Type VII collagen Blistering of intertriginous folds, base of neck, upper back,
lumbosacral region, oral cavity, esophagus, lower genitouri-
nary tract
RDEB pretibial Type VII collagen
RDEB pruriginosa Type VII collagen
RDEB centripetalis Type VII collagen Blistering pattern transitions from acral to truncal over decades
RDEB bullous dermolysis of the Type VII collagen Similar to DDEB bullous dermolysis of newborn but with
newborn increased risk of fatality
Modified from Fine et al. [56]
57
EBS is mainly inherited in an autosomal dominant pattern. EBS usually pres-

ents at birth or shortly after. In the case of localized EBS, later onset during child-
hood or early adulthood may occur. Generally, it is known to cause less scarring,
milia formation, and nail dystrophy as compared to the other types of EB [57].
The subtypes of EBS possess unique characteristics (Table 3.9). The unifying
characteristic of all JEB is enamel hypoplasia, which may be localized or general-
ized [58]. More unique features of Herlitz versus non-Herlitz JEB are displayed in
Table 3.9. DEB classification is based on the pattern of inheritance, autosomal
dominant or recessive. Dominant DEB (DDEB) consists of blistering at birth,
milia, nail dystrophy, and atrophic scarring [57]. Recessive DEB (RDEB) ranges
from mild to severe disease presentation. Neither type is associated with enamel
defects. Additional subdivisions of DDEB and RDEB are named according to
location of involvement (Table 3.9).
In general, to distinguish between the types and subtypes of EB, the structural
level of blister formation, pattern of inheritance, immunohistochemistry and elec-
tron microscope findings, and phenotype are considered. Diagnosis can be per-
formed postnatally or prenatally. Postnatal diagnosis relies on immunofluorescence
antigenic mapping (IAM) and transmission electron microscopy (TEM) [58].
IAM of punch biopsy samples and TEM of shave biopsies, both obtained from
nonblistered skin that has undergone rotary assault [57]. IAM provides informa-
tion on location and pattern of antigen staining. TEM may indicate the structural
abnormality. Prenatal diagnosis consists of DNA mutational analysis from chori-
onic villi samples.
Wound care is an important component of management and may consist of artificial
skin equivalents, cadaveric allografts [59], dermal substitutes, and other wound dress-
ings. More novel therapeutic options are gene therapy, stem cell therapy, allogeneic
fibroblast injections [60], and infusion of recombinant protein [61]. Management of
extracutaneous manifestations includes ophthalmic care for corneal lesions, dilation
of esophageal strictures, gastronomy if poor nutrition, nightly wrapping of digits, sur-
gical degloving procedures, Dual Energy Xray Absorptiometry (DEXA) and, British
Medical Journal (BMJ) scans to screen for osteoporosis.
Summary
Diagnosis and treatment of the underlying etiology are fundamental in caring for
patients with atypical wounds. Despite the armamentarium of treatment options,
atypical wounds remain problematic for health care providers. Prolonged healing of
chronic wounds has a negative impact on the patient, their caregivers, and society as
a whole. These wounds are not only a health burden, but also a psychosocial and
economic problem as well. For these reasons, optimal treatment must be pursued
when atypical wounds are encountered.
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Chapter 4
Nutrition and Wound Healing
Giuseppe Benati and Maria S. Bertone
Keywords Nutrition • Malnutrition • Wounds and protein-calorie supplementation

Ulcers and pharmaconutrients • Enteral nutrition • Oral supplements
Introduction
Malnutrition is generally considered to be one of the main factors in dealing with

prevention and treatment of wounds. Over the course of many years, nutritional
supplementation with protein and calories or specific nutrients (vitamin C, zinc,
selenium, etc.) have been proven to be involved in maintaining skin integrity and
promoting wound healing.
However, even today, the literature is contradictory and the gap between clinical
experience and evidence-based literature is still wide. Researchers have found it
difficult to prove that there is a direct relationship between nutrition and wounds.
Nutrition may represent one of the etiological factors in wounds, but the variables are
many and evidence of nutritional effectiveness is not easily demonstrable. Despite
G. Benati, M.D. (*)

Clinical Nutrition Service, Geriatric Department,
Morgagni Pierantoni Hospital,
Forlì 47100, Italy
e-mail: g.benati@ausl.fo.it
M.S. Bertone, CD
Department of Dermatology, Wound Healing Unit Research,
University of Pisa, Pisa, Italy

64 G. Benati and M.S. Bertone
Step 1. Formal screening test for malnutrition.

Step 2. Complete nutritional assessment.
Step 3. Elaboration of a nutritional treatment and follow-up.
Fig. 4.1 Recommended nutritional procedure in wounds patients
the large number of etiological variables, many studies consider a small number of
patients and their statistical significance may be questionable. Moreover, few inter-
vention studies are currently available and ethical problems with double blind pla-
cebo trials often occur, because avoidance of nutritional treatment can be considered
non-ethical. Finally, the nutritional status definition varies in different studies and
data analysis can be very difficult (different anthropometric data, laboratory param-
eters, dietary intakes and combinations). Although nutrition is therefore considered a
crucial factor in many guidelines, actual evidences make difficult for wound manage-
ment operator approach to nutrition. The purpose of this chapter is to conduct a
thorough analysis of evidences on nutrition and ulcers, so that diagnosis and treat-
ment of malnutrition should be always considered in wound management (Fig. 4.1).
Malnutrition and Its Consequences in Wound Healing
Malnutrition literally means bad or faulty nutrition. Malnutrition is a state of nutri-

tion in which a deficiency or excess (or imbalance) of energy, protein, and other
nutrients causes measurable adverse effects on tissue/body form (body shape, size
and composition) and function, and clinical outcome [1]. It can arise from a number
of medical conditions that differ in both severity and cause (disease-related malnu-
trition) and is also influenced by social, economic, and institutional factors operat-
ing in community, hospital and nursing home settings [2]. A range of terms are used
to describe malnourished status; these include ‘overnutrition’, due to an excessive
food consumption relative to requirements, and the converse, ‘undernutrition’,
where intake is inadequate. A simple parameter to define nutritional status is body
mass index (BMI, weight to square of height ratio): A patient with BMI below 18.5
is considered underweight and a BMI <15 is associated with significant morbidity
and mortality. Loss of weight of 10% of the usual weight in 6 months or 5% in
1 month are indicative of malnutrition. When BMI is ³25 kg/m2 patient may be
considered overweight, and when it is ³30 kg/m2 obese.
Obesity has increased over the last years. People with a high fat mass (mainly
with a central distribution) have an increased risk for comorbidity. Over-nutrition
carries health risks, generally associated with vascular disease, diabetes mellitus,
arthritis and specific types of cancer. Furthermore, in obese older adults, there can
be reduced functional status and decreased quality of life [3]. These patients are at
a high risk for pressure ulcers and leg ulcers. Goals for treatment of obese patients
include management of wound drainage, prevention and resolution of infections,
healing of pressure ulcers, protection of peri wound skin and enhancement of
4 Nutrition and Wound Healing 65
Table 4.1 Role of macro and micro nutrients in wound healing

Energy Cell metabolism, collagen formation, nitrogen retention,
angiogenesis
Proteins Fibroblast proliferation, collagen production, angiogenesis
Micronutrients (vit. A, E, C, Zn) Inflammation, immune response, collagen formation
patient’s comfort. However, data about the efficacy of a specific nutritional therapy
in obese patients with wounds are still lacking [4, 5]. Patients with diabetic foot and
lower extremity wounds are very difficult to treat and are very frequent. However
few data about nutrition and wounds in these patients are present in literature.
Undernutrition has been associated with a number of adverse outcomes in hospi-
talized patients: these include increased risks of morbidity and mortality, increased
sepsis incidence and length of hospital stay [6]. Chronic undernutrition can lead to
the deterioration of vital organ systems, resulting in impaired wound healing. Two
important keys are reduced nutrient availability for healing and oedema, which
impairs nutrient diffusion [7]. Malnutrition is recognized as one of the major sys-
temic risk factors for pressure ulcer development. Both poor nutritional intake and
poor nutritional status are involved in increasing the risk [8].
Total body nutrition involves a complex physiological and biochemical balance
that is affected by numerous factors. The microcellular environment is the final
pathway for important external factors. The final metabolic event, i.e. wound heal-
ing, is the result of a myriad of processes and each step in this process requires a
stable biochemical environment, which is the result of adequate intake and pro-
cessing of nutrients. Macro and micronutrients have an important role in each
phase of wound healing (Table 4.1): from hemostasis/inflammation to proliferative
process and wound remodeling. Adequate energy amount is important for cell
metabolism, collagen formation, nitrogen retention and angiogenesis. Proteins
have an important role in fibroblast proliferation, collagen production and angio-
genesis. Specific amino acid like arginin could promote protein synthesis, stimu-
late immune function and cell proliferation. Micronutrients like vitamins A, E, C
and zinc are important in inflammation and immune response modulation and col-
lagen formation.
The main factors in the process are nutritional intake and access, the psychology
of the patient, social elements, environmental factors and disease processes.
It is important to develop sensitivity to the relationship between total body nutri-
tion and wound nutrition. Each step in the process of healing (inflammation, prolif-
eration, remodeling) is ultimately precursor-dependent upon circulating amino
acids, lipids and carbohydrates [9]. Impaired nutrition can not only alter the modu-
lation of deposition of collagen, fibroblast proliferation, and hydroxyproline con-
tent, but can also impair immune function and oxygen transport. Growth factor
synthesis is also dependent upon adequate nutrient status [10].
Protein deficiency can suppress angiogenesis, thereby altering capillary regen-
eration in the proliferation phase. Malnutrition may alter fibroblast proliferation as
well as collagen synthesis, thereby altering the rate and stability of wound healing.
Even altered lipid levels, important in membrane stabilization and inflammation,

have been shown to adversely affect wound healing [10].
Collagen synthesis requires 1 kcal/g of synthesized collagen and any alteration in
the availability of precursor amino acids or energy substrate will affect collagen
deposition. Each 500 mg of granulation tissue requires 0.5 kcal/ mg for production.
In wounds where there is excessive lactic acid production due to injury or low
availability of oxygen, the lactate is recycled in the pyruvate cycle and the tissue
becomes energy inefficient [11].
Nutritional Screening and Diagnosis of Malnutrition
The process of nutritional care may be broken down into a series of steps: nutri-
tional screening, formal nutritional assessment, formulation of a nutritional care
plan and reevaluation of the care setting, with reformulation of the care plan or ter-
mination of therapy. In the effort to treat malnutrition and its consequences we need
to pass through this entire sequence.
Nutritional assessment and screening represent a comprehensive approach aimed
at screening for the presence or the risk of malnutrition or creating a nutritional care
plan and monitoring the adequacy of the therapy [12].
Patients with wounds and high malnutrition risk or undernourished are likely to
be diagnosed and treated when a formal nutritional risk assessment tool is adopted.
There are many valuable tools that have been developed and validated: Subjective
Global Assessment [13], Mini Nutritional Assessment [14], Nutrition Risk Screening
[15] and Malnutrition Universal Screening Test [16]. All are quick and easy to per-
form, acceptable for patients, with low costs and high levels of reproducibility and
validity. Some authors have demonstrated that these tools may predict patient clini-
cal outcome [17]. This leads us to recommend the adoption of a formal screening
test for malnutrition, whatever it may be: in essence, the most suitable and most
readily applicable.
Formal nutritional evaluation is usually carried out when patients are at risk or
potentially with malnutrition at the screening test and is usually completed by a
specific team dedicated to nutritional problems. Nutritional assessment standards
should focus on: estimation of nutritional requirements; comparison of nutrient
intake with estimated requirements; providing appropriate nutritional intervention,
based on appropriate feeding guidelines such as dietetic counselling, oral supple-
ments, tube feeding; monitoring and evaluation of nutritional outcome, with reas-
sessment of nutritional status at frequent intervals (depending on clinical evaluation).
A multitude of other techniques (indirect calorimetry, body composition analysis,
etc.) are not so useful in clinical practice because of their costs and difficulty of use.
However, appropriate recommended intakes is one the main issues of nutritional
assessment. It is generally accepted that 30–35 kcal/kg body weight for individuals
under stress must be provided and that 1.25–1.5 g protein/kg body weight daily
should be offered [18].
Table 4.2 Macronutrients optimal intakes in pressure ulcer patients

Energy 30–35 kcal/kg/day
Proteins 1.25–1.5 g/kg/day
Water 30 ml/kg/day or 1–1.5 ml/kcal consumed
Macronutrients and Micronutrients in Healing
During the process of wound healing an array of nutrients meet the needs of tissue
metabolism and also provide for the synthesis of its new structural components.
These nutrients can be divided into two major categories: macronutrients, which
include glucose, fatty acids and protein, and micronutrients, which can be subdi-
vided into vitamins, trace elements and minerals. Table 4.2 shows macronutrient
optimal intakes in wound patients.
The presence of a stage/category over two wound increases energy demands by
30–50% and protein demands by at least 50% above normal needs.
The management of protein energy malnutrition also requires at least a 50%
increase in calories and a doubling of protein intake to restore lost lean mass.
Optimum nutrition should be initiated immediately in the presence of any hyper-
catabolic state and also in an already compromised host with pre-existing malnutri-
tion or chronic illness, especially in the frail elderly [19].
The ideal distribution of calories is: carbohydrates (complex form) 55–60%, fats
(polyunsaturated) 25%, and protein (high biologic value) around 20%.
Carbohydrates are utilized in a number of aspects of healing besides being con-
sumed for energy. The matrix is composed of proteoglycans and glycosaminogly-
cans, which are made from polysaccharide chains linked to protein. Lactate is a
metabolic byproduct of glucose. The increase in lactate which is produced by all
wound cells activates the genetic expression [20].
Protein intake corresponds best to wound repair. An intake of 1.5 g/kg/day
appears to be the ideal value [21, 22]. Clinical studies suggest that beta-
hydroxy-beta-methylbutyrate, a natural metabolite of the essential amino acid
leucine, could protect lean mass from stress-related damage and enhance pro-
tein synthesis [23].
Protein with increased concentrations of essentially and conditionally essential
amino acids has a higher biologic value. Arginine and glutamine are nitrogen-rich
amino acids, which contain a variety of elements that are important in reparation
[24]. Both arginine and glutamine are considered to be conditionally essential amino
acids since endogenous production does not appear to be sufficient to keep up with
demand during the “stress response”.
The essential omega-6 fatty acids, linoleic and linolenic, are required for both
cell membrane formation and prostaglandin production [25]. A diet enriched eicos-
apentanoic acid and gamma-linoleic acid is associated with a significant lower
occurrence of new wounds (p < 0.05) in critically ill patients [26].
Water, which constitutes about 60% of adult body weight, may be the most impor-
tant nutrient of all [27]. It is distributed in the body in three fluid compartments
(intracellular, interstitial and intravascular). Fluid requirements are met with 30 ml/
day unless medically contraindicated. Additional fluids are needed for patients with
draining wounds, emesis, diarrhea, elevated temperature, or increased perspiration.
Dehydration is a risk factor for development of wounds and the water needs of
patients with stages 3 and 4 pressure ulcers are very high.
Micronutrients function primarily as cofactors in biochemical reactions and, as
such, are critical to all of the activities of macronutrients. Protein synthesis cannot
continue without adequate quantities of vitamin B6, zinc and copper [28].
Collagen synthesis will be impaired with deficiencies in vitamin C, iron and cop-
per. Vitamin B12, folate and zinc are essential for nucleic acid metabolism and are
essential in the healing wound with rapid cellular proliferation.
There are various forms of nutritional supplements available on prescription to
treat malnutrition and promote wound healing [29].
The effects of high energy protein supplements enriched with arginine, zinc
and antioxidants are very important in pressure ulcer healing in old patients
[ 30-32].
Zinc is an important trace element for the more than 70 metal enzymes
involved in the immune response, inflammation and cell growth. Neither bio-
chemical zinc status nor dietary intake appeared to be related to pressure ulcer
risk. It has been demonstrated however that zinc supplementation may improve
nitrogen balance, thyroid, pancreatic and liver function, as well as the immune
response [33, 34].
Since the 1960s many authors have demonstrated the presence of ascorbic
deficiencies in geriatric and neurological patients. In 1974 Taylor and others showed
that a 1 g supplementation of vitamin C was able to induce a significant reduction
in ulcer surface area [35]. In the group treated with ascorbic acid there was a mean
reduction in pressure-sore area of 84% after 1 month compared with 42.7% in the
placebo group. These findings were statistically significant (P < 0.005). In contrast,
Bergstrom and others did not find any correlation [36].
The Perspective of Wound Care Managers
Despite all the evidence and the confirmed importance of malnutrition in patients
with wounds, malnutrition risk is still under-diagnosed. We recently investigated
nutritional practice among Italian health professionals dealing with wound
patients. A questionnaire about nutritional attitudes and routines was distributed
to 400 wound care managers. Three hundred eighty-eight participants completed
the questionnaire, of whom 68% were nurses, 30% doctors, 2% other health pro-
fessionals. The prevalence of malnutrition in PU patients was estimated >5% of
their patients by 67% of participants. Most of the professionals (93%) declared
that at least one nutritional parameter was considered at their first evaluation. The
considered nutritional index respectively for doctors and nurses is shown in
Table 4.3 Considered nutritional index in 388 Italian wound managements experts
Nurses (%) Doctors (%) Significance level
Actual weight 3 10 P < 0.005
Delta weight 7 13 P < 0.005
Screening tool 2 4 NS
Table 4.3. Of the total of professionals who completed the questionnaire a large
percentage declared that oral supplements, tube feeding, parenteral nutrition
(45%, 64% and 64%, respectively) were prescribed in less than 5% of their
patients. The reasons for such a low treatment rate were the absence of an ade-
quate management of home artificial nutrition and its reimbursement by the Italian
Healthcare System.
Knowledge of the importance of nutrition in prevention and treatment of wounds
is widespread in professionals; this is especially true for nurses as compared to
doctors.
Nutritional instruments for an early diagnosis vary for different professionals;
the number of treated patients is low, with a large discrepancy between attitudes and
reported practice. This shows that implementing good clinical practice may be
difficult to accomplish.
New Perspectives
In conclusion, there is considerable evidence that nutritional intervention is a cru-

cial factor in prevention and treatment of wounds. Easy measurements such as
weight and height or formal screening instruments for malnutrition are still uncom-
mon in clinical wound managing practice and the malnutrition risk is still unrecog-
nized. It is desirable that new and convincing evidence on nutrition and wounds be
obtained. Intervention trials with adequate statistical power should be the starting
point; the end points of such studies should lie in the laboratory demonstration of
the specific role of nutrient supplementation in wound healing and the achievement
of hard end targets such as improving the quality of life, reduced need for hospital-
ization or institutionalization, reduced incidence of infection. At the same time,
recommended intakes in relation to the severity of wounds are still not well known.
Finally, a proper cost analysis of nutritional intervention in wounds is still lacking.
Strategies to increase awareness of the importance of malnutrition in wound patients
should be adopted: there is a genuine need to ensure that nutritional screening and
appropriate nutritional therapy are provided to all patients with wounds.
Acknowledgements We are grateful to the “AIUC Study Group on Nutrition and Pressure Ulcers”
who provided support to collect data shown in “The perspective of wound care managers”:
Emanuele Cereda, Milano (Italy); Guido Ciprandi, Roma (Italy); Marco Masina, Bologna (Italy);
Carlo Pedrolli, Trento (Italy); Oreste Sidoli, Parma (Italy); Giorgio Vertsonis, Bologna (Italy).
References
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leic acid and antioxidants in the prevention of new pressure ulcer formation in critically ill patients
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9. Smith HJ, Wyke SM, Tisdale MJ. Attenuation of proteasome-induced proteolysis in skeletal
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65:277–83.
10. Cereda E, Gini A, Pedrolli C, Vanotti A. Disease-specific, versus standard nutritional support
for the treatment of pressure ulcers in institutionalized older adults: a randomized controlled
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15. De Santi L. Involuntary weight loss and the non healing wound. Adv Skin Wound Care. 2000;
13:11–20.
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220–1.
17. Elia M. Screening for malnutrition: a multidisciplinary responsibility. Development and use of
the Malnutrition Universal Screening Tool (MUST) for adults. Redditch: BAPEN; 2003.
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19. Emery PW. Metabolic changes in malnutrition. Eye. 2005;19:1029–34.
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predicts mortality and length of hospital stay in acutely ill elderly. Br J Nutr. 2006;95:325–30.
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vitamin C and zinc in patients with pressure ulcers: a randomized controlled trial. Clin Nutr.
2005;24:979–87.
22. Stratton RJ, Green CJ, Elia M. Disease-related malnutrition: an evidence based approach to
treatment. Am J Clin Nutr. 2004;79:1128–9.
23. Shenkin A. The key role of micronutrients. Clin Nutr. 2006;25:1–13.
24. Stratton RJ, Ek AC, Enger M, Moore Z, Rigby P, Wolfe R, Elia M. Enteral nutritional support
in prevention and treatment of pressure ulcers: a systematic review and meta-analysis. Ageing
Res Rev. 2005;4:422–50.
25. Schols JM, Heyman H, Meijert E. Nutritional support in the treatment and prevention of pres-
sure ulcers: an overview of studies with an arginine enriched oral nutritional supplement.
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KN. What is subjective global assessment of nutritional status? JPEN. 1987;11:8–13.
27. Allison S. Malnutrition and disease related outcomes. Clin Nutr. 2000;16:590–3.
28. Posthauer M. Hydration: an essential nutrient. Adv Skin Wound Care. 2003;18:32–3.
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30. Whitney JD. Overview: acute and chronic wounds. Nurs Clin North Am. 2005;40:191–205.
31. Stratton RJ, Green CJ, Elia M. Scientific criteria for defining malnutrition. In: Disease-related
malnutrition: an evidence-based approach to treatment. Cambridge: CABI Pub; 2003. p. 1–34.
32. Bergstrom N, Braden B. A prospective study of pressure sore risk among institutionalized
elderly. J Am Geriatr Soc. 1992;40:747–58.
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ulcers. J Am Geriatr Soc. 1991;41:357–62.
34. Komarcevic A. The modern approach to wound treatment. Med Pregl. 2000;53:363–8.
35. Chernoff RS, Milton KY, Lipschitx DA. The effect of a very high-protein liquid formula on
decubitus ulcer healing in long term tube fed institutionalized patients. J Am Diet Assoc.
1990;90:A-130.
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new method based on an analysis of controlled clinical trials. Clin Nutr. 2003;22:321–36.
Chapter 5
Measurement of Wound Healing
and Tissue Repair
Visweswar Bhattacharya, Nilesh K. Agarwal,

and Siddhartha Bhattacharya
Keywords Wound management • Dimensions of wound • Wound resurfacing

Reconstructive modalities of wound • Tissue repair
Introduction
Wounds can be broadly of two types, acute and chronic. Acute wounds result fol-
lowing trauma or excisional surgery. Chronic wounds are those which do not heal
within 6 weeks. The cause of chronic wounds varies depending upon the genesis of
wounds, its depth, the involvement of underlying structures, primary wound care
and tissue handling. Whatever may be the cause, the basic reason is inadequate
circulation.
Common etiologies are trauma, foreign bodies, infection, postoperative dehis-
cence, thermal, chemical and electrical burn, diabetic ulcers, pressure sores, second-
ary to varicose vein, trophic changes following spinal injury or peripheral nerve
involvement. Often the trauma victims, with bony and soft tissue injuries, seek ini-
tial management in orthopaedics. The soft tissue remains under assessed and either
sutured under tension or left open. Sometimes foreign bodies are not adequately
cleaned. Later they are referred to reconstructive surgeon. This leads to enormous
functional, sociopsychological and economical burden to the patient. Such situation
can be avoided if a combined team approach is adopted during the primary
management.
V. Bhattacharya, MS, MCh, Ph.D., FICS (*) • N.K. Agarwal, M.S., M.Ch. • S. Bhattacharya
Department of Plastic Surgery, Institute of Medical Sciences, Banaras Hindu University,
B33/14-16, Gandhi Nagar, 221005, Naria, Varanasi, Uttar Pradesh, India
e-mail: visweswar1@rediffmail.com; plasticandeye@gmail.com; gotu29@rediffmail.com

74 V. Bhattacharya et al.
The assessment of a wound is done by what tissue constituents are lost and
what tissues are exposed. This has most significant importance in perspective of
resurfacing and method of reconstruction. This is the importance of measurement
in reconstructive surgery of any wound. It has direct bearing with the selection of
the type of tissue to be used for resurfacing.
In many situations there may be a gradual cessation of blood flow, due to con-
sistent pressure as in decubitus ulcer, trophic ulcer, leading to necrosis. Circulation
may be also hampered in acute infections as in intense cellulites, necrotizing
fascitis, etc. Hence it is necessary to precisely identify these factors and take
necessary timely measures to prevent and cure the wounds. All the above situa-
tions cause structural and functional changes in the constituents of tissue, lead-
ing to a wound. Thus a clear understanding of the natural human tissue and their
functioning is essential for a treating specialist who encounters a wound and
plans to cure it.
Wound Measurement
In acute wounds all the devitalized tissues are debrided and then the exact three
dimensional measurement is done from skin up to the bone. Accordingly the lost
tissue is replaced by like tissue. Sometimes the wound apparently may look small
but might have wide undermining with overlying devascularization. Such under-
mined tissue should be primarily excised judiciously till the bleeding margin is
reached. Then the actual measurement of the wound is made. Otherwise soon the
evidence of necrosis followed by infection will set in leading to increase morbidity,
hospitalization and expenditure.
Two dimensional measurements in the form of surface area can be done by
measuring its linear dimensions with a tape or ruler. However, this two-dimen-
sional method assumes that the wound has a geometric surface shape, for exam-
ple a rectangle (length × width), a circle (diameter × diameter) or an oval
(maximum diameter × maximum diameter perpendicular to the first measure-
ment). An alternative method of calculating wound surface area is based on the
formula for an ellipse (length × width × 0.785). A three-dimensional mould of
the wound can be created by taking a cast of the wound cavity using a saline or
alginate filling.
In chronic wounds, there is reduced vascularity in the immediate surrounding
area with induration. The base of the wound is adhered to the underlying vital
structures. They may be associated with exposed neurovascular bundle, tendons,
bone, joints or hard wear used by orthopaedician to stabilize the bone (Fig. 5.1).
In these situations also the unhealthy tissue is excised to create the real defect that
will require reconstruction. After accurately measuring the wound, the individual
structures are assessed regarding their continuity e.g. vessels, nerves, tendons and
bone. If there is discontinuity, the length is measured and repaired / replaced
accordingly.
5 Measurement of Wound Healing and Tissue Repair 75
Reconstructive Modalities
Based on the above findings, the reconstructive procedure is selected. The recon-
struction may vary from simple skin graft to complex flaps of different constituents.
Fig. 5.1 (a) Exposed

hardware at knee. (b) Flap
dissected and transposed to
the defect. (c) Sutured to the
margins of the defect
covering the hardware. c
(d) Early follow up
Fig. 5.1 (continued)
It is also important to realize that in a given wound some areas may require only
skin graft and the other area, with exposed vital structures, will require a flap
(Fig. 5.2). Therefore measurement of the wound will vary according to the wound
and its requirement of reconstruction.
Skin Graft
Split skin graft is the simplest technique used to cover the large wound where the
whole thickness of the skin is lost without exposure of the vital structures egg.
Following avulsion injuries, thermal burn etc. The usual donor site is thigh. It con-
sists of epidermis and part of the dermis. If the wound size is too large as compared
to the available donor site, meshed grafts are used. Here the sheet grafts are perfo-
rated by Mesh dermatome with 3–4 times expansion of its original size and secured
over the wound. For such defect it is a life saving procedure. The wound heals
faster; it prevents systemic complications and achieves functional result. If the
defect is small or over face, hand etc. then a full thickness skin graft is preferred
consisting of epidermis and full thickness of dermis. The donor area is sutured
primarily. In any case the exact measurement of the defect and graft should match
to achieve the appropriate result.
Flaps
The major reconstructive challenges are those wounds where the underlying vital
structures are exposed. Such defects are resurfaced by composite tissue consisting
of different constituents. Such composite tissues are called ‘flaps’. The flaps can be
of different varieties namely, fasciocutaneous, adipofascial, fascial, muscle, myocu-

taneous, osteomyocutaneous, etc. The transfer of these flaps necessitates meticulous
planning keeping in mind the dimension of the defect, method of transfer and type
Fig. 5.2 (a) Lateral malleolar

defect with exposed tendon and
bone. (b) Retrograde
fasciocutaneous flap perfused
by distal peroneal perforator.
(c) Part of the wound with
exposed vital structures is
covered by flap and rest by
split skin graft
of vascularity at the base of the flap for adequate perfusion of the tissue [1]. If the
measurement is not accurate, the flap will fall short and there may be necrosis of
part or whole of the flap. Hence the reconstructive procedure will be unsuccessful.
The choice of the flap depends not only upon size of the defect but also on its
contour. A shallow defect requires a thin flap and a deeper contour defect needs
a thick flap. Thus the defect should be properly measured. If this aspect is not
considered, the flap may be unaesthetically bulky or the contour defect will not
be corrected. In both the situations it may require secondary corrective proce-
dure. The successful flap transfer is ensured by ‘planning in reverse’. It is com-
pletely based on the detailed measurement of the defect and flap. Using a piece
of lint to cover the wound, its distal tip is measured. Then all the steps of flap
transfer are worked upon in a reverse direction from insetting of the flap to its
marking on the donor site. Then the flap is marked on the donor site. This is the
fundamental step for the success irrespective of the composition and method of
transfer (Fig. 5.3).
Vascular Basis of Reconstruction
To simplify the understanding, the study of the lower limb vasculature is useful.
There are three major vascular axises in the lower limb below knee, namely anterior
tibial, posterior tibial and peroneal. Several perforators arise from them which course
either through muscles or through septa to reach the deep fascia. Therefore they are
named as musculofascial or septofascial perforators [2, 3]. They fan out on the under
surface of the deep fascia to form the subfascial plexus. Then they perforate the fas-
cia to form the suprafascial plexus. In the proximal 2/3rd of the limb the musculofas-
cial perforators dominate and in the distal 1/3rd, they are mostly septofascial
perforators as the muscle belly continues with the tendon [4, 5]. After perforating the
fascia they form suprafascial plexus which anastomose with the subcutaneous and
then subdermal – dermal plexuses. Thus the continuity of all these segments of net-
work is maintained (Fig. 5.4). Further, the plexus formed by the adjacent perforators
freely communicate with each other providing a continuous dense vascular arcade
throughout the tissue [6, 7]. Therefore starting from muscle to the skin any constitu-
ent alone or in combination can be transferred as a flap to resurface a defect.
All the flaps are based on strong rationality incorporating these vascular arcades
through which the blood perfuses them [8]. The constituent of the flap decides which
vascular networks that have been incorporated. For example, a fasciocutaneous flap
have dense networks which maintains vertical and horizontal vascular continuity
throughout the flap [9]. They are subfascial, suprafascial, subcutaneous, subdermal
and dermal (Fig. 5.5). In adipofascial flap, the subdermal and dermal networks are
not included [10] (Fig. 5.6). In fascial flap, only the subfascial and suprafascial net-
work remains (Fig. 5.7). It is important to understand this fundamental fact because
the safe limit of a flap is directly dependent on these networks. That is why a fascio-
cutaneous flap has larger safe dimension followed by adipofascial flap and then
Fig. 5.3 (a) Planning of inferiorly

based posterior tibial perforator
perfused fasciocutaneous flap by
‘planning in reverse’. (b) The
dissected flap and heel defect.
(c) Flap sutured to the defect and
the donor area split skin grafted.
(d) Flap detached after 3 weeks
and the heal reconstructed
d
PLEXUSES
Subepidermal
Dermal
Subdermal
Skin Subcutaneous
Suprafascial
Subcutaneous Subfascial
Tissue
Fascia
Musculocutaneous
Artery
Septocutaneous
Artery
Internal Artery
Fig. 5.4 Showing the different vascular networks of the composite tissue (flap)
fascial flap. In myocutaneous flap even the underlying muscle is incorporated. One
must also appreciate that thicker the flap less is the mobility and transfer of the tissue
has to be calculated accordingly. Therefore that has bearing on the measurement of
flap while planning. Similarly a flap with broader base will have less mobility. With
advanced knowledge of the perforators, the base can be substantially narrowed with
much greater mobility [11]. This allows increased reach of the flap to reconstruct
distant defects (Fig. 5.8).
The vascularity of flaps and its relation to dimension has two aspects: (a) the
knowledge of type of vessels on which the pedicle flap is based, (b) area of tissue
(angiosome) that is perfused by the incorporated vessel which can be transferred
safely for reconstruction [12]. Earlier one of the major vascular trunk of the limb
was sacrificed to vascularize the flap. In early 1980s, it was realized that the flaps of
larger dimension can be safely raised on smaller branches of the main vascular
trunk, called ‘perforators’. These perforators were mapped in different regions of
the body by cadaveric study. With the evolution of technology in the form of color
Doppler, audio Doppler, two dimensional and three dimensional CT angiography
etc., these perforators can be precisely identified and measured from fixed bony
landmarks. Their internal diameter can be gauged based on which they are classified.
Ultimately perforator flaps of different constituents have evolved.
Once the vascular supply for a flap is decided, the dimension of the required tis-
sue is calculated depending upon the requirement of the defect. For small to medium
size defect, a single flap is sufficient. For larger defects, two or three simultaneous
flaps may be designed perfused by different perforators (Figs. 5.9 and 5.10). All
these decisions are based on accurate measurements. Otherwise the flap may fall
Fig. 5.5 (a) Large defect

over the shin with exposed
bone. (b) Superiorly based
fasciocutaneous flap
dissected based on proximal
perforator of posterior tibial
artery. (c) Transferred to the
wound. (d) Flap sutured to
c
the margins of the defect
short which will lead to unnecessary dissection towards the base of the flap and
suturing under tension. Ultimately the vascularity will be compromised leading to
failure of the procedure.
Common Flaps and Practical Tips
(a) In case of fasciocutaneous flap, the margins are incised 1 cm beyond the flap
making done after “planning in reverse”. The incision should be beveled espe-
cially where multiple tissue constituents are incorporated. This allows all the
layers to fall in one plane at the margin. Subsequently they are kept together as
one unit by interrupted 3/0 chromic catgut stitches. This prevents shearing
movement between individual tissue planes during dissection and transfer of
the flap. Thus the composite vascular network is preserved allowing free flow
of blood without interruption.
From the initial suspicion about their clinical utility to a standard technique
in the armamentarium of a reconstructive surgeon, fasciocutaneous flaps
have come a long way. Constituent wise, fasciocutaneous flaps are those,
which include skin, subcutaneous tissue and the deep fascia.. The majority
of moderate size defects extending from knee to sole can be reconstructed
by these procedures. In contrast to earlier beliefs, fasciocutaneous flaps have
a definite vascular system, a suprafascial plexus and a subfascial plexus
anastomosing with the subcutaneous and subdermal plexi. Direct cutaneous,
musculocutaneous and septocutaneous perforators contribute to these plexi.
For better understanding, let us evaluate the reconstructive modalities for
defects at different locations of the lower limb. For defects from knee to
upper two third of leg, antegrade flaps either based on the perforators arising
from posterior tibial artery (on medial side) or flaps from anterolateral
Fig. 5.6 (a) Exposed tendo Achilles with planning of adipofascial flap. (b) Adipofascial flap dis-
sected. (c) Hinged to the defect. (d) Sutured to the defect and skin grafted. (e) 5 years follow up
aspect based on the perforators of the peroneal artery are used. These flaps
are transferred to the defect by transposition or rotation or combination of
both. The defects at lower third of leg to midsole will require retrograde
flaps based on the lower perforators of the above mentioned vascular trunks.
It is essential to incorporate 2–3 sizeable perforators in the pedicle, to ensure

necessary blood supply, irrespective of the nature of the flap. That is how a
non-convential long flap can be designed with safety.
Fig. 5.7 (a) Nonhealing

wound on the volar aspect of
wrist following electrical
injury. (b) A fascial flap
dissected from forearm.
(c) Turned over to the defect.
(d) Sutured to the defect.
(e) Covered with split skin c
graft
(b) Adepofascial flaps are usually defect based turn over flap. Two aspects need
care (i) the donor site skin is undermined with 2–3 fat globules under the
dermis to protect the subdermal plexus. This prevents marginal desquamation
of the sutured donor site. (ii) It should be a gentle hinged to prevent kinking.
A temporary rubber catheter at the base of the flap is a simple measure to
achieve safety. The adipofascial flap is comprised of subcutaneous tissue and
deep fascia. When the skin is removed from the fasciocutaneous flap, it
becomes an adipofascial flap. Thus, it will incorporate two plexuses less than
that of a fasciocutaneous flap, i.e. dermal and subdermal plexuses, and only
the vascular networks associated with the deep fascia and the subcutaneous
tissue supply this flap. A skin graft is applied on the subfascial surface of the
transferred flap as the flap is usually hinged, and the donor site is closed
primarily. It is preferred to apply the skin graft on the subfascial surface of the
flap which has rich vascularity. Although one should incorporate predop-
plered perforators at the base of the flap, one need not always visualize them
during dissection.
The adipofascial flap is the flap of choice on many occasions as it has several
advantages namely, (1) it provides well vascularized tissue over exposed struc-
tures, (2) it is a thin flap and thus does not look bulky, (3) dissection is easy, (4)
it is reliable and durable, (5) postoperative management is simple, (6) it provides
a good gliding surface for tendons, (7) there is minimal donor site morbidity with
a linear scar, (8) nonhairy tissue is transferred, (9) no major vascular trunk is
sacrificed, (10) this does not require any special training or setup, (11) it can be
transferred in various ways, and (12) the option for free flap remains open.
(c) In case of propeller flap or scalitonized perforator flap of any of the above con-
stituent, a small cuff of tissue should be preserved around the perforators to
Fig. 5.8 (a) Meticulous

planning of retrograde
fasciocutaneous flap for
anterior ankle defect.
(b) Flap dissected based on
distal perforator of peroneal
artery. (c) Sutured to the
defect. (d) Twelve years c
follow up
Fig. 5.9 (a) Large defect

over the distal half of the
lower limb. (b) Resurfaced by
double flaps, one superiorly
based and the other inferiorly
based
Fig. 5.10 (a) Large gluteal defect following excision of pressure sore. (b) Two flaps have
been dissected. (c) Advanced to the defect. (d) The flaps sutured in the midline. (e) Three years
follow up
provide additional safety. With specific knowledge of perforators, the fasciocu-

taneous flap from the calf area can be transferred to the distal defects in a single
stage based on skeletonised distal perforators. These flaps are marked and dis-
sected proximal to the defect , skeletonising the distal perforators under loupe
magnification, and transferred to the distal defect in a single stage. Similar prin-
ciple can be adopted for proximal defect based on proximal perforators. An
incision is made on one side of the proposed flap and is dissected in the subfas-
cial plane towards the axial vessel until the perforators are found. The presence
and positionof the perforators are confirmed at the distal leg from the respective
malleoli. After confirming the perforator’s position, the incision is completed
all around and dissection is completed to make it an island flap. The perforators
are totally skeletonised under loupe magnification. Then the flap is transposed
to the defect. The survivability of these unconventional flaps can be explained
in three ways: (1) Adequate perfusion pressure through the incorporated perfo-
rators. This also proves the importance of the perforators. (2) These perforators
form rich supra and sub fascial plexus at the level of deep fascia. (3) These fas-
cial plexuses are orientated axially, thus making the flap behave like an axial
flap. Hence, it is not only the rich plexus of vessels associated with the fascia,
but also their orientation that is responsible for survival of flaps of 3:1 length:
breadth ratio or more. The prime advantages of these flaps are: (1) greater
flexibility in rotating the flap with enhanced reach compared to the peninsular
flap. (2) The procedure is completed in a single stage. However, careful dissec-
tion is necessary in handling the flap and the perforators.
Excessive manipulation may result in perforator spasm or even their avulsion.
Kinking, torsion or pressure due to haematoma may be detrimental. Even
though skeletonization of a perforator allows the flap to move freely 180 on
either side, care must be taken to avoid kinking and torsion of the vessels by
not over-stretching the flap while insetting. With such careful handling, these
flaps have proved to be very useful for resurfacing defects especially of the
distal leg and foot.
(d) Distally based fasciocutaneous or musculoneurofasciocutaneous sural flaps
with neuro-adipo-fascial pedicled is one of the popular flap for reconstruction
of distal limb defect. The flap is perfused by two sources: neurocutaneous arter-
ies from the supramalleolar vascular network as well as distal septocutaneous
perforators of the peroneal artery. The proximal edge of the flap is usually about
8 cm from the popliteal crease, and the lateral edges do not extend beyond the
lateral border of fibula and medial border of the tibia. Incision is made up to the
fascia and the dissection is continued underneath it preserving the sural nerve .
The flap is transferred to the defect as an island flap with neuro-adipo-fascial
pedicled. The constituents of the pedicle are sural nerve, median superficial
sural artery, its accompanying arterial branches and the lesser saphenous vein.
The distal dissection is up to 5 cm above the lateral malleolus to avoid injury to
the perforators. That is the pivot point of the flap. Flap is used to resurface the
Achilles tendon injuries and concomitant complex soft tissue defects around
heel, ankle and the distal tibia. Advantages of this flap are (1) long pedicle
enables a great arc of rotation (2) no major artery is sacrificed (3) surgical
procedure is relatively short (4) donor site can be primarily close for smaller
flaps (5) suitable for patients even with an external fixator.
(e) Adepofascial extension of Fasciocutaneous flap is a useful method to fill the
moderate size contour defects. Fasciocutaneous flap with adepofascial exten-
sion is a technique utilizing principles of both fasciocutaneous flap and adepo-
fascial flap for moderate size contour defect . In selective situation where there
is contour defect of moderate size with uninfected bed, a fasciocutaneous flap
with adepofascial extension is a suitable choice. To raised the adepofascial
extension a lazy ‘S’ skin incision is made from the middle of the distal margin
of the proposed fasciocutaneous part. The skin is undermined keeping two
globule of subcutaneous fat to protect the subdermal plexus. The distal incision
is deepened through the subcutaneous tissue and deep fascia. The required ade-
pofascial flap is raised by dissecting between deep fascia and muscle till the
distal margin of the fascicutaneous unit. Then the two lateral skin incision of the
fasciocutaneous flap are given and the flap is elevated in continuity up to the
required length. The adepofascial extension part is gently folded under the dis-
tal fasciocutaneous unit and secured by few marginal stitches. The flap is then
transferred to the defect and skin margins are sutured. The donor site of the
adepofascial extension is closed primarily with a drain and that of the fasciocu-
taneous unit is skin grafted.
(f) Fascial flaps are chosen for non contoured defects e.g. over shin of tibia, exposed
tendo Achilles, distal tibia, etc. A fascial flap of a smaller dimension can be used
as a random pattern defect based hinge flap. When one or two sizable perfora-
tors are incorporated in the base, larger flaps can be safely used. A skin graft is
applied on the subfascial surface of the transferred flap and the donor site is
closed primarily. For reconstruction of moderate size defects, a turnover fascial
flap is a simple and reliable method. The flap is horizontally hinged with a verti-
cal base for midtibial defect. The flap is distally based and hinged for defect on
the distal leg, around the ankle and tendo Achilles. Audio Doppler study and
prior marking of the perforators are useful for planning. However, it is not nec-
essary during dissection to visualize the perforators incorporated at the base of
the flap. The maximum safe length of the distal perforator based flap is 14 cm.
In case of a random flap the length is about 8–10 cm depending upon the length
of the base. The fascial flap can be considered as the flap of choice on many
occasions as it has several advantages namely:
(1) It provides a well vascularized tissue over exposed structures. (2) It is a single
stage procedure (3) It is a thin flap, (4) Dissection is easy and operating time is
less. (5) It is reliable and durable. (6) It provides a good gliding surface over the
tendons. (7) It can be easily tailored according to type and location of the defect.
(8) It can be transferred in various ways. (9) Post operative management is sim-
ple. (10) There is minimal donor site morbidity with a linear scar only. (11) No
major vascular trunk is sacrificed. (12) It does not require any special training or
set up. (13) Non hairy tissue is transferred. (14) The option for free flap remains
open. This procedure provides gratifying results in majority of moderate dimen-
sion lower limb defects at non weight bearing areas. It meets most of the require-
ments of reconstruction in a single stage. Therefore whenever feasible this simple
method is justified. The only prerequisite is availability of the healthy tissue of
required dimension adjacent to the defect and undamaged perforators.
Certain common precautions are to be applied to all these flaps: Whenever a

flap is moved more than 90° over a pivot point, acute twist should be prevented. This
can be achieved by raising a little longer flap or by narrowing the pedicle towards the
perforators along the main vascular axis. Avoid suturing under tension by adequate
dissection and transfer. A drain under the flap and firm dressing prevents haematoma
and adds to the well being of the flap. Low molecular dextran infusion, during flap
dissection and for 2 days postoperatively, as an adjuvant therapy, is beneficial.
Post Operative Care: It is as important as surgery. The flap needs to be inspected

every day, at least the distal most part. The steps to be ensured are:
(a) Light dressing.
(b) Avoid pressure over pedicle.
(c) Limb to be kept elevated by 20–30°.
(d) Stitches are removed around 10–12 days.
Instructions to the patient during follow up
(i) Keep the flap clean to prevent infection
(ii) Keep it moist by use of moisturizer
(iii) Protect from trauma
(iv) Inspect twice a day
(v) Explain that different types of sensations will appear after at least 6 months
starting with protective sensation. Therefore till then one has to be careful. The
final sensations appear atleast after 1 year.
(vi) Use soft sole shoes in case of flaps on the ankle, dorsum of foot, on the heel
and sole.
(vii) Visit for monthly check up for first 6 months and 3 monthly for 2 years.
During evaluation
(a) The functional and aesthetic aspect is considered both for the recipient and
donor sites.
(b) Hospital stay is calculated.
(c) Expenditure in calculated.
Conclusion
Acute and chronic wounds of different etiologies pose significant challenge to the
reconstructive surgeons. It is important to choose the appropriate procedure of
resurfacing keeping in mind the nature and location of the defect. Adequate under-
standing of vascularity and meticulous execution is the key to the success. The
locoregional flaps of different constituents and combinations are of enormous value
and popular in managing moderate size defects. The free flaps also require similar
accurate measurements for the defect and the flap for resurfacing successfully. Thus
through out the wound management there is necessity of measurement for definitive
assessment of the wound and planning for resurfacing them.
References
1. Bhattacharya V, Deshpande SB, Watts RK, et al. Measurement of perfusion pressure of perfo-
rators and its correlation with their internal diameter. Br J Plast Surg. 2005;58:759–64.
2. Carriquiry C, Costa A, Vasconez LO. An anatomic study of the septocutaneous vessels of the
leg. Plast Reconstr Surg. 1985;76:354–61.
3. Cormack GG, Lamberty BGH. The fasciocutaneous system of vessels. In: The arterial anat-
omy of skin flaps. Edinburgh: Churchill Livingstone; 1986.
4. Haertsch PA. The blood supply to the skin of the leg. A post-morterm investigation. Br J Plast
Surg. 1981;34:470–7.
5. McCarthy JG, editor. Plastic surgery, vol. 1. Philadelphia: WB Saunders Company; 1990.
6. Ponten B. The fasciocutaneous flap: its use in soft tissue defects of the lower leg. Br J Plast
Surg. 1981;34:215–20.
7. Tolhurst DE, Haeseker B, Zeeman RJ. The development of fasciocutaneous flap and its clinical
applications. Plast Reconstr Surg. 1983;71:597.
8. Bhattacharya V, Watts RK, Reddy GR. Live demonstration of microcirculation in deep fascia
and its implication. Plast Reconstr Surg. 2005;115(2):458–63.
9. Bhattacharya V, Watts RK. Ipsilateral fasciocutaneous flaps for leg and foot defects. Ind J Plast
Surg. 2003;36:30–5.
10. Bhattacharya V, Sunish G, Adil BS. Reconstructive implications of adipofascial flaps in limb
defects. Eur J Plast Surg. 2007;30:169–75.
11. Bhattacharya V, Reddy GR, Goyal S, Kumar U. Skeletonised retrograde distal perforator
island fasciocutaneous flaps for leg and foot defects. J Plast Reconstr Aesthet Surg. 2007;60:
892–7.
12. Taylor GI, Palmer JH. The vascular territories (angiosomes) of the body: experimental study
and clinical applications. Br J Plast Surg. 1987;40:113–41.
Chapter 6
Pressure Ulcers in Neurologically
Compromised Patients
Anbananden Soopramanien and Shivram Singh
Keywords Neurological disability • Neurological compromise • Neurologically

impaired • Stroke • Spinal cord injury • Multiple sclerosis • Motor neurone disease
Brain injury • Pressure ulcer • Flap surgery
Introduction of Neurological Conditions
People suffering from the conditions listed below are at high risk of developing
pressure ulcers:
• Multiple Sclerosis (MS) is a very disabling condition affecting essentially young
women, between 20 and 40 years of age. In 2009 the London School of Tropical
Hygiene [1] calculated that there were around 100,000 people in the UK living
with MS. Patients with MS can present with impairment in cognitive function,
motor function, sensation and continence. Many are wheelchair-users.
• Motor Neurone Disease (MND) is a progressive and fatal disease affecting
mostly men after the age of 50. Their muscles become progressively weak pre-
venting them ultimately from walking, using their upper limbs or even talking.
There are approximately 1,000 new cases each year in the UK. Over the course
of the disease they can become bed-bound or wheelchair-dependent and are at
risk of damaging their skin.
A. Soopramanien, M.D., Ph.D., FRCP (*)

The Glenside Hospital for Neuro-Rehabilitation,
South Newton, Salisbury, Wiltshire, SP2 0QD, UK
Centre of Postgraduate Medical Research and Education,
University of Bournemouth, Christchurch Rd, Bournemouth, Dorset, BH1 3LT, UK
e-mail: asoopramanien@btconnect.com
S. Singh, FRCS
Department of Plastic Surgery, Birmingham Hospital NHS Trust,
Birmingham, UK

94 A. Soopramanien and S. Singh
• Brain Injury can be traumatic, vascular or tumoural. In the UK approximately

135,000 people (mostly young adults) are admitted to acute hospitals each year as a
result of brain injury. Patients can experience problems of physical disability, speech
and memory. A proportion will have double incontinence. After the onset of brain
injury preventative measures must be applied to avoid the skin from breaking down.
• Stroke is the largest cause of disability. 150,000 people are affected each year in
the UK. A few of them will have a repeat stroke. One third of stroke sufferers live
with a disability. Stroke patients may have issues with cognition, motor power,
continence and sensation. Their skin, vulnerable in the acute stage and rehabili-
tation phase, is at risk of breaking down.
• Spinal Cord Injury: the cumulative incidence for traumatic and non-traumatic
causes is 40 per million. It is estimated that there are 40,000 people living in the
UK with a spinal cord injury. Such patients would experience problems with lack
of mobility, paralysed limbs, impaired or absent sensation, incontinence (although
most would be adequately managed). One problem that has come to light recently
is the increased incidence of pressure ulcers with aging (physiological aging and
also time elapsed since injury). Amongst factors responsible is skin thickening,
changes in blood flow (see section on diabetes below and the other chapter on
diabetic foot ulcer in this book), contractures, depression, weight gain and inac-
tivity, physiological and metabolic disturbances below the level of injury [2].
• Other conditions include: Cerebral Palsy (CP), of which there are 113,500 peo-
ple in the UK; Huntington’s disease; Muscular Dystrophy; Parkinson’s disease,
Alzheimer’s; Diabetes.
We draw from our extensive experience of spinal cord injury to cover topics like
physiopathology, incidence, prevalence, sites of pressure ulcers; wound assessment,
measurement and treatment. Post- healing mobilisation is of great importance in
this population group. In the concluding section, we reflect on what can be done to
improve the situation.
Physiopathology
Sir James Paget [3] highlighted the various factors that contribute to the develop-
ment of pressure ulcers with a common pathway from prolonged pressure being
ischemia leading to cell death and necrosis [4]. Pressure is exerted on the skin, sub-
cutaneous tissue, muscle and bone by the weight of an individual against the surface
beneath. The pressure is often in excess of capillary filling pressure (~32 mm of
Hg). Tissues are capable of withstanding enormous pressure for short periods, but
prolonged time and pressure above the capillary filling pressures initiates a sequence
of ischemia, tissue necrosis and ulceration. The external surfaces that cause the
increased pressure usually are mattress, wheelchair, ill-fitting cushion, bed rails,
tight garments or shoes, or even a coin in a pocket!
Friction and shear forces compound the effect of increased, prolonged pressure.
Maceration in patients with incontinence predisposes to further injury, contamination
6 Pressure Ulcers in the Neurologically Compromised Patients 95
and infection. The three prime components: pressure, shear and stress are the main
cause for microcirculatory occlusion that results in ischemia leading to anoxia and
release of inflammatory mediators. Anoxia causes cell death resulting in necrosis
and subsequently ulceration [4].
Muscle damage occurs early in the stages of pressure ulcers as compared to other
tissues. Due to their increased demand for oxygen, irreversible changes may occur
in muscles after as little as 2 h of uninterrupted pressure. The skin, in comparison
can withstand direct pressures up to 12 h. Hence, by the time an ulcer is present in
the skin surface, significant damage of underlying muscle is already done and results
in a cone shaped ulcer.
Recent advances in the theory of tissue damage have suggested a reperfusion
injury as the reason for chronicity of ulcers. Restoration of blood supply to an isch-
emic area causes reperfusion injury leading to more damage, and enlargement of
ulcer. This is common in paraplegic patients who with good intentions are turned
from side to side in order to relieve pressure exerted on bony prominences. The
products of the reperfusion injury, such as oxygen free radicals, along with the
mediators of inflammation, have been implicated in the cell death and necrosis. In
patients with normal mental state, sensation, and mobility, the pressure ulcers are
unlikely to develop as compared to the neurologically impaired. The “non-neuro-
logically compromised” patients automatically shift from one area of increased
pressure to another frequently before the effects of prolonged pressure occur. The
neurologically compromised patients, who have loss of sensation combined with
inability to avoid long pressure by moving, are more prone to skin breakdown. In
addition, in these cases chronic contractures, spasticity, disuse atrophy, under nour-
ishment and poor general condition may lead to large ulcers.
Patients with diabetes develop pressure ulcers [5–7] (mostly in the legs and feet)
as a result of neuropathy (inability to adequately feel the lower extremities), periph-
eral arterial disease (PAD, responsible for a mismatch in the blood supply), and
stiffening of the plantar soft tissues as people age, and changes in the gastrocne-
mius-soleus muscle complex, (leading to contractures in the Achilles tendon and
contact on the ground being made with the forefoot). These factors explain the
development of ulcers in the forefoot in mobile patients. Another element is size of
shoes [8] as “most feet of elderly people with or without diabetic neuropathy do not
fit ordinary casual footwear”, since the feet were generally broader than the foot-
wear available.
Incidence of Pressure Ulcers
What is the incidence or prevalence of pressure ulcer in the neurologically compro-

mised patients? Available figures do not distinguish between these patients and
others. Even then reliable figures do not exist. A European pilot survey noted that
up to one in five acute care patients suffered some degree of tissue damage [9].
Others report that as many as 1 in 4 patients with pressure ulcers developed them in
hospital [10].
Reported prevalence rates range from:

4.7–32.1% for hospital populations
4.4–33% for community care populations
4.6–20.7% for nursing home populations
23 out of 103 patients developed at least one pressure ulcer after an average stay
of 6.4 days [11]. In the Greater Glasgow Health Board, 8.8% of patients had a pres-
sure ulcer. Those aged 70 or over accounted for 70% of skin breakdown [12].
England and Wales do not conduct a national pressure ulcer survey. For this
reason, data that could be used to illustrate important trends in practice and clinical
outcome are not available. An audit carried out by Phillips [13] showed that more than
half of all the ulcers recorded in the UK during 2005–2007 were acquired in hospital.
An unpublished audit on the Duke of Cornwall Spinal Treatment Centre in Salisbury
(UK) in 2008, showed that 26% of patients referred for rehabilitation within 6 weeks
of their injuries had already contracted a pressure ulcer on admission [14]
A study of the prevalence of pressure ulcers in a community sample of Spinal
Injury patients [15] suggests that smoking and pre-existing conditions were associ-
ated with increased prevalence. Nearly one quarter of participants had pressure
ulcers at the time of the survey. Periodic weight lifts and daily inspection of skin for
pressure ulcer damage were not associated with decreased prevalence in the sample.
However, those who inspected skin daily tended to detect pressure ulcer damage
early. These findings correlate with the study from Coggrave and Rose [16], which
showed that a brief pressure lift failed to raise tissue oxygen in the trochanteric
areas. They established that the lift must be at least 1 min 51 s in duration each time
for proper tissue oxygenation in the trochanteric area. Gyawali et al. [17] suggests
that intermittent electrical stimulation redistributes pressure and promotes tissue
oxygenation in loaded muscles of individuals with spinal cord injury.
Loss of feeling was critical as patients were unable to appreciate pain when an
ulcer was developing. Risk of developing an ulcer increased with age. The duration
of the paralysis was equally important. After discharge from hospital, many patients
developed skin breakdown because of poor facilities at home, inappropriate advice
from those who look after them and physiological changes in their body.
In summary, just doing pressure lifts is not a passport to avoiding pressure ulcers
occurring. Many other factors are at play: general condition, muscle bulk and tone,
body weight, fat content and skeletal frame; nutrition, age, spasticity, psychology,
skin moisture, friction, shear and arterial pressure also need to be taken into consid-
eration when planning treatment.
Sites of Pressure Ulcers
40% of pressure ulcers are in the sacral area, 40% are in the heels, the other sites in
the lower limbs would be foot, ischium, trochanter; other areas are legs or thighs
(e.g. when a catheter tubing rubs against the skin causing friction and pressure)
(Figs. 6.1, 6.2, 6.3, 6.4, 6.5, and 6.6).
Fig. 6.1 Sacrum – a sacral

ulcer
Fig. 6.2 Forefoot – in this

case tight footwear and
sweating has led to this ulcer
Fig. 6.3 Heel – this is

another example of the
effects of a tight-fitting shoe,
in addition to vascular
problems
Fig. 6.4 Lateral malleolus –

the contours of a shoe or the
lateral malleolus rubbing
against a hard surface in bed
or wheelchair as a result of
spasticity, can lead to this
type of skin damage
Fig. 6.5 Ischium – a coin in

a pocket, poor seating or
cushion can lead to ischial
ulcers
Fig. 6.6 Trochanter –

patients in the lateral position
and bedridden can develop
trochanteric ulcers
Other Sites
See Figs. 6.7 and 6.8.
Recurrence of Pressure Ulcers
Most recurrent ulcers were severe, located at the same anatomical site and reoc-
curred within 4 months, suggesting that they were more likely incomplete healing
of the initial affected area.
Fig. 6.7 Knee ulcer (from

tight jeans)
Fig. 6.8 Scrotum –

inappropriate clothing, plastic
materials (responsible for
sweating and friction),
urinary tract infection and
indwelling urethral catheters
can cause ulcers in the
scrotum
Assessment, Measurement
Clinical assessment: Inspection (colour, changes observed, presence or not of ery-

thema, necrosis, and excoriation), palpation (temperature, blanching of erythema),
examination (sensation, degree of hydration, tissue consistency). Thus we would be
able to distinguish between:
• Normal Reactive Hyperaemia – a visible effect of localized vasodilatation (redness).
The area will blanch with fingertip pressure and
• Abnormal Reactive Hyperaemia – excessive vasodilatation and induration
(edema) in response to pressure. The skin appears bright pink red and the redness
lasts 1 hour to 2 weeks.
In either case, patients will be kept on bed rest until the hyperaemia clears.
In a suspected or established pressure ulcer, the consensus is to use the
classification of European Pressure Ulcer Advisory Panel grading system
(EPUAP) [18].
• Grade 1: intact skin with non-blanchable redness of a localized area usually
over a bony prominence. Darkly pigmented skin may not have visible blanch-
ing; its colour may differ from the surrounding area. The area may be painful,
firm, soft, warmer or cooler as compared to adjacent tissue. Grade I may be
difficult to detect in individuals with dark skin tones and indicate “at risk”
persons.
• Grade 2: partial thickness skin loss involving epidermis, dermis, or both. The
ulcer is superficial and presents clinically as an abrasion or blister. Surrounding
skin may be red or purple.
• Grade 3: full thickness skin loss involving damage to, or necrosis of, subcutane-
ous tissue that may extend down to, but not through underlying fascia.
• Grade 4: extensive destruction, tissue necrosis, or damage to muscle, bone, or
supporting structures with or without full thickness skin loss. Extremely difficult
to heal and predisposed to fatal infection.
The extent of damage will be assessed through clinical examination, use of a
sterile probe (to assess depth), serial good quality photography (to monitor progress
of treatment), plain x-rays or MRI or CT scan, with or without contrast if there is a
sinus (to rule out osteomyelitis and involvement of underlying tissues).
There are software programmes available today that will allow precise mea-
surement of areas and volumes (when needed), and monitoring of pressure
ulcers remotely so that the plastic surgical or rehabilitation teams can rationa-
lise their resources and remotely offer their expert services to more patients. The
use of remote monitoring – telemedicine – must be encouraged so specialist
skills may be available without the patient having to travel long distances, and
to promote the role of trained, but non necessarily medical doctors, to provide
first line care.
Preventative Measures
In bed, in the acute stage, patients are “risk assessed” for the occurrence of pressure
ulcers using scales like the Waterlow or Braden. These scales are tools that should
by no means replace clinical involvement. Patients need to be log rolled (also called
30° tilt) every 3 h, and each time the skin is checked. In rehabilitation and later
stages, the patients would be turned every three hours and the skin monitored. If the
skin is at risk, they could be put on a pressure-relieving mattress and a cushion
would be used in the chair; whilst providing additional layers of protection, these
devices do not dispense with the need for position changes and regular skin inspec-
tion. All patients should have a cushion, chosen to suit individual needs.
The National Institute of Clinical Excellence (NICE) guidelines [19] make a

series of evidence-based recommendations that focus on the holistic care of the
patient. Unfortunately intervention is more often reactive than preventative.
NICE suggest that assessment of risk (of pressure ulcers) is carried out within
6 h of admission, yet just four (less than 10%) of the hospitals surveyed [20]
had obtained data whether this actually happened. Just six hospitals (less than
15%) audited the presence of a preventative care plan for those with identified
risk or with existing ulcers; 60% of patients should have had the above plan but
did not.
Schoonhoven noted that a percentage of patients with grade 3–4 sacral
wounds, were not provided with any cushion and sat up for 7 h or more. 50% of
patients with active ulcers on the sacral/coccygeal or ischial regions sit out
between 2 and 7 h.
More recently, the importance of height and dimension of the chair when patients
are seated, have been highlighted. However, as the availability of variable height
chairs was limited to the hospitals audited, this has not been considered alongside
safe cushions for this purpose.
In the seated position, optimum pressure is as important as the nature of the sup-
port surface, as some wheelchair cushions offer good pressure relief but poor pos-
tural support and vice versa.
Interface pressure analysis does not provide complete information about the
effectiveness of pressure relief manoeuvres. Measures of tissue perfusion may help
to establish more effective strategies. Relief achieved by standard wheelchair push-
ups may not be sufficient to recover tissue perfusion compromised during sitting;
alternative manoeuvres may be necessary.
The dynamic seating system provided effective pressure relief with sustained
reduction in interface pressure adequate for complete recovery of tissue perfusion.
Dynamic Seating System is an active wheelchair seating system that dissipates
energy resulting from user extensor thrusts (in high level injuries with extensor
spasm). The system provides support during episodes of controlled, voluntary
motions, while dissipating energy during involuntary thrusts.
Differences in perfusion recovery times between subjects with spinal cord
injury and controls raised questions about the importance of changes in vascular
responses to pressure after spinal cord injury. The risk of developing pressure
ulcer was 4.3 times greater in complete paraplegia patients than in any other type
of spinal cord injury. Complete paraplegic patients who do not practise standing
period regularly, or who have compliance issues have a greater risk of suffering
pressure ulcers.
Patients with spinal cord injury and pressure sores suffer from anaemia and
serum protein alteration that fell within the range of metabolic alteration of chronic
disorders and neoplastic diseases. The alterations depend on a decreased utilisation
of iron stores in the reticuloendothelial system, and on inhibition of the hepatic
synthesis of albumin. Iron treatment should be avoided because of the risk of
hemochromatosis.
Traditionally interface pressure measurement between the person’s skin and the
seat or mattress has been carried out using sensors. These days, pressure mapping
provides an improvement to the sensors previously used. The answers provided by
the system should be interpreted with caution, for reasons explained above; many
other factors account for the skin to be at threat. Pressure mapping offers a good
visual aid to show the comparative pressures between different surfaces, which is
useful when trying to convince the patient to change equipment.
Treatment and Post-Healing Mobilisation
Medical treatment is the mainstay of treatment options. Many may heal with conser-
vative measures and less than 10% of the patients require surgical intervention. Key
principles in the conservative management include pressure reduction procedures,
adequate debridement of the necrotic and devitalised soft tissue including underlying
devitalised bone, control of infection followed by meticulous wound care.
In the neurologically impaired patients the following important issues need to be
addressed:
(a) Spasticity should be controlled with appropriate medication; in some instances,
surgical intervention may be required.
(b) Optimise nutritional status with adequate intake of calories, proteins and vita-
mins. A normal balance can be achieved with oral and or parenteral nutrition.
Restoring near normal nutritional status reduces the risk of developing pressure
ulcers. The use of the Malnutrition Universal Screening Tool (MUST) is highly
recommended [21].
(c) A normal and positive nitrogen balance of 1.0–2.0 g/kg/day [22, 23].
(d) Dealing with urinary or faecal incontinence to prevent wound contamination
and subsequent infection.
(e) Wound care with specialised agents that help in debridement and dissolution of
necrotic tissue. Recent introduction of Topical Negative Pressure (TNP) method
has revolutionised the conservative management of pressure ulcers.
Surgical Treatment
A radical wound debridement with excision of the bursa, necrotic and osteomyelitic
bone is necessary prior to the planning of reconstructive procedure. The goals of
reconstruction are improvement of patient hygiene and appearance, prevention or
resolution of osteomyelitis/infection, reduction of fluid and protein loss through the
wound, prevention of further malignancy (Marjolin ulcer).
Reconstruction is defined by various factors like anatomic site of ulcers, previous
scars or surgeries and surgeon’s preference and expertise. A vascularised tissue is
placed in the wound to eliminate dead space, enhance perfusion, decrease tension
on the wound closure and provide a source of padding especially over the prominent
bony areas.
The commonest choices of flaps are the musculocutaneous and fasciocutane-
ous flaps. Rarely a free flap from a distant site could be harvested for cover of the
ulcer and the dead space. Drains placed in the wound prevent immediate haema-
toma and seroma formation that may lead to wound problems of infection and
dehiscence.
In general, chronic stage 3 and 4 pressure ulcers require reconstruction. The deci-
sion to intervene surgically is usually done by a multidisciplinary team who provide
patient social support, medical optimisation and postoperative rehabilitation and
physiotherapy. A clear understanding of management of postoperative care is impera-
tive in order to prevent further recurrence that remains very common in many cases.
The site of an ulcer determines the selection of the type of flaps. Fasciocutaneous
flaps are shown to withstand more stress, shear and pressure as compared to the
muscle flaps. The tolerance to ischemia in muscle flaps is relatively low as com-
pared to other flaps. Grade 1 and 2 pressure ulcers are treated with conservative
dressings and/or Topical Negative Pressure dressings, following a thorough debri-
dement and optimising nutrition and medical treatment.
Preoperative preparation involves control of spasms either by medication, pos-
ture management or surgery, arranging for pressure reducing mattresses or cush-
ions, treating uterine tract infections (UTI) as appropriate, and managing faecal and
urinary incontinence.
The examples of surgical options of flaps for various sites are
(a) Ischial pressure ulcer: Gluteal thigh flap
(b) Sacral defects:
– Gluteal thigh flap, Gluteus musculocutaneous muscle flap, Inferiorly based
rotation flap.
– Superior & inferior Gluteal artery flaps based on the Gluteus maximus
muscle/sliding flaps
– V–Y Gluteal advancement flaps
(c) Trochanteric pressure ulcer: Tensor fascia Lata (TFL flap)
(d) Multiple ulceration/recurrences (where no local flap options are available, the
options are fillet flap/amputation/free flap.
Mobilisation After Healing
The problem is different for the ambulant patient compared to the wheelchair-user,
as the former will be encouraged to stand. In either case, all skin marks must have
disappeared and account must be taken of the fact that the patient has been on bed
rest for a long time, and may struggle with postural hypotension. The process of
mobilisation must be gradual and may necessitate the use of sympathomimetic
amine drugs, 30 min before mobilisation. The Duke of Cornwall Spinal Treatment
Centre (DOCSTC) [24] provides a detailed protocol for mobilisation.
If the ulcer is sacral, it is often preferable to get the patient to directly sit in a
chair, instead of the bed. The act of mobilisation involves the multidisciplinary team
(physiotherapists, occupational therapists, nurses) to ensure that causes having led
to the ulcer are identified and eliminated. In addition, the appropriate clothing,
transfer methods, wheelchair and cushion must be selected. Pressure mapping will
be carried out to identify any areas of high pressure.
The patient will stay in the chair for a very short time the first day. Over the fol-
lowing days, they will slowly increase their time to build up the pressure tolerance.
Each time the skin will be monitored, and if there is any concern they should go
back on bed rest.
Conclusion
A significant proportion of patients are at risk of developing pressure ulcers in the

hospitals, and in the community. The Waterlow or Braden score is often routinely
carried out on admission of a patient, after which little happens. There appears to be
a lack of understanding of the importance of initiating preventative measures, or
paying attention to detail (choice of wheelchair, clothing or cushion). In certain
centres, patients would be well looked after whilst they are in bed, but would be
allowed to sit in a chair with an established ulcer! At times there may not be any
cushion on offer in the chair. There is much to do in these areas.
For many years patients have been instructed to carry out pressure lift, but
research suggests that these lifts may be too short to allow re-oxygenation of tissues
in bony prominences. Long lifts may exert too much pressure on the shoulders, and
cause other types of damage – given that paraplegics develop shoulder pain and
degeneration through years of lifting and transferring. Alternative methods of pres-
sure relief exist and are the topics of current studies.
It is essential to reduce the incidence of pressure ulcers in hospitals offering care
for the acutely ill and in the community. Measures must therefore be taken using
modern media available, including the Internet and social networks to provide edu-
cation to professionals, patients and caregivers so that there is more awareness of
these issues that impact on quality of life of individuals at a high economic cost to
society. It is vital to ensure good physical and mental health and appropriate
nutrition.
Once an ulcer is suspected or established, the patient will be on strict bed-rest
and managed by an expert team, preferably a multi-disciplinary team of specialists
in rehabilitation, working in partnership with tissue viability specialists and plastic
surgeons. They will identify the factors that have led to the ulcer and decide on the
best treatment options, conservative or surgical, taking into account the challenges
of impaired motor power and sensation. Research of new methods to treat ulcers
will include the use of fibroblast growth factor [25].
Once the ulcer has healed, the mobilisation is a slow process to ensure long-
lasting benefits of all the efforts invested, and prevent recurrence. It is a long road
but one that must be crossed so patients with spinal injuries and consequent pressure
ulcers get the best care.
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Chapter 7
Complications of Wound Healing
Somprakas Basu and Vijay Shukla
Keywords Wound healing • Complications • Corrupt matrix • Biofilm • Collagen

disorders and wound infection
Introduction
Wound healing is an organized, four-phased system involving hemostasis,

inflammation, proliferation and maturation. If injured tissue follows these phases,
the wound will close. However this process is far from simple. In itself, wound heal-
ing involves a huge orchestration of cells and biochemical molecules in an environ-
ment of constantly changing signaling processes, temporally and spatially. A variety
of physiological events and environmental agents can divert this healing cascade,
alter the wound bed environment and stall healing. Moreover complications can
also arise from uncontrolled physiological processes of healing in response to envi-
ronmental stimuli, patient’s past and present health status as well as a combination
of these factors. One important point to be kept in mind is that it is the entire patient
not just the wound site that suffers from and responds to the stress of a chronic
wound or a complication. A wound and its healing mechanism is not a process inde-
pendent of the patient. Thus attention to nutrition, immunity, treatment of systemic
diseases and an overall well being of the patient including a good quality of life is
so very important for a holistic approach toward wound management.
S. Basu, M.B.B.S., M.S. • V. Shukla, M.B.B.S., MCh (Wales) (*)

Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University,
Varanasi, Uttar Pradesh 221005, India
e-mail: vkshuklabhu@gmail.com

110 S. Basu and V. Shukla
Perhaps the most important wisdom of the surgeon is that many kinds of disor-
dered healing can better be avoided in time. Liver disease can be treated, steroid
hormone excess can be corrected or specifically treated with vitamin A, and nutri-
tional deficits can be anticipated and avoided or treated/supplemented. Familial
tendencies to scar formation can be thwarted at an earlier rather than later, often
hopeless, stage. Burn contractions across susceptible joints can be anticipated and
prevented by splinting and traction. However, what we do not have is a prevention
or cure for a vast variety of conditions of excessive repair, conditions that sur-
geons do not ordinarily recognize as complications of wound healing. A vast
amount of human misery is due to hypertrophic scar formation in arteries (arterio-
sclerosis), joints (arthritis), and ocular disease, especially those involving the
scarring of the cornea. Surgery may contribute to the prevention as well as cure of
these disorders.
The complications of wound healing can be grouped under three broad head-
ings: (1) delayed or non-healing, (2) poor healing and (3) uncontrolled healing.
The first condition is a non-healing chronic wound which may be due to disrup-
tion in the normal physiological processes of repair. It can be due to an abnor-
mally extended inflammatory phase without progress to the proliferative phase,
commonly seen in wounds for example infected wounds or lower extremity
venous ulcers. It may also be due to failure of neovascularization or failure of
epithelization or even an arrest in wound contraction. The second broad compli-
cation may be due to an inappropriate inflammatory phase which leads to poor
proliferation and inadequate collagenisation, or inadequate polymerization, cross-
linking of collagen, defective collagen synthesis due to genetic predisposition, or
even in some metabolic states or drug use, so commonly seen in patients undergo-
ing steroid therapy and in patients with uncontrolled diabetes mellitus. It may
also occur from a poor proliferative phase leading to maturation arrest as seen in
the venous ulcer due to local nutritional imbalance, inadequate oxygen available
for healing, and edema. The wound tends to breakdown easily and recur. The
third group contains uncontrolled collagenisation which may lead to excessive
scarring, hypertrophic scars and keloids. Here it needs to be emphasized that
wound healing is a continuous process with temporal merging of the different.
They are so closely interrelated with the maintenance of a perfect balance that at
times a defect in a particular physiological step may lead to arrest or disorder in
multiple pathways resulting in an overlap of outcome, e.g. a wound in a patient
with uncontrolled diabetes may be infected, may have poor neovascularization or
poor wound contraction, all in the same wound. Hence effort should be made to
broadly diagnose what is the principal problem and how it can be tackled or pre-
vented and not as problems of various phases of healing. Although it is easier to
acknowledge that a weak scar can be due to protein deficiency or poor cross-
linking of collagen, the goal of management should be the patient as a whole and
not the wound.
7 Complications of Wound Healing 111
Normal Repair of Wound

Before we attempt to understand the various disorders of repair, it is important that
we should look at the process of normal repair of a wound. Wound healing is an
obligate response to various types of stimuli. These stimuli may be injury, an antigen-
antibody reaction, a thrombus, a bacterial or viral infection, and possibly some other
stimulus. In the case of injury, the healing sequence is initiated by activation of tissue
complement or by the coagulation cascade. The admixture of platelets and thrombin
produces platelet derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1),
epidermal growth factor (EGF), and fibroblast growth factor (FGF) that have the
capacity to stimulate replication of fibroblasts and vascular endothelial cells in the
area of injury [1]. Serotonin is also released, which, together with histamine (released
by mast cells), induces a reversible opening of the junctions between the endothelial
cells, allowing the passage of neutrophils and monocytes (which become mac-
rophages) to the site of injury. Activated tissue complement fractions also attract
leukocytes. The polymorphs and lymphocytes which enter the wound seem to be
basically devoted to preventing infection. Macrophages, on the other hand, seem to
contribute significantly to the normal healing of injuries. The admixture of mac-
rophages with fibrin, foreign body, endotoxins, and bacteria activates macrophages to
release transforming growth factor alpha (TGF-alpha) and transforming growth
factor beta (TGF-beta), vascular endothelial growth factor (VEGF) and FGF that
account for most of the subsequent cellularity of wounds [2]. Macrophage factors
also account for at least some of the stimulus to epithelization. The marginal basal
cells at the edge of the wound migrate across the wound, and, within 48 h, the entire
wound is epithelized. In short, the macrophage encodes the events of injury and
translates them to signals for repair and thus coordinates the whole process in a
smooth and sequential manner. At the end of the normal inflammatory reaction to
injury, the elements of proliferation, namely, fibroblasts, new vessels, and hypertro-
phic epithelial cells, are all present and ready to deposit the extracellular connective
tissue matrix, collagen, fibronectin, and proteoglycans. The stimuli to initiate colla-
gen synthesis and deposition seem to relate to the anatomy, or spatial relationships of
the site of injury. All wounds are characterized by a site at which injury and
inflammation are at their extremes. At that site, partial pressure of oxygen, pO2, is
low while pH and partial pressure of carbon dioxide, pCO2, are high. Relatively low
levels of oxygen tension stimulate replication induced by growth factors. Oxygen
tensions either above or below this range seem to inhibit cell replication. When
enough cells have accumulated, lactate levels increase, thus stimulating the forma-
tion of enzymes for collagen synthesis. These enzymes are not found in cells at higher
pO2 and lower environments of lactate for longer periods of time.
Fibroblasts synthesize and excrete collagen monomer that is then polymerized to
provide the strength needed to maintain tissue continuity. Numerous biochemical
steps are required for biosynthesis of collagen monomer as well as for polymerization.
The first collagen fibers accumulated outside the cell is in the form of a gel the mate-
rial resulting from lysis by extracellular enzymes of fibroblastic and leukocytic ori-
gins. As the wound matures, it remodels and replaces the original gel with larger,
stronger fibrils and fibers. Wound contraction is a pronounced feature in wounds left
to close by secondary intention. The cells responsible for wound contraction are
called myofibroblasts, which resemble fibroblasts but have cytoplasmic actin filaments
responsible for contraction. A wound continuously undergoes remodeling to try to
regain the state similar to that prior to injury. It regains 70–80% of its original tensile
strength at 3–4 months postoperative, and 90–95% at the end of 12 months. The
remodeling continues throughout the lifetime of the patient. The following steps sum-
marize the process of wound healing into a vascular response and cellular responses.
Vascular response
• Initial vasoconstriction as a direct response to trauma
• Exposed sub endothelial tissue activates coagulation and complement cascades
• Platelet adhesion and aggregation causes clot formation
• Degranulation of platelets releases growth factors and chemotactic factors
• Inflammatory response due to histamine and 5-hydroxytryptaminen(5HT) release
produces:
– Vasodilatation
– Increased capillary permeability
– Margination of neutrophils
Cellular response
• Migration of neutrophils, macrophages and lymphocytes
• Macrophages produce growth factors leading to migration of fibroblast and
keratinocytes.
• This causes cellular proliferation with three components:
– Epithelialization
– Wound contraction
– Fibroplasia
• Epithelial barrier is important to prevent infection and to maintain fluid balance

• Migration of keratinocytes require presence of granulation tissue
• When epithelial cover is complete, contact inhibition prevents further epithelial
growth
• Contraction due to myofibroblasts can account for up to 80% reduction in wound
size
• Fibroplasia due to procollagen production by fibroblasts
• Intra/intermolecular bonds form the collagen fibers (triple helical quaternary
structure)
• Extracellular matrix contains fibronectin and glycosaminoglycans
• It regulates collagen synthesis and cellular differentiation
• Simultaneous angiogenesis
• Proliferation is followed by remodeling

• Maximum collagen production occurs at 20 days
• Maximum wound strength at 3–6 months
• Initial collagen production disorganized
• Remodeling lines it up with stresses in skin
• Reduced vascularity and cellularity
Complications of Wound Healing: Delayed Healing/Non-healing
In healthy individuals with no underlying inhibitory factors an acute wound should

heal within 3 weeks with remodeling occurring over the next year or so. If a wound
does not follow the normal trajectory of healing, it may become arrested in one of the
stages and the wound becomes chronic. Chronic wounds are thus defined as wounds,
which have “failed to proceed through an orderly and timely process to produce
anatomic and functional integrity, or proceeded through the repair process without
establishing a sustained anatomic and functional result” [3]. Resulting from a loss of
balance in the physiological mechanisms of healing, a wound may enter into a phase
of delayed healing or in extreme situation, non-healing. Conventionally, a period of
6–8 weeks has been accepted by various authorities as the cut off time, beyond which
the wound is labeled as chronic/non-healing. A number of causes of delayed healing/
non-healing are well known, e.g. wound infection, persistence of foreign body or
bacterial proteins, chronic irritation and trauma, and ischemia, of which the wound
infection is the commonest cause of wound related morbidity. These wounds cause
severe emotional and physical stress as well as create a significant financial and
nutritional burden on patients and the whole healthcare system as a whole. In chronic
wounds, the actual “wound” area is more than the lesion that is visible; this is to say
that pathological damage occurs to tissues around the area where skin integrity is
evidently disrupted. Venous ulcers are generally surrounded by a fibrotic process
called lipodermatosclerosis, with which is healing difficulties are associated [4]. A
callus is almost always present around neuropathic ulcers in the patient with diabe-
tes. It is arguable that this plays a role in the pathogenesis of non-healing [5]. Tissue
beneath and around ulcers is also affected by the fundamental abnormality leading to
the ulceration, whether venous hypertension or pressure. Once a wound is defined
chronic the underlying causes should be identified.
Local and systemic factors that impede wound healing:
Local factors
• Inadequate blood supply
• Increased skin tension
• Poor surgical apposition
• Wound dehiscence
• Poor venous drainage
• Presence of foreign body and foreign body reactions
• Continued presence of micro-organisms

• Infection
• Excess local mobility, such as over a joint
Systemic factors
• Advancing age and general immobility
• Obesity
• Smoking
• Malnutrition
• Deficiency of vitamins and trace elements
• Systemic malignancy and terminal illness
• Shock of any cause
• Chemotherapy and radiotherapy
• Hyperbilirubinemia, uremia
• Immunosuppressant drugs, corticosteroids, anticoagulants
• Inherited neutrophil disorders, such as leucocyte adhesion deficiency
• Impaired macrophage activity (malacoplakia)
Epidemiology
Chronic wounds mostly affect people over the age of 60 years. The incidence is about
0.78% of the population and the prevalence ranges from 0.18% to 0.32% [6]. As the
population ages, the number of chronic wounds may be expected to increase [7].
Types
The majority of chronic wounds usually fall into three predominant categories:
venous ulcers, diabetic ulcers, and pressure ulcers. A small number of wounds that
do not fall into these categories may be due to radiation induced or ischemic causes.
Venous Ulcers
Venous ulcers, which usually occur in the legs, account for about 70–90% of chronic
wounds [8] and mostly affect the elderly. These the result of unrelieved venous
hypertension caused by improper function of valves that exist in the veins to prevent
blood from flowing backward. Valve dysfunction may occur due to damage result-
ing from deep vein thrombosis or congenital aplasia. Microvascular ischemia results
from the venous trapping of blood and combined with reperfusion injury due to
activation of trapped leucocytes and consequent tissue injury, leads to formation of
these ulcers.
Diabetic Ulcers
Diabetes mellitus, another major cause of chronic wounds, is increasing in preva-

lence [9]. Patients with diabetes commonly get neuropathy a sequel of which is
diabetic foot disease and wounds. This cohort has a 15% higher risk of amputation
[8]. Diabetes causes neuropathy, which inhibits nociception and the perception of
pain. Thus these patients may not initially notice small wounds in their legs and feet,
and may therefore fail to prevent infection or repeated injury [10]. Further, diabetes
causes immune compromise and microangiopathy thus preventing adequate oxy-
genation of tissue leading to ischaemia and poor healing [10]. Excess pressure also
plays a role in the formation of diabetic ulcers.
Pressure Ulcers
These are another leading type of chronic wounds [7], which usually occur in
people with conditions such as paralysis, coma or unconscious states that inhibit
movement of body parts that are commonly subjected to pressure; such as elbow,
heels, shoulder blades, and sacrum [11]. Ischaemia occurs when pressure in the
tissue is greater than pressure in the capillaries which leads to restriction of
blood flow into the area [7]. In such circumstances, when there is shear stress on
tissue, breakdown may occur. The presence of fistulas and incontinence states
favours tissue breakdown resulting in pressure ulcers. Muscle tissue, which
needs more oxygen and nutrients than skin does, suffers from the worst effects
from prolonged pressure. As in other chronic ulcers, reperfusion injury damages
tissue.
Predisposing Factors
In addition to poor circulation, neuropathy, and difficulty in movement, other fac-

tors that contribute to chronicity include systemic illnesses, old age, and repeated
trauma. Comorbid ailments that may contribute to the formation of chronic wounds
include vasculitis, immune suppression, pyoderma gangrenosum, and diseases that
cause peripheral ischemia [8]. Immune suppression can be caused by illnesses or
drugs used over a long period, for example steroids [8]. Emotional stress can also
negatively affect the healing of a wound, possibly by raising blood pressure and
levels of cortisol, which lowers immunity [12].
An important factor that may contribute to chronic wounds is old age. The skin
of older people is more easily damaged, skin capillaries are fragile and older cells
do not proliferate as fast and may not have an adequate response to stress in terms
of gene upregulation of stress-related proteins. In older cells, stress response genes
are over expressed when the cell is not stressed, but when it is, the expression of
these proteins is not upregulated by as much as in younger cells.
Comorbid factors that can lead to ischemia are especially likely to contribute to
chronic wounds. Such factors include atherosclerosis, edema, sickle cell disease,
and arterial insufficiency-related illnesses [8].
Repeated physical trauma plays a role in chronic wound formation by continu-
ally sustaining the inflammatory cascade. The trauma may occur by accident, for
example when a leg is repeatedly bumped against a wheelchair rest, or it may be due
to intentional acts. Heroin users who lose venous access may resort to ‘skin pop-
ping’, or injecting the drug subcutaneously, which is damaging to tissue and
frequently leads to chronic ulcers [13]. Children who are repeatedly medically
examined on account of a non healing wound have been known to be victims of a
parent with Munchausen syndrome by proxy, a disease in which the abuser may
repeatedly inflict harm on the child in order to receive attention. [14]
Pathophysiology
Chronic wounds may affect merely the epidermis and dermis, or all the way to the
fascia [6]. Such wounds may result from the same causes as do acute wounds, such
as surgery or accidental trauma, or they may form as the result of systemic infection,
vascular, immune, or nerve insufficiency, or comorbidities such as neoplasia or
metabolic disorders. The reason a wound becomes chronic is the body’s inability to
deal with the damage produced by factors such as repeated trauma, continued pres-
sure, ischemia, or debilitating illness [6, 10].
Though much progress has been accomplished in the study of chronic wounds,
advances in the study of their mechanism of healing have lagged behind expecta-
tions. This is partly because animal studies are difficult, animals do not get chronic
wounds, since they usually have loose skin that quickly contracts, and they normally
do not get old enough or have contributing diseases such as neuropathy or chronic
debilitating illness. Nonetheless, researchers at present understand some of the
major factors that lead to chronic wounds, among which are ischemia, reperfusion
injury, and bacterial colonization.
Ischemia is an important factor in the development and persistence of chronic
wounds, especially when it occurs repetitively and/or when combined with the
patient’s old age. Ischemia causes tissue to become inflamed and cells to release fac-
tors such as interleukins, chemokines, leukotrienes, and complement components
that attract neutrophils. While they fight the pathogens, neutrophils also release
inflammatory cytokines and enzymes that damage cells [8]. The production of reac-
tive oxygen species (ROS) to kill bacteria for which they use myeloperoxidase is an
essential role of neutrophils. The enzymes and ROS produced by neutrophils and
other leukocytes damage cells, prevent cell proliferation and wound closure by dam-
aging DNA, lipids, proteins [15], the extracellular matrix (ECM), and cytokines that
normally aid healing leading to a corrupt matrix. Neutrophils remain longer in
chronic wounds than acute wounds contributing to higher levels of inflammatory
cytokines and ROS [16]. Since wound fluid from chronic wounds has an excess of
proteases and ROS, the fluid itself can inhibit healing by inhibiting cell growth and
breaking down growth factors and proteins in the extracellular matrix (ECM).
“Corrupt” Matrix
It has been proposed that a lengthened inflammatory phase causes increased levels
of proteases such as matrix metalloproteases (MMPs), elastase, plasmin and
thrombin, which destroy components of the extracellular matrix (ECM) and dam-
age the growth factors and their receptors that are essential for progression of heal-
ing. Elevated levels of oxygen free radicals that alter these essential components,
have an important role in the pathogenesis a chronic wound [17, 18]. The matrix is
an essential component of the proliferative phase of an acute wound and which is
the main medium of cellular interaction in a healing wound is “corrupted” in a
chronic wound. It has been observed that fibronectin is totally degraded in wound
fluid collected from venous stasis ulcers and is partially degraded in the exudate
collected from chronic diabetic foot ulcers [19, 20]. Interestingly, the addition of
intact plasma fibronectin to chronic wound fluids results in rapid degradation of the
fibronectin; similar results have also been reported with vitronectin [19].
The extracellular matrix plays an important regulatory role in the biological
behavior of epidermal cells. For epidermal cells to migrate from the wound edges
they must be in contact with early matrix containing fibronectin, fibrin and collagen.
Migrating cells move from the wound edge toward the center adhering to the ECM
traveling along the fibrin, fibronectin and collagen strands. It is arguable that degra-
dation of these proteins by high proteolytic activity, contributes to the delays
observed in epidermal resurfacing by keratinocytes as well as poor granulation tis-
sue formation by vascular endothelial cells and fibroblasts. Typically in chronic
wounds the epithelial cells proliferate and bunch up on the wound edges but fail to
migrate. Lack of a normal ECM at the wound edges is now recognized to be the real
problem. Failure of re-epithelialization is therefore an excellent example of a matrix
deficiency state. It is also thought that elastin degradation occur in chronic wounds.
The likely source of elastase is the chronic wound is the polymorphonuclear leuko-
cytes. Elastin degradation or its decreased expression leads to the loss of elastolytic
properties in skin as evident in ageing or scar tissues [21].
Evidence of collagen degradation in chronic wounds has largely been inferred
from data measuring levels of MMPs in chronic ulcer fluid [22–24]. Analysis of tis-
sue biopsies from chronic pressure ulcers has shown that levels of MMP are
significantly elevated and are strongly associated with inflammatory cells when com-
pared with normal skin tissue [25]. Other proteases may also play important roles in
damaging ECM components or growth factors. The serine protease urokinase plas-
minogen activator (uPA) is known to activate a proteolytic cascade, resulting in the
activation of MMPs [26]. The lack of regulation of protease activity in chronic
wounds, e.g. alpha-1 protease inhibitors, tissue inhibitors of matrix metalloproteases

(TIMPs) leads to the degradation of ECM, eventual tissue breakdown, ulceration and
skin stripping frequently seen in venous leg ulcers and pressure ulcers.
The resident cells in a chronic wound (fibroblasts, keratinocytes, and endothelial
cells) go through several cycles of replication, probably reach their limits of prolif-
eration and become senescent. Evidence of poor proliferative potential is obtained
from the experimental observation of fibroblasts cultured from older animals, which
exhibit decreased proliferative potential in vitro [27]. Indirect evidence suggests
decreased proliferative potential of fibroblasts from diabetic ulcers [28, 29] and
venous ulcer fibroblast [30–33] compared to normal fibroblasts though the reasons
are for this are unclear. It is speculated that this involves senescence of the cells
through repeated division. Recent reports in the literature indicate that telomere
shortening may have a probable role [34–36]. Thus the characteristics of a typical
chronic wound are (1) an autonomous state of inflammation (2) a corrupt ECM (3)
poorly responding cells particularly fibroblasts limiting ECM production (4) failure
of re-epithelization due to lack of ECM orchestration and a defective scaffold.
Growth Factor Problems
Growth factors are, in essence, proteins that orchestrate cellular activities. Their
function is dependent on the receptor sites they attach to. They not only affect cell
growth but also play an important role in cell migration, differentiation, and apop-
tosis [37]. During the initial inflammatory phase, platelets release several growth
factors such as platelet-derived growth factor (PDGF), epidermal growth factor
(EGF), insulin-growth factor (IGF-1), and transforming growth factor beta (TGF-
b). Each of these factors and many others are essential for cell migration, prolifera-
tion, and extracellular matrix deposition. In a chronic wound the response of various
resident cells to the growth factors may be different as compared to an acute wound.
That there is a loss of orchestration is evident from various abnormal cellular
responses starting from lack of synthesis of the growth factor leading to its deficiency
in the wound bed, poor receptor sensitivity in the resident cells, rapid breakdown of
these factors and the heightened presence of various blocking ligands derived from
the ongoing inflammation. The excessive amount of tissue MMPs which are present
in a chronic wound may break down the growth factors resulting in poor local con-
centration [38]. It has also been proposed that repeated infection and the resultant
bacteria present in the wound may lead to a deficiency of growth factors. Bacteria
have been shown to produce proteases that degrade growth factors, hence, not allow-
ing a wound to progress to a healing state.
It has been hypothesized that around venous ulcers there exist periulcerous cuffs
that may contain trapped growth factors and so on. Another possible explanation for
the less-than-expected effectiveness of growth factors can be found in emerging
evidence suggesting that resident cells in chronic wounds are unresponsive to the
action of certain cytokines and growth factors. For example, fibroblasts cultured
from venous ulcers are unresponsive to the action of transforming growth factor-
beta 1 (TGF-beta 1) [39] and PDGF [40]. Also, venous ulcer fibroblasts respond to
certain cytokines but not others. Similarly, fibroblasts from diabetic foot ulcers have
shown an unusual response to single growth factors [41]. Some researchers [42]
have suggested that combinations of growth factors are required to stimulate dia-
betic ulcer fibroblasts [41]. For reasons that are not yet clear, some chronic wound
fibroblasts exhibit selective unresponsiveness to the action of certain specific growth
factors. Whether senescence is related to the unresponsiveness of ulcer fibroblasts
to growth factors is unknown. Also unclear is whether other resident cells within
wounds are affected to the same extent. Fibroblasts are relatively easy to culture and
that may be the reason why the available data on this subject are mostly restricted to
this cell type at this time.
Drugs That Inhibit/Delays Wound Repair
The drugs in daily use may affect wound healing. These interfere with specific
phases of wound healing and will affect cells, pathways, growth factors, cytokines
and other important components of the wound healing cascade. Not all are phase
specific and may have a global effect on the wound. They may delay wound healing,
lead to a chronic wound or may result in a poorly healed wound which breaks down
with time and stress. Although the list is very big, we will restrict our discussion to
the commonly used drugs.
Corticosteroids inhibit fibroplasia, vascular proliferation and epithelialization
[43]. They also delay wound contraction and increase the susceptibility to infection.
In the inflammatory phase there is a failure of leukocyte migration (such as poly-
morphs and macrophages) into the wound which diminishes fibroplasia and revas-
cularisation. When applied topically steroids inhibit fibroblast proliferation and
collagen synthesis and may cause peripheral vasoconstriction at the wound inter-
face. Corticosteroids are probably the most common cause of inflammatory arrest in
wounds [44].
Anti-cancer drugs are antineoplastic but not cancer cell specific. They affect
wound healing directly and indirectly. The principal mechanisms of injury irrespec-
tive of the class of drug include myelosuppression, damage of basal keratinocytes
leading to dermal atrophy and causes platelet mediated inflammatory response lead-
ing to micro-thrombi formation [45]. There is also the risk of an extravasation injury
resulting from leakage of the drug into the soft tissue resulting in necrosis and ulcer
formation. The anti cancer drugs kill the young multiplying cancer cells; so also in
wounds that are an area of similar activity.
Hydroxyurea is associated with lower extremity ulcers in patients who are on it long
term or use on high doses. Methotrexate partially blocks DNA and RNA synthesis. It
is 100–1,000 times more cytotoxic to macrophages and T cells than epithelial cells. It
inhibits interleukin-1 (IL-1) and decreases synthesis of interleukin-6 (IL-6) [46].
Smoking is a well established cause of delayed healing. The chemicals in cigarette

smoke include nicotine, carbon monoxide and hydrogen cyanide. Nicotine diminishes
red blood cells, fibroblasts and macrophages and increases platelet adhesiveness and
produces cutaneous vasoconstriction. Carbon monoxide affects the oxygen transport
via hemoglobin and hydrogen cyanide inhibits enzyme systems of oxidative metabo-
lism. Moreover, nicotine stimulates the neutrophilic elastase enzyme and leads to an
unstable matrix. All these factors can affect wound healing at various stages [47].
Antiplatelet drugs are aspirin and the non-steroidal anti-inflammatory drugs
(NSAIDs). These inhibit prostacyclin synthesis, inflammatory mediators derived
from arachidonic acid metabolism, platelet aggregation and acid mucopolysaccha-
ride synthesis in wounds. Inhibition of COX-I increases local ischemia and hypoxia
associated with chronic venous ulcers. There is some evidence that the COX-II
inhibitors delay healing, by inhibition of angiogenesis and reducing scar formation
without disrupting epithelialization in the early phase of wound healing [48, 49].
Antibiotics are often overused in the treatment of acute and chronic wounds.
Penicillin interferes with the tensile strength of the wound by affecting the cross-
linking of collagen. Erythromycin demonstrates anti-inflammatory properties
through the inhibition of leukocyte chemotaxis. MMP activity and angiogenesis
stimulation may also be modulated by various antibiotics [50].
Colchicine is used in the treatment of gout. It reduces granulocyte migration and
cytokine release. It is vasoconstrictive, reduces synthetic function of fibroblasts and
interrupts extracellular transport of procollagen. Increased collagenase activity
causes collagenolysis and inhibits wound contraction [46].
The anticoagulant drugs in general do not affect wound healing. These agents
inhibit the initial phase of wound healing by preventing the coagulation cascade.
However once the coagulation cascade has set in and the inflammatory phase has
started they do not have much role. Vasoconstricting drugs such as adrenaline, nicotine
and cocaine can cause tissue hypoxia affecting the microcirculation. Immunosuppressants
other than the corticosteroids show no evidence of significant inhibition of healing.
They, on the contrary, facilitate healing in wounds related to auto-immune diseases
and vasculitis. However, these drugs may increase the risk of infection in wounds and
should be used with caution.
Therapeutic Intervention to Correct a Matrix Deficiency State
The prime focus of research in this arena is based on the accurate understanding of
the pathophysiology of a chronic wound. The therapeutic approaches till date have
focused broadly on three areas of intervention:
1. Attempts to decrease ECM degradation from ongoing inflammation.
2. Initiation of local production of a healthy ECM.
3. Providing a ready-to-use ECM to the non-healing wound to activate healing.
Decreasing ECM Breakdown
It would require correcting the chronic inflammatory state, by modifying the excess
production of pro-inflammatory cytokines, and the resulting excess MMP produc-
tion, and decreased levels of MMP inhibition [51–54]. It is interesting to note here
that the chronic inflammation is in part caused by an ECM deficiency state and the
ECM deficiency is in turn due in large part to chronic inflammation, a sort of vicious
cycle. Wound dressings that are dynamic and biologically engineered exist. One of
these types is a combination of oxidized regenerated cellulose and collagen
(Promogran®). There is some Johnson & Johnson, UK evidence that it reduces
levels of MMP activity and degrades exogenous PDGF in chronic wound fluid
in vitro; The mechanism of action is thought to act as a competitive substrate for
wound fluid proteases, which spares the PDGF at the expense of degrading the col-
lagen in the dressing [55]. Another product that combines collagen with alginate
Systagenix Wound Management Inc., NY (Fibracol®) has also been developed to
absorb wound exudate and reduce MMP activity by providing collagen as a com-
petitive substrate [56].
Increasing Local ECM Production
Increasing local ECM production requires the ability to turn on the apparently dor-
mant fibroblasts to produce the ECM components. The fibroblasts on the edges of
a chronic wound have been shown to be less responsive to components of ECM
such as growth factors. Current approaches add individual ECM components to
stimulate production or to add fibroblasts themselves, which can produce their own
ECM [57, 58]. The topical ECM components, which have been used for this pur-
pose, are collagen and hyaluronic acid [59]. The purpose of providing collagen is to
provide a matrix scaffold for cell proliferation and migration.
It would be expected that the dual effect of a natural scaffold for cell movement
and the potent biological effect of the collagen molecule would be diminished when
presented in this form. Expediting cell entrance into the wound would eventually
lead to more ECM production. A natural, biologically active matrix would be a
more effective stimulant. Hyaluronic acid, though a known wound stimulant, has
been used with poor success. Theoretically the use of fibronectin is inspiring as
many studies have demonstrated its deficit in the chronic wound. It has also been
shown that fibroblasts exposed to fibronectin proliferate and produce a much greater
amount of ECM than fibroblasts exposed to collagen. It is however not available for
clinical use.
Another currently used approach is the topical application of growth factors,
an ECM component known to be deficient in the chronic wound. It augments
healing by directly stimulating a number of cells in different phases of healing
and indirectly by stimulating the fibroblasts to elaborate new ECM [60]. However,
the problem in the chronic wound is that senescent (unresponsive) fibroblasts are
not very responsive to growth factors. It therefore would seem that the more
chronic the wound and the more unresponsive the fibroblast, the less likely the
response to a growth factor, especially a single growth factor [61, 62]. Topical
platelet derived growth factor is the agent most commonly used especially in the
diabetic ulcers. It has been shown in experimental studies to act by increasing
fibroblast proliferation and ECM production, and re-epithelization of diabetic
ulcers.
Another novel approach is addition of new fibroblasts extracted from human
neonatal foreskins and grown on tissue cultures, to the chronic ulcers. In theory,
these juvenile fibroblasts would make provisional ECM and increase healing rate
[61]. But at present there is no evidence that these new fibroblasts directly turn on
the resident senescent fibroblasts. Their action is short term, corresponding to the
life span of these cells. Therefore, repeated applications are necessary to generate
enough ECM to perpetuate healing.
Adding a Preformed ECM to the Wound
There is good evidence that adding a preformed ECM to a wound, especially a full
thickness wound, can increase healing rate in a difficult to heal wound [63–69]. It
also appears that a complete ECM is more effective than single ECM components.
There are a large variety of preformed ECM type products, most of which are
synthetic. There are however natural acellular matrix also, which are very promis-
ing in chronic wounds. However, all matrices are not the same. There is consider-
able variability in the wound cell response depending on the composition of the
matrix [70, 71]. For example, fibroblasts placed in a fibronectin or fibrin rich sur-
face matrix produces a much greater matrix expression than those placed in a pure
collagen matrix. Several synthesized collagen based matrix are clinically avail-
able. These products offer scaffolding properties but do not have natural collagen,
fibronectin or glycosaminoglycan in place [72–75]. Products prepared from
human skin such as processed cadaver dermis, an example being Alloderm® (Life
Cell Corporation, NJ, USA), are incorporated into the wound for direct epithelial
cell migration. Oasis® (Cook Biotech Inc., IN, USA) wound matrix is a natural
ECM derived from the small intestinal submucosa of the pig intestine. The sub-
mucosal components, which direct the continuous gut mucosal turnover, are basi-
cally identical in composition to human extracellular matrix [64, 74].
Tackling a chronic wound and stimulating healing in it is difficult and is a sub-
ject of ongoing research. Although various promising products have been the out-
come of such research, clinical use of some of these is limited at present by various
factors like cost, availability, inadequate result, repeated application and compli-
ance. At present it would seem most logical to use ECM components earlier on
wounds known by their nature to be difficult to heal rather than wait until there is
evidence of non-healing. This delay may well eliminate the potential beneficial
wound healing effects of the use of the potent healing properties of ECM in matrix
deficiency states.
Wound Infection
Historical Background
The ancient Egyptians were the first society to have trained clinicians treating phys-
ical ailments, including application of various potions and grease to assist wound
healing [77]. Hippocrates of Greece (460–377 BC) used vinegar to irrigate open
wounds and wrapped dressings around wounds to prevent further injury. Galen
(Roman gladiatorial surgeon, 130–200 AD) was first to recognize that pus from
wounds inflicted by the gladiators heralded healing (pus bonum et laudabile; “good
and commendable pus”). Unfortunately, this observation was misinterpreted, and
the link between pus formation and healing was emphasized so strongly that foreign
material was introduced into wounds to promote pus formation-suppuration, and
the concept persevered well into the eighteenth century. The mechanism of wound
healing remained a mystery, as highlighted by the famous aphorism by Ambroise
Paré (French military surgeon, 1510–1590), “I dressed the wound. God healed it.”
Robert Koch (Berlin, 1843–1910) first recognized the cause of infective foci as
secondary to microbial growth, whereas it was Joseph Lister (British surgeon,
1827–1912) and Louis Pasteur (French bacteriologist, 1822–1895) who revolution-
ized the entire concept of wound infection. Lister first recognized that antisepsis
could prevent infection. In 1867, Lister placed carbolic acid into open fractures to
sterilize the wound and to prevent sepsis and by 1871 he had been using carbolic
spray in the operating room to reduce contamination [78]. Alexander Fleming
(1881–1955) performed many of his bacteriological studies during the period of
World War II and is credited with the discovery of penicillin. Sterilization of instru-
ments began in the 1880s as did the wearing of gowns, masks, and gloves, which
were introduced by William Halsted (Johns Hopkins University, 1852–1922) [79].
Surgical Wound Infection: The Worldwide Impact
Surgical site infections (SSIs) account for 14–16% of the estimated two million
nosocomial infections affecting hospitalized patients in the United States [80].
Internationally, the frequency of SSI is difficult to monitor because criteria for diag-
nosis might not be standardized. A survey sponsored by the World Health
Organization demonstrated a prevalence of nosocomial infections varying from 3%
to 21%, with wound infections accounting for 5–34% of the total [81]. In 2002 the
Nosocomial Infection National Surveillance Service (NINSS) reported after a sur-
veyed over the period October 1997 and September 2001 that the incidence of hos-
pital acquired infection related to surgical wounds in the United Kingdom was 10%.
The related costs the UK National Health Service was estimated at one billion
pounds (1.8 billion US dollars) annually. Collated data on the incidence of wound
infections probably underestimate true incidence because most wound infections
occur when the patient is discharged, and these infections may be treated in the
community without hospital notification.
SSIs are associated not only with increased morbidity but also with mortality.
Seventy-seven percent of the deaths of surgical patients were related to surgical
wound infection [82]. Kirkland et al. [83] calculated a relative risk of death of 2.2
attributable to SSIs, compared to matched surgical patients without infection.
The Prevalence of Chronic Ulcers
Chronic wounds are a significant cause of morbidity. Studies in the UK have esti-
mated the point prevalence of leg and foot ulcers to be in the region of 1.48/1,000
population [84] and there is clear evidence that prevalence increases with advancing
age [85] with up to 3.6/1,000 population in those over 65 years old. The annual
prevalence of venous leg ulcers alone has been estimated to be 1.69% in those aged
65 years and over [86].
Potential Wound Pathogens
The microorganisms infecting the wound can be bacteria, fungi, protozoa and
viruses. Bacteria can be cocci, bacilli and spirochetes. The growth and survival of
all bacteria in the wound is dependent upon environmental factors, e.g. aerobes
require oxygen whereas anaerobes are rapidly killed by oxygen. It is important to
note, however, that both aerobes and anaerobes can survive in close proximity to
each other and that some can survive in both conditions; these are known as faculta-
tive anaerobes. The fungi are responsible for superficial infections of the skin, nails
and hair and, although they have been isolated from wounds, they are rarely patho-
genic in this setting [87]. Some types of protozoa are significantly associated with
infected skin ulcers and produce specific skin infections. Viruses do not generally
cause wound infections. However bacteria can infect skin lesions formed during the
course of certain viral diseases. It is important here to remember that different
micro-organisms can exist in polymicrobial communities as occurs in a chronic
wound [88].
MRSA
Methicillin resistant Staphylococcus aureus (MRSA) was first reported in the UK in

the 1980s and remains a cause of concern for the healthcare industry. There are now
many different strains of MRSA affecting a large number of individuals in many
different healthcare settings. The degree to which people are affected ranges in
severity from simple wound colonization, which does not need aggressive treat-
ment, to systemic infection such as bronchopneumonia which may prove resistant
to the best care. Anecdotal evidence suggests that MRSA is no more pathogenic in
a wound than the non-resistant version. However, it is accepted that if a wound is
infected with MRSA it is difficult to manage with antibiotics. As a general rule,
practitioners should follow the local protocol for the management of a wound colo-
nized with MRSA, with ongoing treatment based on clinical signs.
The microflora of leg and foot ulcers is usually polymicrobial and recent studies
using molecular techniques have emphasized their complex ecology [89]. Using
conventional techniques, the mean number of bacterial species per ulcer has been
found to range from 1.2 up to 4.4. [90–92] Staphylococcus aureus and coagulase-
negative staphylococci have been the predominant organisms isolated from both
prospective, purpose-collected samples and retrospective analysis of clinical inves-
tigations. Pseudomonas aeruginosa is another frequently identified organism and
has been found in 7–33% of ulcers [90, 92, 93]. A number of other aerobic species
have also been reported, including Escherichia coli, Enterobacter cloacae, Klebsiella
species, Streptococcus species, Enterococcus species and Proteus species [91, 92–
95]. This list is illustrative of the range of aerobic bacteria that exist in chronic
wounds.
In addition to aerobes, anaerobic organisms are frequently identified in wounds,
although they vary considerably depending on the wound environment. While one
study observed obligate anaerobes in 25% of chronic leg ulcer samples [96],
another found [95] that they constituted only 6% of diabetic foot ulcer (DFU)
isolates. However, a more focused study by Bowler and Davies [94] found anaer-
obes in 73% of non infected leg ulcers and 82% of infected leg ulcers. The most
common isolates are Peptostreptococcus species and pigmented and non-pig-
mented Prevotella/Porphyromonas species [94]. Finegoldia magna (previously
classified as Peptostreptococcus magnus) was found to be present in 19.6%, and
Peptoniphilus asaccharolyticus in 9.8% of non-infected venous leg ulcers [97].
Kontiainen and Rinne [98] found that clinical swabs sent for analysis, presumably
from infected or assumed infected wounds, yielded obligate anaerobic rods
(mainly Bacteroides species) from 12% of ulcers and anaerobic cocci (peptostrep-
tococci) from 8%. Bacteroides, Peptostreptococcus and Prevotella species were
found to be the most frequently isolated obligate anaerobes in mild or moderately
infected DFUs [95].
The continuity of the microbial profile of chronic wounds over time is unclear.
Some believe that the microflora of chronic wounds is a relatively stable entity.
Hansson et al., [97] found that 90% of ulcers that were followed for 4 months, or
until healing, contained at least one resident organism that was isolated from all
monthly swabs. The observation of stable microbial populations was also supported
by the work of Gilchrist and Reed [99], following the observation that once a spe-
cies was present, it generally remained so, with the exception of the transient appear-
ance of P. aeruginosa. However, closer examination of their data shows that 85% of
wounds acquired new aerobes and 45% new anaerobes over the 8 week study period.
More recently Basu et al. reported that the bacterial flora tend to change over time.
It was observed that the initial gram positive skin flora is replaced by gram negative
flora after 8–12 weeks [92]. After initial swabs had been taken, Trengove et al. [96]
found at least one new bacterial group present in subsequent swabs in 82% of
patients, and thus concluded that the microbial populations of chronic wounds alter
over time.
Wound Contamination and the Development of Infection
There are a number of ways in which micro-organisms can gain access to a

wound:
• Direct contact: transfer from equipment or unclean hands of health worker
• Airborne dispersal: micro-organisms deposited from the surrounding air
• Self-contamination: physical migration from the patient’s skin or gastrointesti-
nal tract
Whilst there is no definitive evidence to identify the most common route of entry
for a micro-organism into a wound, direct contact and poor hand-washing tech-
niques of healthcare providers during pre- and post-operative phases of patient care
are considered to be significant factors.
The presence of a micro-organism within a wound does not indicate that wound
infection is inevitable [100]. Protective colonization may play a part whereby some
bacteria produce highly specific proteins that kill or inhibit other, usually closely
related, bacterial species or certain bacteria produce a variety of metabolites and
end products that inhibit the multiplication of other micro-organisms [101]. The
interaction between ulcer and bacteria can be stratified into four levels: contamina-
tion, colonization, critical colonization and infection [102]. Whilst, contamination
and colonization by microbes are not believed to inhibit healing, the line between
colonization and infection can be difficult to define. The term ‘critical colonization’
has been used to describe the stage at which bacteria begin to adversely affect wound
healing. Ultimately the development of an infection will be influenced largely by
the interaction between the virulence of the organism and the immunological status
of the patient; for example, patients considered most at risk are those being treated
with long-term steroids and those receiving chemotherapy. Virulence describes both
the pathogenicity and invasiveness of the relevant micro-organism. A number of
specific factors have also been identified in relation to infection rates in surgical
wounds. These include:
• Presence of an existing chronic infection
• Time interval between skin preparation and surgery
• Nature of the invasive procedure – especially if involving the bowel
• Extent of tissue loss and/or trauma to tissues during surgery
• Poor application of the principles of asepsis at the time of wound dressing
changes
• Presence of devitalized tissue within the wound – necrotic tissue or slough, if
over 50%
• Nature and prolonged presence of exudate not managed by a closed drainage
system.
The factors related to the pathogenic effect of virulent microorganisms which
may facilitate wound infection are:
• Exotoxin production – leads to vigorous stimulation of host immune cells
• Endotoxin release in the blood stream – antigens present in the cell wall which
stimulates T cell subsets to release cytokines that initiate cell and tissue damage
• Bacterial synergy – a mechanism of interspecies interaction whereby the growth
and potency one species is enhanced by the presence of other species through the
provision of unidentified growth factor; the classic example is the necrotizing
soft tissue infection.
• Biofilm production – a microbial colony encased in an adhesive polysaccharide
matrix that is usually attached to a wound surface. Cells in biofilms exhibit
decreased sensitivity to host immunological defense mechanisms, decreased sus-
ceptibility to antimicrobial agents, and increased virulence.
A range of clinical criteria have been used to define infection in chronic wounds.
The Consensus Development Conference on Diabetic Foot Wound Care [103]
agreed that a DFU should be considered infected when there are purulent secretions
or the presence of two or more signs of inflammation (erythema, warmth, tender-
ness, heat, induration). Guidelines for the management of chronic venous leg ulcers
produced by the British Association of Dermatologists and the Royal College of
Physicians [104] recommend that infection should be considered if one of the fol-
lowing is present: pyrexia, increased pain, increasing erythema of surrounding skin,
lymphangitis or rapid increase in ulcer size. It is accepted that chronic wounds by
their very nature may not always display the classic symptoms of infection (pain,
erythema, edema, heat and purulence) and it has been suggested that an expanded
list, including signs specific to secondary wounds (such as serous exudate plus con-
current inflammation, delayed healing, discoloration of granulation tissue, friable
granulation tissue, foul odor and wound breakdown) be employed to identify
infection. [105].
Microbiologically, a critical bacterial load, synergic relationships between
bacterial species and the presence of specific pathogens have all been proposed
as indicators of infection. The presence of microbes per se is not indicative of
wound infection. However, the possibility that a critical microbial load might
directly affect the healing outcome in both acute and chronic wounds has been
considered for several decades, with a direct relationship first being demonstrated
by Bendy et al. as early as in 1964 [106]. Since then, work carried out by many
others has led to the widely-held opinion that non-healing is associated with a
bacterial load of more than 105 bacteria per gram of tissue [107]. The point to
note here is that microorganisms are identified in the deep tissue of all chronic
wounds, yet the role they play and the impact of specific species on wound lon-
gevity are unclear. The distinction between infected and colonized wounds has to
be considered on a clinical basis and not by microbiological analysis due to the
universal colonization of chronic wounds [108]. Microbial analysis can be of
benefit when considered in concert with clinical observations to confirm caus-
ative organisms and their sensitivities, and so enable refinement of antibiotic
regimens [108, 109]. Clinical diagnosis of infection can, however, also be prob-
lematic due to the nature of the wounds and this uncertainty may lead to the
unnecessary use of antibiotics.
Biofilms
In their natural environments, most bacterial and fungal species alternate between
planktonic (free-living) and sessile states in response to environmental stimuli.
Transition to a sessile state frequently occurs in response to stress like nutrient limi-
tation, immune attack leading to adaptive mutation and phase variation. These lead
to genotypic changes which finally favors a genetically-changed subpopulation of
cells that can survive the environmental stress. This is thought to reflect a develop-
mental switch to a more perseverance mode, a situation akin to “the survival of the
fittest”, and the bacterial population forms a protective coat surrounding them for
thriving in adverse situations. Biofilm is thus a population of microbes attached
irreversibly to a biotic and abiotic surface, encased in a hydrated matrix of exopoly-
meric substances, proteins, polysaccharides and nucleic acids [110]. It is a complex
developmental process involving attachment and immobilization, cell-to-cell adhesion
and interaction, micro-colony formation, and formation of a confluent three dimen-
sional biofilm structure [111]. Initially bacteria attach to a moist surface or a sub-
strate where water is abundant. This attachment, called adsorption is influenced by
electrical charges carried on the bacteria. If the association between the bacterium
and its substrate persists long enough (hours), other types of chemical and physical
structures may form which transform the reversible adsorption to a permanent and
essentially irreversible attachment. The final stage is associated with the production
of extracellular polymer substances (EPS). This layer of EPS and bacteria can now
entrap particulate materials such as clay, organic materials, dead cells and precipi-
tated minerals and ions adding to the bulk and diversity of the biofilm habitat. This
growing biofilm can now serve as the focus for the attachment and growth of other
organisms and thus increasing the biological diversity of the community. Thus
broadly speaking, the above process is made up of two phases of colonization: the
attachment phase and the accumulation phase [112]. These two phases are geneti-
cally separable, and multiple gene products have been identified in laboratory strains
as contributors to robust biofilm formation in vitro, including fibrinogen and
fibronectin binding proteins, microbial surface components recognizing adhesive
matrix molecules (MSCRAMM family adhesins), capsular polysaccharide adhe-
sion factors, bacterial DNA, autolysins, teichoic acids, and polysaccharide intercel-
lular adhesins [112].
The formation and maturation of a biofilm involve a number of transitional states,
which have been characterized through genome-wide expression profiling and pro-
teomic analysis. A number of proteins have been identified, which plays specialized
roles in the biofilm phase and a more comprehensive change in gene expression
have been observed which reflect an overall downshift in the metabolic activities
consistent with the perseverance mode [113]. These multicellular colonies also
express genotypic response in the development and spread of antibiotic resistance
in the nosocomial pathogens. The genotypic response can include the de novo emer-
gence of antibiotic resistance through mutation and an enhanced propensity of cells
in biofilms to exchange genetic material bearing antibiotic resistance traits through
intra- and interspecies transfer mechanisms [114].
Thus it is evident that the bacteria in a biofilm behave differently from their free
floating (planktonic) counterparts. The regulation of bacterial gene expression in
response to cell population density, called quorum sensing, is accomplished through
the production of extracellular signal molecules called autoinducers [115]. Biofilm
production is regulated by quorum sensing systems in several pathogens. Far from
being the homogeneous populations usually assumed in planktonic pure cultures,
the microbial inhabitants in a significant sense behave as multicellular assemblages,
continuously interacting with the host cells and among various species which coex-
ist in the biofilm. With maturation the biofilm may remain monomicrobial or may
convert into a polymicrobial colony. It is thought that the presence of more than one
type of microbes is beneficial to the colony as mutual exchange of substances
including genetic elements take place. The isolation of multiple discrete species
from a mature biofilm provides the most compelling evidence of synergism [116].
Studies of polymicrobial biofilms formed by C. albicans and S. epidermidis indicate
that the exopolymeric matrix produced by the fungal species may protect the bacte-
ria against antibiotics, while the bacterial matrix protects the fungi from antifungal
action. This structural complexity allows bacteria to survive the environmental
stress and nutritional dirt [117, 118].
Exactly how the bacteria benefits from the low grade inflammatory response it
maintains at the host-biofilm interface is a matter of ongoing research. It is thought
that if the biofilm is in close proximity to the host tissue, as in a chronic wound, by
inducing an inflammatory reaction it increases capillary permeability of the local
host vasculature and results in a sustained release of the plasma exudate, which per-
colates through the biofilm providing nutrients to the bacteria [119]. Deep within the
biofilm the nutrients are carried by the convective flow of water via water channels.
Those species residing in the outer layer multiply rapidly and undergo rapid turnover.
Comparatively the deeply located bacteria remain dormant or undergo slow multipli-
cation. An attempt to kill the outer bacteria results in rapid multiplication of the
deeper colonies and replenishment. Invasion of the host tissue occurs typically when
the balance between the immunity and the virulence tilts in favor of the bacteria.
Thus they are notoriously difficult to eradicate and are a source of many chronic
infections. According to the National Institutes of Health, biofilms are medically
important, accounting for over 80% of microbial infections in the body [120]. A
mature biofilm can tolerate antibiotics at concentrations of 10–1,000 times more than
are required to kill planktonic bacteria. Bacteria in biofilms are resistant to phagocy-
tosis, making biofilms extremely difficult to eradicate from living hosts [120]. Hence
biofilms colonizing chronic wounds are devastating for the host. They increase the
bioburden of the wound and maintain a low grade inflammation within its margins
resulting in inhibition of proliferation and thus a non-healing ulcer results.
Antimicrobial Tolerance of Biofilms
Inadequate exposure of the microbes to the antibiotics limits their efficacy in deep-
seated biofilm-associated infections leading to chemotherapeutic failure. Trapping of
antimicrobial agents in the EPS is often cited as a universal mechanism underlying

the insensitivity of biofilms to antimicrobial action. In vitro studies also demonstrate
a differential ability of penetration of antibiotics in the biofilms. Thus, although the
penetration of vancomycin and ampicillin is limited, antibiotics like tobramycin,
rifampicin and some antifungal agents demonstrate good penetration by diffusion
through the water channels in the biofilm. However, studies to assess the actual intra-
cellular penetration of the antibiotics into cells within the biofilm matrix are lacking.
For bacteriostatic classes of antibiotics, which depend on host immunity for
microbial kill, the location of bacteria within EPS is thought to effectively sequester
host immune cells. Moreover the bacteria can evade immune detection by repress-
ing key ligands and antigens [121]. However, no definitive studies have addressed to
a systematic difference between the efficacies of bacteriostatic and bactericidal
agents in biofilm-associated infection in vivo.
The elevated expression of efflux pumps in the microbe is a satisfactory explana-
tion by which bacterial cells can exhibit decreased susceptibility to antibiotics.
Specific upregulation of genes involved in antibiotic transport outside the cells, and
their regulators has been observed in studies involving Pseudomonas, E. coli and
Candida biofilms [122–124]. However this is not a global phenomenon and is
restricted to antibiotics which are substrates for efflux transporters.
The altered metabolic activity in the biofilm-associated microbe, which is an
adaptive mechanism of survival in adverse environment, protects the bacteria against
antimicrobial-induced killing. For example, the antibiotic that acts against the cell
wall synthesis is most effective when the microbes are actively multiplying. The
slow replication of bacterial species in the biofilm decreases the therapeutic efficacy
of a drug even when the therapeutic concentration is achieved. However, it may be
different in the case of antibiotic that targets protein synthesis or bacterial RNA
[125]. It is thought that the small colony variants of bacteria induce genetic changes
in the metabolic pathways, a characteristic so often found in the isolates from per-
sistent infections [126]. Hence genotypic adaptations that alter metabolic capacity
and decrease the growth rate may contribute to both antibiotic tolerance and the
persistence of some biofilm-related infections.
Physiologic heterogeneity is another feature that distinguishes bacteria in a
biofilm from their planktonic counterparts. This phenomenon is thought to be due
to a combination of inter-bacterial quorum sensing signals, accumulation of toxic
byproducts and dissimilar local microenvironments (different nutrient and oxygen
grading). Such phenotypic heterogeneity has been linked to the decreased suscepti-
bility of a subset of cells (persisters) in the biofilm to specific antimicrobials. These
so-called persister cells are not resistant to the antimicrobial but instead appear to
escape killing through what is hypothesized to be a transient dormant state [127].
Evidence of Biofilm Forming in a Chronic Wound
It is difficult to certain which infections in a chronic wound are caused by biofilms

since the relationship would appear to be based on mutualism. Increasing evi-
dence based on both in vivo and in vitro research has proposed some criteria
regarding this [128]. Firstly, the invading organism should be associated to a sub-
stratum or a surface (wound). Direct examination of the infected tissue shows
bacteria living in cell clusters or micro-colonies and encased in EPS. Thirdly, the
infection is confined to a particular location, although dissemination may occur.
Lastly, the infection is difficult or impossible to eradicate with antibiotics despite
the fact that the responsible organisms are susceptible to killing in the planktonic
state. Although there are limitations to these criteria, they provide the general
characteristics of a wound infection due to biofilm. A few clinical observations
also aid in the diagnosis of a wound harboring biofilm. If an acute wound develops
by the side of a chronic wound and heals faster than the chronic one, the latter may
be biofilm. Even after the host factors predisposing a chronic wound are success-
fully managed, the presence of a biofilm will promote persistence of the ulcer that
will remain painful and be exudative if the regular use of biocides fails to heal a
chronic wound, clinically biofilm presence should be excluded. The undulating
clinical course of a wound is another good clue. Chronic wounds tend to wax and
wane, at times becoming more painful and exudative, with more tissue necrosis
and then improving slightly over weeks to months. Chronic wounds also seem to
respond to appropriate antibiotics marginally only to deteriorate again once the
antibiotics are withdrawn. This general clinical picture of chronic wounds is very
similar to other human infectious diseases e.g., prostatitis, sinusitis, endocarditis
and other biofilm-based diseases of humans also have the same undulating, chronic
clinical picture.
Confirming the Diagnosis of Wound Infection
If, after careful assessment, it is apparent that the wound is infected, it is important
to confirm this and identify the causative organism(s) and possible sensitivities to
antibiotics. Wound swabbing is the most common sampling method used through-
out the world although its clinical value has been questioned by a number of authors
[88, 129]. It has been suggested that routine swabbing, such as at weekly intervals
or at the time of frequent dressing changes, is neither helpful nor cost effective
[130]. In purely financial terms, a negative wound swab costs from £15 to £25 per
swab – dependent upon the health setting in which it has been obtained – and each
requested antibiotic sensitivity will cost an additional £5 per set per organism. The
relative sensitivity accuracy of the various common techniques of obtaining samples
for the laboratory are curettage (75%), needle aspirate (69%) and culture swabs
(60%) [90]. Thus the culture swabs are notoriously inaccurate for obtaining the true
impression of the bacterial species of the wound. In fact in most situations it is the
bacterial species in superficial layer of the biofilm which is represented by a
superficial culture swab. These organisms may not represent the invasive species
residing in the deeper parts of the biofilm, which are responsible for wound infec-
tion. Probably the best assessment of a biofilm colony is provided by tissue biopsy
and culture. Classically after a thorough debridement, a sample tissue biopsy should
be taken from the edge of the wound (the center contains only the saprophytes),
preferably from the undermined margins. Culture of this tissue sample provides
information of the polymicrobial nature of the biofilm. In cases where the bacterial
growth is fastidious, identification of bacterial DNA/RNA, which is so abundant in
the biofilm matrix, is possible by the use of polymerase chain reaction (PCR).
Culture based methodologies for the identification and quantification of microor-
ganisms, although a convention are not without shortcomings. Many microorgan-
isms have been overlooked in these studies due to the inability to grow them in
culture. Presently denaturing gradient gel electrophoresis (DGGE) is a method for
separating similar sized DNA fragments based on sequence [131]. Bacterial com-
munity diversity (i.e. number of different species) can be estimated from the number
of bands in gels from each species. PCR based identification of bacterial DNA/RNA
material is also a refined method of accurately diagnosing the different species of
microbes associated with the biofilm. Although a scanning electron microscopic
examination provides a high quality documentation of the biofilm, it does not pro-
vide information regarding the species.
Therapeutic Strategies Against Biofilm
The recent interest research into biofilms has spawned a number of new treatment
strategies for controlling biofilms. It is already known that bacteria in a biofilm
communicate with each other via diffusible signal molecules in a process termed
quorum sensing. The quorum sensing regulates biofilm formation, an important
mechanism, which can be manipulated through the use of quorum sensing inhibi-
tors to inhibit biofilm formation and function. This strategy of jamming communi-
cation is now moving towards application. One example of such inhibitors is the
brominated furanones that block quorum sensing by acyl homoserine lactones, a
class of signal molecules used by some Gram-negative bacteria. Furanone-based
quorum sensing inhibitors have been shown to increase antibiotic sensitivity of
Pseudomonas aeruginosa biofilms.
Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis)
use cyclic peptides as quorum sensing signals. One such 33-kDa autoinducer pro-
tein called RNAIII-activating protein (RAP) induces phosphorylation of the target
of RAP (TRAP), which is important for various cellular functions including toxin
synthesis. A synthetic peptide termed bacterial RNAIII-inhibiting peptide (RIP)
interferes with the normal reception of these signals and inhibits phosphorylation of
TRAP, and has shown efficacy in combating biofilm infections including those
caused by MRSA in a number of animal models [132, 133].
Iron is an important ion in the development of biofilms. The availability of iron appears
to govern the rate of staphylococcal biofilm development on venous catheters. It is also
required to stabilize the Pseudomonas aeruginosa biofilm. The use of natural or synthetic
iron binding compounds like lactoferrin or those which disrupt iron metabolism like gal-
lium compounds could be a way of limiting biofilm formation in a wound [134].
One attractive strategy for biofilm control is to target the gelatinous matrix. If the
EPS that hold the biofilm together could be disrupted or degraded, the biofilm would
disperse and its innate defenses would be subverted. This may be realized through the
use of enzymes. For example, a hexosaminidase that specifically cleaves the primary
extracellular polymer of many staphylococcal biofilms has been recently described.
This enzyme, termed Dispersin B, has demonstrated remarkable efficacy in disrupting
and removing Staphylococcus epidermidis biofilms. An enzyme cocktail could be
considered for topical wound treatment as a way of loosening and removing biofilm.
Weak electric fields enhance the efficacy of antibiotics against bacterial biofilms.
This phenomenon, termed the “bioelectric effect,” has been tried with efficacy as an
experimental technique. Because of the ready accessibility for the placement of
small wires or coils, a chronic wound may be a relatively straightforward context in
which to test this technology. Similarly low frequency ultrasonic waves have shown
to improve antibiotic penetration in biofilms [135, 136].
The use of certain drugs which are specific against some bacteria, and which
have the potential for penetrating biofilms are if interest. These drugs can be used
with the combination of other specific antibiotics, e.g. rifampicin and quinolones or
beta-lactams or even fusidic acid against Staphylococcus biofilm [137]. Another
novel strategy is slow release of silver from hydrogel coated dressings. Liposomes
containing amikacin, for better penetration and delivery, has also been used with
success in experimental models [138].
Most clinicians would agree to the removal of a foreign body which may act as the
substrate for the biofilm. Such a foreign body may an implant or even a slough or a
sequestrum. Surgical debridement is an important step for the removal of slough,
sequestrum or even a biofilm-coated polypropylene mesh from a nonhealing wound.
Other potent antibiotics with better biofilm penetration are being tried with success
but as yet experimental and investigational are dalbavancin, telavancin (lipoglycopep-
tides), tigecycline (glycylcycline), daptomycin (lipopeptides), quinupristin-dalfopris-
tin (streptogramins) and linezolid (oxazolidinone) [139–143].
Fungal biofilm-associated infections are notoriously difficult to treat systemically
with antifungal agents. This poor in vivo efficacy is not surprising, given the in vitro
insensitivity of biofilms formed by various fungal species to antifungal agents [144].
However, agents of the echinocandin class (caspofungin, micafungin, anildafungin)
exhibit significantly superior in vitro killing of fungal cells in biofilm [145]. Lipid-
based formulations of amphotericin B have also proven effective in vitro assays of
biofilm activity. Presently antifungals of the echinocandin class and amphotericin B
lipid formulations represent the best available options for managing fungal biofilms.
Complications of Wound Healing: Poor Healing/Improper

Healing
Disorders of Collagen Synthesis and Polymerization
Collagens account for one-third of the body’s proteins, are the biggest protein family
of connective tissue and the extracellular matrix. The collagen family has various
physiological functions. This super-family includes 28 different types. These groups

can also be classified into subtypes according to their structural and functional prop-
erties. The collagen structure is frequently characterized by the Gly-Pro-Hyp tripep-
tide. Glycine makes up one-third of all the amino acid residues, and the repetitive
structure consists of many other amino acids [146]. Its unique triple-helix structure
is important for many cellular functions such as adhesion and extracellular matrix
activation and for enzymatic functions such as hydroxylation of collagen’s Lys and
Pro residues. Collagen is usually found in fibril form in the extracellular matrix. Each
collagen chain consists of approximately 1,000 amino acid residues. Fibril-forming
collagens are types I, II, III, V, and XI, and these types of collagens are most common
in the body [147]. Type I collagen is the most abundant protein in humans [148]. The
collagen biosynthesis requires eight specific post-translational enzymes [149].
The defects of collagen synthesis comprise a wide group of healing disorders,
which affect various steps of collagen synthesis. The transfer of an oxygen molecule
for hydroxylation of proline and lysine residues is essential for polymerization of
collagen and its secretion from the cell [150]. This essential step is mediated in the
presence of vitamin C as a cofactor. The scorbutic wounds are fragile and tear apart
easily. Since the deposition of collagen is deficient and defective the scar formation
is weak, unable to bear the stress and strain of the normal skin and eventually breaks
down. Microscopically, large vacuoles of amorphous material collect within
fibroblasts. Administration of vitamin C restores healing to normal in an astonish-
ingly short period of time, presumably because collagen precursor has accumulated
and is awaiting only the presence of this one critical cofactor.
Protein deficiency states and not mere starvation, leads to poor collagen deposi-
tion. The amino acids methionine and lysine appear to be particularly critical for
collagen synthesis. Correction of protein energy malnutrition as a whole and not
only particular amino acid supplementation is essential for correcting collagen syn-
thesis defects.
Lathyrism in animals is due to ingestion of b-aminoproprionitrite, which inter-
feres with the condensation of adjacent lysine molecules in collagen monomers and
subsequently prevents the formation of a covalent bond between molecules. The
lysine-lysine bond is responsible for a major portion of collagen fiber strength. This
condition illustrates the severity of defects of collagen polymerization and cross-
linking. Several congenital enzymatic disorders that prevent collagen polymeriza-
tion have also been found, e.g., dermatosparaxis and lysyl hydrozylase deficiency
[149]. In these conditions, a variety of consequences of “soft collagen,” including
poor repair, are seen. A cross-linking defect due to penicillamine and/or copper
deficiency has also been found.
Inadequate repair is always a danger in uremia, liver failure with jaundice, diabe-
tes, and infection. Repair is quantitatively impeded by uremia. Uremic patients on
regular dialysis have better wound healing potential than those who remain uremic
for a prolonged time in the postoperative period. The induction of obstructive jaun-
dice may or may not impair healing. However, liver failure certainly plays a
significant part of the disorder. These patients also are susceptible to nutritional
deficiencies [151].
It is well known that poorly controlled diabetes causes poor wound healing and
an increased rate of wound infection. In the case of severe ketoacidosis with calorie
loss from osmotic diuresis, the primary aim in the postoperative period is essentially
the control of hyperglycemia. Diabetic patients also have wound problems related
to neuropathy and leukocyte defects. Atherosclerosis, more common in diabetics,
limits perfusion, particularly of the lower extremity. Leukocytes from diabetics have
decreased ability to migrate to the site of injury, and hyperglycemia itself (more
than 250 mg/dl) may inhibit phagocytosis of bacteria.
Genetic Disorders Affecting Collagen Metabolism
The large super-family of collagen and the genetic control of its metabolism, and
the number of second messenger systems involved are so vast that a small genetic
defect can affect its metabolism in some form or the other. The critical roles of col-
lagens are identified with the diseases that are caused by the mutations on the genes
coding them, among which are Marfan syndrome, cutis laxa, some subtypes of
Ehlers-Danlos syndrome, Alport syndrome, Bethlem myopathy, some subtypes of
epidermolysis bullosa and Knobloch syndrome among the others. Interestingly all
types of collagen genetic defects, although give rise to defective collagen metabo-
lism, do not necessarily affect wound healing, indicating specific mechanism of
collagen function being controlled by the specific collagen genes and the mutant
collagen expression is different at different places.
Marfan syndrome is an autosomal dominant, multisystemic disease of the con-
nective tissue [152]. It affects approximately 1 in 5,000 to 1 in 10,000 individuals
and results from the mutations in the FBN1 gene, which encodes the fibrilin-1 [153].
More than 500 FBN1 mutations have been determined for this syndrome. Its char-
acteristics are aortic dilatation that progresses with aortic valve failure, mitral valve
prolapse, lens dislocation, long and slim body and long limbs, arachnodactyly, chest
deformations, defects in skeletal system, skin, nervous system and lungs, and some-
times scoliosis [154, 155]. Surgical repair of the dissecting aneurysm is plagued by
soft connective tissue which fails to hold sutures. Cutis laxa is also a heritable dis-
ease in which the skin is thick, easily stretched, and hangs in pendulous folds. Its
manifestations tend to increase with age. Hernias, especially ventral hernias, hoarse-
ness of voice, dislocations, arthritis of the hips, and intervertebral disc disease are
common [156]. The basic defect is in the elastic tissue, and histologic examination
of the skin shows fragmentation of elastic fibers.
Ehlers-Danlos syndrome affects all races and both genders. At least 10 subtypes
are classified based upon genetic, biochemical and clinical features. The syndrome is
characterized by fragile and tender skin, easy bruising and atrophic injury. In approx-
imately half of the cases, mutations in the COL5A1 and COL5A2 genes encoding the
alpha1 and alpha2 chains of type V are defined [157]. Type IV Ehlers-Danlos syn-
drome is caused by 19 different mutations in the COL3A1 gene coding for type III
procollagen [158]. Type VI is a result of the homozygote and heterozygote mutations
in the lysyl hydroxylase gene. The skin of type VI patients is hyperelastic, bruise
easily and heal with difficulty [159]. In type VII the defect is in the process of trans-
formation of procollagen to collagen; inadequate procollagen amino protease activity
is observed. Types I and III result from decreasing lysyl hydroxylase activity [157].
Other types like the types V, VII and X are rare and infrequently reported.
Alport syndrome is caused by deletion mutations in COL4A5 and COL4A6
genes or COL4A3 and COL4A4 genes [160]. Eighty percent of Alport syndrome
cases are X-linked and its prevalence is 1/5,000. This syndrome affects some of the
basal membranes and is characterized by renal failure, loss of hearing and lens
abnormalities. The pathognomonic feature is the loss of type IV collagen network
formed by alpha3, alpha4 and alpha5 (IV) chains [160].
Epidermolysis bullosa is a heterogeneous group of heritable disorders. It has
been classified under three anatomic groups: epidermolytic, junctional, and dermo-
lytic types. The clinical features include blistering of the skin and mucous mem-
branes as a result of a small injury. Defects in at least 10 genes are responsible for
the different forms of this disease [161].In dominant simplex type, mutations in the
K5 or K14 genes cause basal cell damage and blistering. The recessive simplex type
is marked by muscular dystrophy, which results from abnormal plectin and skin
fragility syndrome from PKP1 mutations. The junctional type results from the lami-
nin 5, type XVII collagen and alpha-6-beta-4 integrin gene mutations, while the
dystrophic type is related with mutations in the type VII collagen gene [162].
Knobloch syndrome is an autosomal recessive disorder characterized by high
myopia, vitreoretinal degeneration, occipital bone damage, and congenital encepha-
locele. Pathological mutations in the COL18A1 gene at 21q22.3 have been identified
as the genetic defect in this disease [163].
Bethlem myopathy is an autosomal dominant disease that is characterized by
muscle weakness and distal joint contractures [164]. The type VII collagen, which
plays a role in connecting the cells and extracellular matrix, is affected by mutations
in its genes. It is a relatively milder disease with slow progression.
There are many other forms of genetic defects affecting various soft tissue and
bones or cartilages of the body, like the fibroblast growth factor receptors, which
when affected can give rise to a variety of diseases depending on the type of recep-
tor involved. Similar genetic defects may be seen with various matrix proteins,
receptors and cytokines. The detailed discussion is beyond the scope of this chapter.
However it is only the major defects which manifest as wound healing problem or
if healed result in a poor scar.
Complications of Wound Healing: Excessive Healing
When a wound heals, a scar takes its place. Simple tissues such as fat, connective
tissue, and epithelium regenerate, but the skin, being a complex organ derived from
two germ layers, heals by the formation of a predominantly fibrous scar. If the injury
sections or destroys the papillary dermis, a scar will always be formed. Depending
on the degree and nature of injury this scar may be inconspicuous or disfiguring.
Examples of disfiguring scars include keloids, widened scars, and hypertrophied

scars. The difference between a keloid and a hypertrophic scar is that a keloid con-
tinues to enlarge beyond the original size and shape of the wound, while a hypertro-
phied scar enlarges within the confines of the original wound. Although both can be
red and raised, keloids continue to grow and hypertrophied scars tend to regress over
time. Initially, hypertrophied scars can be raised, red, pruritic, and even painful. With
time they become pale and flat. Hypertrophied scars appear worse at 2 weeks to
2 months. Keloid scars tend to remain red, pruritic, and painful for many months to
years. Although both can recur after surgical excision, the recurrence of a keloid is
more common. Widened scars are wounds that separate during the healing process,
usually in response to lines of tension perpendicular to the wound edges.
In general, between the two the hypertrophic scars are more responsive to treat-
ment, although both can recur after excision. Keloids has predilection for particular
race and ethic population and are 5–15 times more common in darker pigmented
people. Keloids are more prevalent in younger patients (10–30 years) and the pro-
pensity for keloid formation can be genetically transmitted as an autosomal domi-
nant or recessive trait [165]. Widened scars can occur in persons of any age. Widened
scar formation occurs with no predilection to sex or ethnicity. No inheritance pat-
tern is associated with the risk for scar widening. Widened scars are most com-
monly found to involve the arms, legs, and abdomen. Sun tanning can contribute to
scar widening through attenuation of the tissues.
Conclusion
Wound healing is an amazingly complex biological event orchestrated most per-

fectly in a normal physiological environment. It is when the intricate balance of this
complex process is lost a situation of non-healing, poorly healing or excessive heal-
ing is reached.
In the last decade the threat of biofilm has been appreciated as the single most
important complication of wound healing. Since microbes are ubiquitous, bacterial
biofilms form on open wounds very quickly and is the principal cause of chronicity
of an ulcer since they are notoriously resistant to killing. Advancing technologies
though effective, are not yet adequate in dealing with this threat. As the biological
mechanisms of biofilm and its metabolism unfold, options in the treatment also
increase. In order to completely counter this wound healing problem, further
research is required in the better understanding of the formation, biological proper-
ties, defenses, and metabolism of a biofilm.
Fast track researches in the management of chronic wound due to corrupt matri-
ces and growth factor deficits are on the way. Various novel therapeutic strategies
are already available. However it needs to be cost-effective to reach the multitude of
patients mainly in the developing world where the burden of wound complications
is much higher. A better understanding of the pathology of a non-healing wound is
the pre-requisite before starting treatment with these technologically advanced
products, and to prevent unnecessary use in a wasteful way.
Lastly management of disorders of wound healing is a shared responsibility-

research input in the laboratory needs to match its clinical use. The patient-friendly
aspect of wound care, compliance and cost are important areas, which need support
of health care managers.
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Chapter 8
Epidemiology of Wounds
David J. Margolis
Keywords Chronic wounds • Epidemiology • Venous leg ulcer • Diabetic foot

ulcer • Diagnosis • Risk factors
Introduction
Chronic wounds of the lower extremities are an important health concern. They
have been estimated to cost more than $1 billion per year worldwide and $12 million
in the USA [1]. The prevalence of lower extremity chronic wounds has been esti-
mated to be between 0.18% and 1.3% in the adult population [2–4]. These wounds
are most likely associated with venous disease, arterial insufficiency of the lower
extremity, diabetes, and local pressure. Further, these wounds are associated with
aging and diabetes. Given the aging populations of most developed countries and
the epidemic of type II diabetes, it is fair to assume that the number of individuals
living with a chronic wound or the consequences of a chronic wound is going to
increase. For example, from 1980 to 2008, the number of diabetic Medicare
beneficiaries aged 65 or older increased from 2.3 million to 7.4 million [5]. The goal
of this chapter is to describe the basic epidemiology of two chronic wounds, venous
leg ulcer and the diabetic foot ulcer.
D.J. Margolis, M.D., Ph.D.

Departments of Biostatistics and Epidemiology and Dermatology, University of Pennsylvania,
901 Blockley Hall, 3400 Street, Philadelphia, PA 19104, USA
e-mail: margo@mail.med.upenn.edu

146 D.J. Margolis
Venous Leg Ulcer
The most common lower extremity wound is likely associated with venous disease
[4, 6]. Venous leg ulcers are thought to account for 40–70% of lower extremity
chronic wounds, with a disproportionate percentage of individuals with venous leg
ulcers being elderly or female [6–8]. It has been estimated that between 500,000 and
2 million Americans have a venous leg ulcer [2, 6, 9]. However, the richness of the
epidemiologic literature on this type of wound is poor as compared to the diabetic
foot ulcer (see below).
Consistent case definition has been problematic. Physiologically, individuals
with venous leg ulcers are believed to have chronic ambulatory venous hyperten-
sion [10, 11]. Clinically, the diagnosis of a venous leg ulcer is usually made in any
individual with a chronic wound in the “gaiter area” of the lower extremity and
other clinical signs compatible with venous abnormalities (e.g., varicose veins,
venous blush, lipodermatosclerosis, etc.). While some opine that the gold stan-
dard diagnostic test for this condition is a duplex ultrasound, this test is not always
consistent with clinical examination [12]. Health care providers often make the
diagnosis of a venous leg ulcer based on the presence of a chronic wound in the
“gaiter area” of the lower extremity and in a patient with other clinical signs com-
patible with venous abnormalities (e.g., varicose veins, venous blush, lipoderma-
tosclerosis, etc.). The gaiter area of the leg is located between the lower third of
the calf and 1 in. below the malleolus. Individuals with chronic wounds in this
area have an adequately functioning lower limb arterial system (e.g., ankle bra-
chial index of >0.80, presence of a palpable distal limb pulse) and abnormalities
of the venous system of the lower extremity. In reality the clinical diagnosis of a
venous leg ulcer probably represents a spectrum of diseases manifesting as a com-
mon clinical syndrome that, as a group, can be treated and prevented with similar
therapies. In fact, some authors argue that because most patients are not evaluated
for abnormalities of ambulatory venous pressures, the more correct diagnostic
term is “a leg ulcer” on an individual who has adequate lower extremity arterial
blood flow [13–16]. These ulcers tend to occur on the leg while diabetic foot
ulcers occur on the foot.
As noted, venous leg ulcers are often associated with clinical findings consistent
with venous disease. Varicose veins are thought to be prevalent in at least 50% of the
adult population. The more severe findings of chronic venous insufficiency are present
in about 10% of the adult population [12]. Overall, the prevalence of venous leg ulcers
is between 0.6% and 2.0% [16–18]. They are generally noted to be present in about
1–2% of the population over 65 years of age [16–18]. The peak prevalence of this dis-
ease occurs after the age 60 [16–18]. These wounds are more often present in women
[16–18]. Additionally, about 15–45% will have a past history of deep venous thrombo-
sis and more than one half will have findings of deep venous reflux [17]. These indi-
viduals also suffer from many other medical problems. For example, 20% will have
depression, 30% hypertension, 25% osteoarthritis, and 10% diabetes [19]. Finally, a
significant number of individuals with a venous leg ulcer have a diminished quality of
8 Epidemiology of Wounds 147
life as manifested by impaired mobility, depression, feelings of social isolation, fear,

anger, anxiety, negative self-image, lost time from work, and adverse effects on finances
[20–23].
Diabetic Foot Ulcer and Lower Extremity Amputation
About 170 million people currently are afflicted with diabetes. It is estimated that
by 2025, 300 million persons worldwide will have diabetes and by 2030, 360 mil-
lion persons, equaling a worldwide prevalence of more than 4% [24–27]. The preva-
lence of diabetes among those over 65 years of age was last estimated by the Centers
for Disease Control and Prevention (CDC) using 2007 data from the National Health
Interview Survey to be 19.1% among those aged 65–74 and 17.6% for those more
than 75 years of age. These estimates are based on self-reports and are thought to
underestimate the true prevalence by a third (www.cdc.gov/diabetes/statistics/prev/
national/figbyage.htm). In the CMS population of beneficiaries with at least
12 months of continuous enrollment in Medicare Parts A and B Fee-for-Service
(FFS), the prevalence of diabetes was 27.1% in 2007 and 27.9% in 2008, which is
about one-third higher than previous CDC prevalence estimates. For those
beneficiaries who were 65 years of age or older, the prevalence was 28.0% in 2008.
As has been previously reported, these rates vary by geographic location (Fig. 8.1).
More than 16 million people in the United States have diabetes mellitus. In 2008,
within the US Medicare population there were about 8.9 million participants who
had diabetes mellitus [28].
Those with diabetes are expected to suffer from several associated medical com-
plications such as renal disease, cardiac disease, and retinopathy. In addition,
between 10% and 15% of those with diabetes can expect to develop a foot ulcer at
some point in their lives [29–31]. Lower extremity amputation (LEA) is less com-
mon but it is an extreme complication associated with diabetes and foot ulcer. Both
foot ulcer and amputation vary by geographic location [32]. Diabetic foot ulceration
can develop because of acute or chronic cutaneous compromise of the skin, arterial
insufficiency, peripheral neuropathy, or a combination of these factors [33]. In fact,
approximately 20% of diabetic patients with foot ulcers will suffer from inadequate
arterial blood flow to their lower extremity. This is also called peripheral arterial
disease and for this chapter will be abbreviated as PAD. In addition, about 50% of
those with diabetes and a foot ulcer will display peripheral neuropathy, and about
30% will display a combination of both conditions (i.e., neuroischemia) [30, 33, 34].
The prevalence of PAD among those with foot ulcers has recently been shown to be
increasing and some have opined that PAD is now the major reason for a foot ulcer in a
patient with diabetes either primarily as PAD or in combination, neuroischemia [30].
Wounds on the feet of those with diabetes are likely the most studied chronic
wound. Many studies have been published on the epidemiology of these highly
related complications and they are well reviewed elsewhere (see [30] and [35]).
148 D.J. Margolis
The annual incidence of foot ulcer is around 2% for those with type II diabetes
and the prevalence varies between 2% and 12% depending on geographic loca-
tion [28]. Individuals with diabetes are over ten times more likely to have a non-
16.1 - 22.2
22.3 - 25.8
25.9 - 29.0
29.0 - 33.7
33.7 - 45.1
5.2 - 6.7
6.7 - 7.4
7.5 - 8.3
8.3 - 9.5
9.6 - 12.9
Fig. 8.1 The percentage of diabetes as compared to the full population (a), and percentage of
those with diabetic foot ulcer (b) and lower extremity amputation as compared to those with dia-
betes (c) in 2008 beneficiaries of the Medicare Parts A and B Fee-for Service population
0.8 - 1.3
1.4 - 1.6
1.7 - 2.0
2.1 - 2.5
2.6 - 3.6
traumatic amputation than those not afflicted with diabetes. Among those with
diabetes, the annual incidence of lower extremity amputation varies between
about 2–30 per 1,000, again depending on location, ethnicity, and race. In 2003,
the CDC noted that foot ulcers occurred, based on the National Hospital Discharge
Survey, with a frequency of 8 (ages 65–74) to 11 (age 75 and greater) per 1,000
hospital discharges of those with diabetes. These rates have been shown to
exhibit variation by age, gender, ethnicity/race, and health care region. These
estimates are somewhat limited because outpatient care or chronic care facilities
were not surveyed, individuals with venous leg ulcers may have been counted,
and individuals with more than one hospitalization could have been counted
more than once.
Similar estimates were recently established from the CMS population Figure 8.1.
In this population, the 2008 point prevalence for foot ulcer in beneficiaries with dia-
betes was 8.0%. The annual incidence of foot ulcer that year was 6.0% [36]. The
prevalence of foot ulcer in the sub-population with diabetes and PAD was 18.6% in
2008 [28]. The incidence of diabetic foot ulcer that year was 13.1% in the PAD sub-
group. The prevalence of foot ulcer among those in the sub-population of Medicare
Parts A and B beneficiaries with diabetes varied by race; 8.0% for those categorized
as white, 8.7% for those categorized as black, 4.2% for those categorized as Asian,
8.6% reporting Hispanic ethnicity, and 9.6% for those categorized as North American
Native [28].
A preexisting foot ulcer is the major risk factor for LEA in those with diabetes.
Between 40% and 80% of LEAs in diabetics are preceded by a foot ulcer [37–39].
150 D.J. Margolis
The CDC estimated in 2005 that the rate of LEA was 5.3–5.6 per hospital dis-
charge per 1,000 individuals with diabetes. Since 1997 the rate of LEA had been
slowly decreasing. In general, non-traumatic LEAs are at least 10 times more prev-
alent in those with diabetes than with any other concomitant medical illness [26,
40, 41]. In the US, nearly 80,000 LEAs are performed in diabetics each year [42,
43]. In 2005, the overall rate of hospital discharge for LEA was about 4.3 per 1,000
persons with diabetes in contrast to a rate of about 3 per 10,000 in the general
population [27, 42, 44–47]. Similar estimates were recently established using the
CMS population of beneficiaries described above. In this population, the yearly
point prevalence for CMS beneficiaries with diabetes was 1.8% in 2008 [28] (see
Fig. 8.2). The annual incidence of LEA among those with diabetes was 0.4% (i.e.,
4 per 1,000) that year [36]. The prevalence of major amputation in 2008 was 1.3%
and minor amputation was 0.46%.The prevalence of LEA in those with diabetes
and PAD as compared to beneficiaries enrolled in Medicare Parts A and B FFS was
5.9% that year [28]. As with foot ulcer, the annual incidence of LEA in those with
diabetes and peripheral arterial disease was higher than those without PAD, 1.8%
in 2008. These rates varied by age, gender, race/ethnicity, and geographic location.
For example, in 2008, the prevalence of LEA was 2.3% for male diabetics and
1.3% for female diabetics. By race/ethnicity, the prevalence of LEA among those
with diabetes based on race was 1.5% for those categorized as white, 3.4% for
those categorized as black, 0.70% for those categorized as Asian, 2.6% reporting
Hispanic ethnicity, 4.0% for those categorized as North American Native, and
1.5% for those categorized as other. However, incidence appears to be trending
down [36].
Diabetes mellitus is a significant illness both from an individual point of view
and a societal perspective. LEAs have a profound effect on quality of life, and are
associated with increased health care costs and an increased risk of mortality [26,
41, 42, 44, 48, 49]. In fact, within 5 years of having an LEA about 60% of those with
diabetes will die [48]. In 2008 alone, 13.6% of all Medicare beneficiaries with a
pre-existing LEA died [36].The burden of this disease is further demonstrated by
the costs associated with caring for those with this illness. In 2007, the CDC esti-
mated that in the US total costs related to diabetes totaled $174 billion, including the
social cost of intangibles such as pain and suffering, care provided by nonpaid care-
givers, medical costs associated with undiagnosed diabetes, and diabetes-attributed
costs. Just the health care costs associated with foot ulcers and LEAs can be quite
high and add significantly to this overall estimate. In 1995, Medicare claims for
diabetic foot ulcers exceeded $1.4 billion [50]. In the UK, the costs were $1.6 bil-
lion [51]. Among CMS beneficiaries who had a prevalent diabetic foot ulcer, the
mean reimbursement for all CMS-medical services was $35,100 in 2008 [52]. The
mean cost of caring for those with a prevalent LEA was $54,100 [52]. As the num-
ber of individuals in our population with diabetes increases, so likely will the num-
ber of individuals with an LEA.
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Research and Quality; 2010.
Chapter 9
Histopathology of Wounds
Mariya Miteva and Paolo Romanelli
Keywords Non-healing ulcers • Biopsy • Histology • Light microscopy • Vasculitis

Vasculopathy • Immunohistochemistry • Special stains • Markers
Introduction
Chronic wounds are a health problem of enormous socioeconomic impact affecting

very large numbers of patients worldwide [1]. There are several issues with under-
standing and treating chronic wounds. First of all establishing a precise chronic
wound model in animals is very difficult despite attempts to create sloughy wounds
[2]. Second of all, interpolating the results of different clinical trials is not always
feasible since difficulties are encountered in comparing study design variables as
well as variations occur in the definition of a chronic ulcer, choice of age groups and
approaches to patient identification. The true overall prevalence of chronic wounds
is usually underscored since it does not include people who are self-caring [3].
Finally, the limitations in understanding chronic wounds include also the fact that in
many cases clinicians are reluctant to perform repeated biopsies in non-healing
ulcers due to lack of straightforward results from the histological reports, rarely
going beyond “consistent with chronic ulcer”. Moreover, there are no systematic
reviews in the literature regarding histopathology of chronic wounds and data could
M. Miteva, M.D. • P. Romanelli, M.D. (*)

Department of Dermatology and Cutaneous Surgery,
University of Miami Miller School of Medicine,
1600 NW 10th Ave., 33136 Miami, FL, USA
e-mail: promanelli@med.miami.edu

156 M. Miteva and P. Romanelli
be extracted mainly from clinical reports and trials. Therefore, most of the informa-
tion here on histopathology and Immunohistochemistry of chronic wounds comes
from our own experience as part of our wound pathology service in this centre. We
believe that through elucidating the different aspects of wound pathology further
insightful progress in both understanding of the pathogenesis and in the clinical
management of chronic wounds would be achieved.
A chronic wound has been defined clinically as an ulcer that has not healed
within 3 months and with a slow reduction in wound size (£1 mm/week) [4]. Chronic
wounds have diverse etiologies, with more than 90% of them falling into three cat-
egories: venous ulcers, pressure sores and diabetic ulcers [1, 2].
Venous Ulcers
Venous ulcers are the most common type of ulcer encountered in the daily practice.
Although the exact pathogenesis of venous ulcers is unknown, several theories exist.
Most commonly venous hypertension, or increased ambulatory venous pressure, is
thought to be the causal in the development of venous ulcers [5, 6]. Venous hyper-
tension leads to capillary distention and widening of endothelial pores that allow the
leakage of macromolecules, release of toxic metabolites, proteolytic enzymes, free
radicals and cytokines resulting in the formation of pericapillary fibrin cuffs. All
these events are implicated in the self-amplifying cascade of impaired healing
[7, 8]. In venous ulcers particularly, venous hypertension results in preferential
accumulation of leukocytes in the diseased leg, leading to oxygen stress and subse-
quent vessel injury, thus aggravating the condition further [9]. Another concept
states that the trapping of white blood cells in capillaries leads to areas of local
ischemia as well as the release of proteolytic enzymes that cause endothelial dam-
age [7]. Falanga hypothesized that growth factors may be trapped in a pericapillary
matrix cuff and are therefore not available to stimulate healing [10]. Mustoe et al.
summarized all these concepts within a novel hypothesis which claims the persis-
tence of chronic wounds to be largely attributable to the co-existence of three major
factors: (1) cellular and systemic changes of aging, (2) repetitive ischemia-reperfu-
sion injuries (often in the setting of underlying ischemia) and (3) bacterial coloniza-
tion resulting in inflammatory host response [1, 2]. All these features can be
identified in the biopsy of a venous ulcer (Fig. 9.1a). Punch biopsies and incisional
biopsies are advocated because they permit more tissue to be examined. In our
experience, in general the best site for biopsy is the margin of the ulcer because it
enables the comparison between the ulcerated area and the peripheral skin.
Furthermore, the base usually shows non-specific features of dermal necrosis and
dense inflammation with secondary vascular damage to the underlying vessels
which simulates vasculitis or vasculopathy and is referred to as pseudovasculitis or
pseudovasculopathy [11]. However, in cases where malignancy is suspected, a
biopsy from the ulcer bed is necessary to exclude sarcomatous development as this
is the most common site where these tumors arise (see section “Other types of
9 Histopathology of Wounds 157
Fig. 9.1 A Hematoxylin and

Eosin (H&E) stained a
specimen of a venous ulcer:
(a) low power (×2
magnification) – ulcerated
epidermis with adjacent
hyperplasia; edema, dermal
fibrosis, inflammatory
infiltrate and clusters of blood
vessels in the upper dermis;
(b) high power (×4
magnification) –
aneurysmatic distortion of the
blood vessels
chronic wounds”). The most common features of venous ulcers include ulcerated
epidermis covered by a layer of fibrin with or without noticeable clumps of bacteria
that can be highlighted by the Gram’s stain. The adjacent epidermis is usually acan-
thotic (epithelial hyperplasia) with few if any apoptotic keratinocytes and shows
serum crust with neutrophils, parakeratosis and spongiosis. If there are increased
apoptotic keratinocytes, increased mitotic activity and atypia in a hyperplastic epi-
dermis from an ulcer, this necessitates the exclusion of evolving squamous cell car-
cinoma (SCC). The papillary dermis shows edema and subepidermal dilated
capillaries (aneurysmal dilatation) [12] as well as clusters of newly formed vessels
as a sign of stimulated angiogenesis due to the chronic ischemia (Fig. 9.1b).
Fig. 9.2 Immunohisto-

chemistry for PTAH highlights
in blue the pericapillary fibrin
cuffs and intraluminal fibrin
thrombi (×10 magnification)
The blood vessels show plump endothelial cells and many of the small vessels
which are confined to the ulcerated area show features of fibrinoid necrosis of the
wall and/or pericapillary fibrin deposition referred to as fibrin cuffs. We support
previous statements that these cuffs form due to the chronic inflammation rather
than the increased venous pressure as we have observed them also in other cases of
chronic wounds in which there is increased inflammation and no evidence of venous
hypertension in the etiology. There may be focally present small thrombi. A stain
that highlights fibrin thrombi used extensively in our lab is PTAH (Phosphotungstic
Acid Hematoxylin) (Fig. 9.2). The presence pericapillary cuffs, fibrinoid necrosis of
the wall with no concomitant inflammation of the vessel wall as well as the restric-
tion of the involvement to the area of ulceration with characteristic sparing of some
vessels is a feature of pseudovasculatis/pseudovasculopathy which distinguishes it
from primary vasculitis/vasculopathy. In the dermis there is increased red blood cell
(RBC) extravasation and deposition of hemosiderin, patchy or diffuse moderately
dense perivascular and interstitial lymphohystiocytic infiltrate with occasional pres-
ence of mast cells, plasma cells, neutrophils and eosinophils. A study reported an
increased number of mast cells in the surrounding skin of venous ulcers and deple-
tion of mast cells in the ulcer base [13]. The deep reticular dermis and the subcuta-
neous fat show features of lipodermatosclerosis (LDS) which is characterized by
lipomembranous (membranocystic) fat necrosis and septal fibrosis [14]. According
to Herrick et al. [15], the extensive fibrotic changes in the dermal and subcutaneous
tissue of LDS are due to excessive accumulation of extracellular matrix and are in
favor of healing rather than tissue destruction. In conclusion, in recent years, two
new concepts have been proposed using novel Immunohistochemistry markers that
seek to explain the basis if venous ulcers. The first one showed that while there is
little to no Tumor necrosis factor alpha (TNF-a) typically present in normal skin of
patients with chronic venous ulcers, there is a significant amount of TNF-a present
in the macrophages of the stroma within the ulcers themselves. In addition, ulcers
of longer duration have more TNF-a present than those of shorter duration,
illustrating the relationship between local TNF-a and chronic venous ulcers [16]. In
the era of biological treatment this may have important implications for treatment of
refractory venous ulcers [17]. The other concept showed that the immunohis-
tochemical marker D2-40 (which identifies podoplanin and is a sensitive stain for
lymphatic endothelia) highlighted in venous ulcers significantly more dermal lym-
phatic vessels per unit width than controls (compensatory lymphangiogenesis) as
well as significantly higher percentage of lymphatic vessels with collapsed lumina
probably related to the constant overload due to the lymphedema.
Ulcers Resulting from Arterial Hypertension
Hypertensive leg ulcers were first described by Martorell and are sometimes
referred to as Martorell’s ulcers or more commonly as hypertensive ischemic leg
ulcers [18]. Patients usually present with a long history of poorly controlled hyper-
tension though such ulcers may also develop in patients with good control of the
hypertension. It is important to be understood that hypertensive ulcers are neither
venous nor arterial ulcers [19] and patients who have a history of vascular disease,
peripheral arterial occlusive disease, chronic venous insufficiency or have Doppler
evidence of venous or arterial disease are excluded. These are usually shallow,
superficial, not deep, lower extremity ulcers on the lateral side above the ankle
which are exquisitely painful with central necrosis limited in depth to the dermis
and surrounded by a red purpuric margin spreading regularly and centrifugally.
The histology is considered non-specific [19]. However, one study reported consis-
tent histologic findings in 16 hypertensive ulcers out of 22 studied [20]. These
findings comprised of (1) internal elastic lamina reduplication, (2) hyaline change
and (3) luminal narrowing. There was no evidence of vasculitic process or vascul-
opathy. Moreover, staining for fibrin was present within the necrotic inflammatory
slough in the ulcer base (which is a common feature of the ulceration process, as
described above) but in no case was there pericapillary fibrin deposition such as
found in venous ulcers. The hyaline change of the vessel wall (Fig. 9.3) is due to
deposition of plasma proteins, lipids and basal membrane material which can be
demonstrated, using Periodic Acid Schiff (PAS) (for basal membrane) to highlight
the thickened walls. In our experience, the histology of hypertensive ulcers shows
the non-specific features of any ulcer found in its wound bed i.e. inflammatory
slough and granulation tissue, inflammation and hemosiderin deposition. However,
in biopsies of hypertensive ulcers obtained from ulcer margins, hyalinization of the
wall and a much reduced lumen/wall ratio are found. It is confirmatory to do the
PTAH stain for fibrin which is negative in these cases as compared to venous ulcers.
In hypertensive ulcers, the sub-endothelial hyalinization of the wall is the actual
cause of the narrowing and ischemia. Verhoeff-van Gieson stain for elastics may be
used to highlight the internal elastic lamina reduplication and elastin deposition in
the arterioles and small arteries. It should be noticed that similar features are seen
in other ulcers particularly in diabetic ulcers because of underlying arteriosclerosis.
Fig. 9.3 Hypertensive ulcer:

hyaline arteriosclerosis and
intimal thickening of the
arteriolar wall; H&E, ×10
magnification
Many times patients with chronic ulcers present with several co-morbidities which
account for the polietiologic nature of the ulceration and such ulcers are referred to
as “of mixed etiology”.
Diabetic Foot Ulcer
Patients with Diabetes mellitus (DM) are particularly prone to develop foot ulcers
[21]. The pathogenesis of the diabetic foot ulcers (DFU) is multifactorial with sev-
eral mechanisms being particularly important for that: persistent hyperglycaemia
and oxidative stress that lead to cellular dysfunction of the epidermal cells and
fibroblasts of the matrix [22], increased production of metalloproteinases, oxidative
stress damage, reduced host immunity and increased bacterial damage and suscep-
tibility to infections as well as decreased levels of neuropeptidases which are impor-
tant for the chemotaxis and growth factor production. Neuropathy and ischemia,
two common complications of diabetes mellitus, are the primary underlying risk
factors for the development of DFU and for its complications [23]. Perhaps one
third of DFU have a mixed neuropathic and ischemic etiology. Arteriosclerosis
occurs at younger age in persons with DM than the general population [24].
Furthermore, collateralization and angiogenesis are insufficient to overcome the
loss of blood flow through narrowed or occluded arteries leading to severe ischemia
of the limb followed by amputation [25]. A recent theory [26] suggested that in the
diabetic foot there is peripheral sympathetic denervation with loss of vasoconstric-
tion, which results in increase of the flow of blood through the arterio-venous shunts
and what then develops is “capillary steal” in which blood is shunted away from the
capillaries through these vessels. This results in reduced nutrition to the skin and
ulceration [26]. In our experience the histology of DFU shows the conventional
Fig. 9.4 Diabetic foot ulcer.

(a) H&E of small arterioli a
with abnormal intimal growth
and atherothrombosis (in
arrow); ×4 magnification;
(b) PAS stain highlights the
thickened basal membrane of
small arterioli
non-specific features of any chronic non-healing ulcer with some additional features
such as: (1) thickened basal membrane and sub-endothelial hyalinization of the ves-
sel wall due to increased hydrostatic pressure and chronic inflammation (Fig. 9.4b).
This pattern is analogous to the one observed in hypertensive ulcers. (2) Some of the
vessels show abnormal intimal reaction (growth) and atherothrombosis leading to
luminal occlusion (Fig. 9.4a). A special stain called Factor VIII can be used to high-
light the platelet thrombi that stain negative for PTAH which labels fibrin thrombi.
(3) Sometimes a DFU presents histologically with broad ulcerated area showing
features of perforation of collagen and elastic tissue through the surface. The latter
can be confirmed by applying special stains such as Masson Trichrome for collagen
and Verhoeff-van Gieson for elastic fibers. This histologic feature is clinically
known as mal perforans. However, perforation of ulcers non-related to DM has also
been reported [27]. In conclusion, as in all other cases of non-healing ulcers,
clinico-pathological correlation is essential.
Pressure Ulcers
Pressure ulcers occur as areas of tissue necrosis that result from external physical
compression, shear forces and friction. One study found the overall pressure ulcer
prevalence to be 14.8%, with a nosocomial pressure ulcer prevalence of 7.1% [28].
Individuals who are unable to relieve themselves of long periods of pressure par-
ticularly at the bony prominences, are at increased risk for developing necrosis and
ulceration. Typically such patients are elderly, neurologically impaired, or hospital-
ized with acute illness and frequently suffer with impaired nutrition. Anatomically,
the sacral region is by far the most common area for pressure sores to develop. It
accounts for over 70% of all occurrences, with sacral (46%) and ischial (26%) loca-
tions being most common [29]. The severity of pressure ulcers depends on many
factors, including the duration of pressure, friction and shear forces, and the person’s
ability to heal. The National Pressure Ulcer Advisory Panel (NPUAP, www.npuap.
org) developed the following staging system for pressure ulcers:
Stage I – The skin is intact with changes in color, temperature, or texture.
Stage II – The epidermis, and sometimes a portion of the dermis, is stripped
away, leaving a shallow wound.
Stage III – There is loss of the full thickness of the skin with underlying subcu-
taneous tissue exposed.
Stage IV – The wound is deep enough to expose muscle, bone, and tendons.
A tissue biopsy should always be performed of wounds that do not demonstrate
clinical improvement despite adequate care and wounds in which tissue invasion
by bacteria is suggested. By using a simple Gram stain the biopsy allows distinc-
tion between simple surface contamination and the more serious tissue invasion
that could not be revealed by the common practice of swabbing the wound surface
for culture. Furthermore, cases of ‘Decubital candidosis’ [30] due to Candida
albicans have also been described. They are related to the prolonged bed rest and
the long-term use of antibiotics. A skin biopsy is a useful and simple tool to prove
PAS positive yeasts in the horny layer. Most of the biopsies performed in non-
healing ulcers are prompted by the concern to rule out malignancy as malignant
degeneration, most commonly SCC of non-healing pressure ulcers has been
reported with an estimated incidence of 0.5% [31]. Furthermore pressure ulcer
Fig. 9.5 H&E stained

specimen of a chronic
pressure ulcer from the sacral
area shows epidermal
hyperplasia with fibrinoid
necrosis of the papillary
dermis, dilated vessels and
thickened collagenous
stroma; ×4 magnification
carcinomas have aggressive behavior as previous studies have reported an 80%

2-year mortality rate in patients presenting with local disease who are treated only
with surgical excision [32]. The histopathology of early pressure ulcers shows
dilation of the vessels of the upper dermis and swollen endothelial cells. A perivas-
cular round cell infiltrate is seen in the papillary dermis. There is vascular engorge-
ment, platelet aggregate formation, and perivascular hemorrhage. Subepidermal
bulla may form if the process is acute. As the disease progresses, the epidermis
and adnexae become necrotic, which is a reason for slow re-epithelization in pres-
sure ulcers. There is abundant fibrin and a sparse neutrophilic infiltrate within the
dermis. In advanced stages, there is full-thickness necrosis of the dermis and a
black eschar develops [33]. One study looked at the histopathology of pressure
ulcers from 20 patients using light and electron microscopy [34]. Surgical biopsy
specimens from the ulcer edge, ulcer margin, and adjacent normal, non-ulcerated
skin showed four general types of ulcer histopathology. Morphological features
common to all ulcer groups were a dense fibrin matrix, occluded blood vessels,
numerous inflammatory cells, and a range of cellular viability from functionally
normal appearing cells to cells in all stages of necrosis. Our experience is in agree-
ment with these findings. One additional feature we have encountered in biopsies
from chronic non-healing pressure ulcers is atypical decubital fibroplasia which is
characterized by atypical, enlarged, degenerated fibroblasts with abundant baso-
philic cytoplasm, in the deep dermis and subcutis. There is usually fibrinoid
necrosis, reactive fibrosis, neovascularization, fat necrosis and ectatic vessels [35,
36]. Dilated vessels are a relatively consistent finding in pressure ulcers probably
reactive to the chronic ischemia and impaired tissue nutrition (Fig. 9.5). However
it is still unclear why there is impaired neovascularization in poorly healing pres-
sure ulcers if they show strong expression of vascular endothelial growth factor in
different regions of the ulcer [37]. Another interesting finding is the presence of
mast cells in the stroma of pressure ulcers [38]. However, the role of this finding
is unknown yet. We use Giemsa or Toluidine blue to clearly highlight the mast
cells and their granules.
Calciphylaxis
Calcific Uremic Arteriolopathy (CUA) is a syndrome of vascular calcification and

skin necrosis commonly associated with chronic renal failure related to secondary
hyperparathyroidism and calcium-phosphorus abnormalities. In rare cases, calciphy-
laxis has been reported in patients with normal renal function but with liver disease,
chronic immunosuppression, chronic inflammatory conditions, proteins C and S
deficiency, malignant neoplasms and warfarin use [39]. According to one study pri-
mary hyperparathyroidism, malignancy, alcoholic liver disease, and connective tis-
sue disease were the most common reported causes for calciphylaxis among
nonuremic patients [40]. Cutaneous calciphylaxis manifests with non-inflammatory
retiform purpura which is the hallmark of cutaneous microvascular occlusion syn-
dromes. The pathogenesis of this condition is not fully elucidated. Some recent stud-
ies suggest that vascular calcification occurs through a common pathway involving
nuclear factor kB activation resulting in transdifferentiation of vascular smooth mus-
cle cells into osteoblast like cells [41, 42]. Renal failure as well as all above men-
tioned non-uremic conditions can activate this process through various factors and
regulators including specific proteins. For instance the matrix Gla protein which is a
known inhibitor of the bone morphogenic protein-2-mediated vascular calcification
is suppressed in calciphylaxis [39]. This transformation of smooth muscle cells into
osteoblast like cells is followed by subsequent calcification and expression of osteo-
pontin. Osteopontin is a bone matrix protein produced by osteoblasts. Immunostaining
of calcified vessels in calciphylaxis patients on dialysis demonstrated the presence of
osteopontin and several other bone-related proteins while non-calcified vessels
remained negative for osteopontin [43]. Histology of calciphylaxis usually shows
calcium deposition within the walls of small subcutaneous arteries, arterioles which
can be either in the intima or media although medial deposition is more characteristic
of calciphylaxis. The arteriolar calcification is often superimposed by intimal
fibroblastic hyperplasia causing marked luminal narrowing and contributing to the
ischemic necrosis in calciphylaxis [44]. A spectrum of histologic changes can be
observed from vessels with fine calcifications without fibrosis (Fig. 9.6a) to vessels
with calcification and proliferating endothelial fibroblasts to vessels with both
calcification and endovascular fibrosis and stenosis [45]. Additional features such as
giant cells, microvascular thromboses, calcific panniculitis, extravascular
calcifications with infiltrate of neutrophils and macrophages may be present as well.
We have encountered a case of calciphylaxis which presented on histology with only
diffuse hemorrhagic infiltration of the reticular dermis and subcutis (Fig. 9.7) and
this was confirmed to be clinically appreciated by the clinician as diffuse violaceous,
hemorrhagic plaques in the area where an eschar subsequently developed. In our lab
we use both the special stain von Kossa for calcium and the Immunohistochemistry
for osteopontin to highlight the osteoblast like evolution of the smooth muscle cells
of the vessel walls (Fig. 9.6b). In conclusion, of note is that the vascular calcification
in calciphylaxis is sometimes difficult to differentiate from Mockenberg’s medial
calcific stenosis and atherosclerotic vascular disease. Both calciphylaxis and medial
a b
Fig. 9.6 Calciphylaxis, H&E, 4× magnification. (a) A small artery in the subcutis with circumfer-
ential calcification of the media (b) the same artery stains positive with osteopontin
Fig. 9.7 Calciphylaxis,

H&E, ×4 magnification. This
is an early presentation of
evolving calciphylaxis with
diffuse hemorrhage (red
blood cell extravasation) in
the deep reticular dermis and
subdermis which was
appreciated clinically as
violaceous ecchymotic
plaques
calcific stenosis share medial location of the calcium deposits. However, the calci-
fication in Mockenberg’s disease is not accompanied by fibroplasia and inflammation
as in calciphylaxis. In comparison to atherosclerotic vascular disease, calciphylaxis
lacks smooth muscle proliferation, lipid laden macrophages or connective tissue
extracellular lipid deposits [44].
Fig. 9.8 Vasculopathy: a

case of cryoglobulinemia,
H&E, ×10 magnification. All
vessels show hyalinized walls
and occluded lumina by thick
non-cellular material
(precipitated cryoglobulins)
Vasculitis and Vasculopathy as a Primary

Cause of Chronic Ulcers
Vasculitis and vasculopathy are two vascular diseases which are characterized by
vascular damage (such as vessel leakiness, edema, vessel destruction in the form of
necrosis of the endothelium and fibrinoid deposition within the vascular lumina or
vascular walls). However, the difference is that the diagnosis of vasculitis requires
two criteria: vascular damage and inflammatory infiltrate whereas in vasculopathy
there is vascular damage – most commonly this includes pathologic alteration of the
vessel walls, embolic phenomena and occlusion but usually no inflammation as a
primary pathogenetic factor. It has been shown that in fact, platelet and lymphocyte
activation is present in vasculopathy (livedoid vasculopathy), whereas the levels of
inflammatory mediators are in a normal range. In cutaneous small vessel vasculitis,
the high serum levels of proinflammatory cytokines suggest that they are actively
involved in the pathogenesis [46]. Most common cases of vasculitis related ulcers
we encounter as part of the wound pathology service are from patients with leuco-
cytoclastic vasculitis which presents histologically with fibrinoid necrosis of the
small vessels and nuclear dust (from the destroyed nuclei of the neutrophils).
Vasculitis in all its types (depending on the size of the affected vessels, the infiltrate
pattern and its composition etc.) is well a studied disease and there is significant
amount of available literature for review. We will therefore discuss briefly only
some relevant features of vasculopathy.
Vasculopathy means vascular injury without features of vasculitis. The most well
known prototype of this disease is the coagulopathy pattern seen in cryoglobuline-
mia (Fig. 9.8). The histologic pattern in type I is characterized by the presence of
pink eosinophilic material (precipitated cryoglobulins) deposited subjacent to the
endothelium and throughout the vessel wall as well as within the vessel lumen
resulting in thrombus like appearance. In type II and III there may be inflammatory
infiltrate and the pattern may be similar to leucocytoclastic vasculitis. In biopsies
from another common vasculopathic condition referred to as livedoid vasculopathy
which presents with livedoid lesions with focal ulcero-necrotic evolution on lower
extremities, there is segmental hyalinization, endothelial swelling and a various
number of thrombi of dermal vessels as well as mild lymphocytic perivascular
infiltrate [47]. There are many underlying conditions that need to be investigated if
the clinical and histologic features are suggestive of a primary vasculopathic pro-
cess. Laboratory workup needs to include complete blood cell count, Rheumatoid
factor, Erythrocyte sedimentation rate, Cryoglobulins, Lupus anticoagulant test,
Anticardiolipin antibodies, Fibrin and degradation products, Factor V Leiden,
Antithrombin III, Proteins C and S, Serum fibrinogen, Antinuclear antibodies
(ANA), anti-ds DNA, complement C3, C4 levels, Thrombin time, APTT, pANCA,
cANCA, viral antibodies and so on. A very important finding that needs to be
emphasized again is the fact that features reminiscent of vasculitis and vasculopathy
can be observed in specimens from any chronic ulcers, especially in the vessels
underneath the slough or the eschar as well as in the wound bed. They are focal,
segmental and are related to the chronic ischemia and inflammation and are not of
etiologic relevance to the ulcers, i.e. they represent secondary non-specific epiphe-
nomena related to the vascular injury. Therefore, they should not be misinterpreted
as features of a primary systemic vasculitis or vasculopathy. An interesting pre-
sentation of vasculopathy is the Rheumatoid arthritis associated vasculopathy
described by Magro and Crowson [48] that is considered an active vasculopathy
encompassing lymphocyte-dominant, neutrophil-rich and granulomatous vasculitis.
Rheumatoid factor positivity and anti-Ro and anticardiolipin antibodies were
described as co-factors contributing to vascular injury in some cases.
Malignancy and Chronic Ulcers
There is a subset of chronic leg ulcers that do not improve with adequate treatment
over time and therefore should be investigated for the presence of a neoplasm mas-
querading as a non-healing leg ulcer. This is particularly true for any ulcer that starts
to change suddenly in any of the following patterns: develop an exophytic mass;
irregularities in the ulcer bed or a thickening, a “rolling up” of the edge of the ulcer
[49]. Other clinical features suggestive of a malignant process arising within an ulcer
are: abnormal granulation, protracted course of the ulcer despite appropriate treat-
ment, spreading of the ulcer, extensive granulation tissue, unusual pain and abnormal
bleeding [50, 51]. Yang et al. [52] studied 43 patients with 55 malignant skin lesions.
Tissue biopsies were obtained from the ulcerated lesions that suggested malignancy
or were not responding to appropriate treatment. 53 malignant lesions on the legs
were indentified, giving a frequency of malignant ulcers of 4.4 per 100 leg ulcer
patients, or 2.2 per 100 leg ulcers. Seventy-five percent of the malignant ulcers were
Fig. 9.9 H&E, ×4

magnification. A focus of
well differentiated SCC in a
chronic venous ulcer
basal cell carcinoma (BCC) and 25% were SCC. Combemale et al. [50] studied 80
patients, accounting for 85 tumors with a female: male ratio of 2.5:1 and a mean age
of 75 years. Eighty-eight percent of the ulcers were of venous origin and their mean
duration was 27.5 years. Clinical findings included abnormal granulation tissue in
76% of cases, absence of healing in 14% and unusual extension in 6%. Histologically,
98% of the tumors were SCC, among which 82% were very well or well differenti-
ated and 18% moderately or poorly differentiated. The two remaining cases were
basal cell carcinomas (BCC). The overall death rate was 32%; it was higher when
lymph-node (66%) or visceral metastases (83%) were present. They concluded that
malignant transformation of chronic leg ulcers of vascular origin is mainly encoun-
tered in elderly patients and manifests as an abnormally vegetating lesion, which
may be occasionally bilateral. Malignant transformation usually occurs towards
well-differentiated SCC and only exceptionally towards BCC. The high death rate,
especially in metastatic cases, is at least partly due to delay in diagnosis. Baldursson
et al. [53] matched 10,913 patients with a diagnosis of venous leg ulcer from the
Swedish Inpatient Registry with registrations of SCC of the lower limb recorded by
the Swedish Cancer Registry, and found 33 cases of non-melanoma skin cancer.
After scrutinizing the pathology and case records, 17 cases of SCC were considered
as being certainly secondary to venous leg ulcers, whereas in six cases of remitting/
relapsing ulcers the connection was probable. The relative risk calculated on 17 cases
was 5.80. The median duration of the ulcer before the diagnosis of cancer was
25 years. They concluded that SCC is a complication of chronic venous leg ulcers,
although the absolute risk is very small. In our experience the Marjolin’s ulcer (which
is a malignant neoplasm complicating a chronic non-healing ulcer of long duration –
usually of more than 3 years) is not that rare as reported. We have seen cases of
chronic ulcers presenting with features of SCC on histology (usually well differenti-
ated SCC, Fig. 9.9) as well as cases of BCC (Fig. 9.10), melanoma and sarcomas.
Fig. 9.10 H&E, ×4

magnification. A case of
ulcerated nodular BCC
presenting as a chronic
non-healing ulcer on the leg
The evolution of a BCC is usually slow and it may present as an ulcerated mass on
the extremities that can be misdiagnosed as a chronic venous ulcer and surgical treat-
ment is delayed [54]. One important message is that while carcinomas usually arise
from the border epithelium, including adnexal epithelium, sarcomas most commonly
develop in the center of the ulcer [55]. If malignancy is suspected, it is important to
obtain several biopsies, including the wound base.
Other Types of Chronic Wounds
Apart from the most common types of ulcers discussed above, less common types
of non-healing ulcers we encounter under the microscope include Pyoderma gan-
grenosum which is a diagnosis of exclusion, factitial (self-inflicted) ulcers, infectious
ulcers and radiation ulcers. It should be borne in mind that any ulcer showing fea-
tures of dense inflammatory infiltrate, including granulomatous pattern and/or pseu-
doepitheliomatous hyperplasia and diffuse necrosis should be investigated with
special stains for fungal and acid fast positive mycobacterial organisms (such as
PAS, Gomori methenamine silver (GMS), Acid Fast Bacillus (AFB) and Fite) to
exclude infectious disease. Radiation ulcers are a clinical challenging as they pres-
ent histologically with necrosis of the pilosebaceous follicles and eccrine glands
where the re-epithelization starts from. Squamosus eccrine metaplasia or syrin-
gosquamous metaplasia has been reported due to radiation [56, 57]. (Fig. 9.11)
Other features of non-healing radiation ulcers include: ulcerated epidermis, atrophic
adjacent epidermis, swollen hyalinized collagen showing irregular eosinophilic
staining with scattered atypical stellate fibroblasts (radiation fibroblasts) (Fig. 9.12a).
Fig. 9.11 Radiation ulcer,

H&E, ×10 magnification.
Squamous eccrine metaplasia
due to radiation – atypical
cells with hyperchromatic
nuclei, increased nuclear/
cytoplasm ratio and
prominent nucleoli line up
the ductal lumina of affected
sweat glands
Fig. 9.12 Radiation ulcer,

H&E, ×4 magnification. (a)
Collagenous fibrotic stroma
with many atypical (bizarre)
stellate fibroblasts (arrow);
(b) vascular injury (extensive
fibrinoid necrosis of the vessel
walls)
The vessels show features of hyalinization of the vessels walls, some of the vessels
reveal extensive fibrinoid necrosis and/or occluded lumina by thrombi (Fig. 9.12b).
The deep reticular dermis is usually fibrotic. It is always important to exclude evolv-
ing malignant process (SCC, BCC and sarcomas).
In conclusion, it is crucial to biopsy chronic non-healing leg ulcers for four
reasons:
1. it is always important to exclude underlying malignancy which can be diagnosed
only histologically;
2. histology is a fast and simple method to diagnose infectious etiology which may
go clinically unrecognized for a long time;
3. in most cases histology allows to differentiate vasculitis from vasculopathy
which requires different management approach and
4. more information about the pathogenesis of non-healing ulcers can be obtained
through the use of new emerging immunohistochemistry markers which may
help the implementation of target oriented treatment modalities.
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Chapter 10
Measuring Pressure in the Diabetic Foot
Thanh Dinh, Aristidis Veves, and Francesco Tecilazich
Keywords Diabetic foot ulcer • Peak plantar pressure • Shear pressure • Total con-
tact cast • Diabetic shoes
Introduction
Over the last few decades, a decreasing trend has been observed in such compli-
cations of diabetes as kidney and eye diseases. This is the result of improved
understanding of diabetes. By contrast, the incidence of lower extremity ampu-
tations resulting from diabetic foot ulceration continuities to increase [1].
Diabetic foot ulcerations continue to greatly burden patients’ lives as well as the
healthcare system and remain the leading cause of hospitalization in the diabetic
patient.
The etiology of diabetic foot ulcers has been postulated to be the result of mul-
tiple factors, mainly, the presence of peripheral neuropathy, foot deformities and
minor trauma. In 1979, Brand [2] put forth the idea of “repetitive moderate stresses”
as the underlying forces responsible for tissue breakdown. Two decades later, in a
T. Dinh, D.P.M. (*) • A. Veves, M.D., D.Sc.

Surgical Department, Beth Israel Deaconess Medical Center, Harvard Medical School,
West, BIDMC, 1 Deaconess Road, Boston, MA 02215, USA
e-mail: tdinh@bidmc.harvard.edu
F. Tecilazich
Surgical Department, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, MA, USA

176 T. Dinh et al.
multi-center study, Reiber et al. further described the triad of peripheral neuropathy,
foot deformity, and minor trauma as the underlying preceding factors in the devel-
opment of 63% of foot ulcers [3].
Following the development of a foot ulcer, foot pressure evaluation and modula-
tion plays a vital role in the treatment and ultimate resolution of the ulcer. Since the
insensate foot is unable to detect pain signals, it is theorized that unmitigated pres-
sures results in continued trauma to the wound, with subsequent failure to heal the
wound in a timely fashion. Thus, pressure reduction in the area of ulceration, com-
monly referred to as “off-loading” is an essential component of the treatment regimen.
There exists a variety of methods to measure the pressures in the diabetic foot as well
as a number of techniques to diminish these pressures with varying success rates.
Early identification of persons at risk for foot ulceration is of central importance
in any plan for diabetes care and amputation prevention. Thus, evaluation of foot
pressure in the diabetic foot is of paramount importance prior to the development of
foot ulceration. Foot pressure measurement to determine “loss of protective” sensa-
tion in the diabetic foot is considered a standard screening tool for evaluation of
patients at risk for the development of diabetic foot ulcers. Patients who are unable
to detect the pressure exerted by the device used are deemed to have loss of protec-
tive sensation. Thus, these patients are likely to develop foot ulceration and should
be provided with proper education in early detection of foot trauma as well as
screened on a regular basis by a health care provider.
Evaluation of Peripheral Neuropathy
Detection of Peripheral Neuropathy
The detection of peripheral neuropathy can be performed effectively with a number

of different non-invasive, in office techniques. The identification of patients with
peripheral neuropathy is an important clinical examination, as the presence of
peripheral neuropathy often necessitates the initiation of preventative strategies to
prevent foot ulceration.
Tuning Fork
The conventional tuning fork is an easy and inexpensive method of assessing vibratory
sensation. The tuning fork is struck, resulting in vibration generated. The tuning fork is
then applied to the patient’s skin and the test is considered positive when the patient
loses vibratory sensation whereas the examiner continues to detect the sensation.
While simple to perform in a clinical setting, the tuning fork is not the most
accurate test to assess vibration threshold (VT). In one study, a standardized method
of assessment of VT, with a C128-Hz tuning fork, was compared to VT measured
10 Measuring Pressure in the Diabetic Foot 177
with a biothesiometer in normal patients free of neurological disease at four sepa-

rate bony points. The biothesiometer was found to be more accurate compared to a
timed tuning fork. However, the accuracy of both declined with patient age, espe-
cially for patients older than 50 years.
Semmes-Weinstein Monofilament (SWM)
Cutaneous pressure perception measured by SWM is widely considered to be the

ideal screening instrument for neuropathy and risk for ulceration because of its
simplicity, sensitivity, and low cost [4–10]. There are three types of filaments: 4.17,
5.07 and 6.1 (1, 10 and 75-g force, respectively). In a pivotal study, Birke et al. [11]
examined all three types of the SWM in a group of 72 patients with Hansen’s dis-
ease and 28 patients with diabetes mellitus, and concluded that the 5.07/10-g
monofilament was the best indicator of protective sensation. SWM testing involves
the use of a 5.07 monofilament applied in a perpendicular fashion to a patients skin
until it buckles, for a 1 s interval, resulting in 10 g of pressure exerted.
The rationale of the monofilament test is to measure the patient’s ability to sense
a point of pressure. Typically, the monofilament is initially applied to the patient’s
hand with the eyes open to establish the sensation that should be felt. Thereafter, the
monofilament is tested on different areas of the foot [12]. While conceptually sim-
ple, there is no consensus on the number of testing sites or criteria for determining
protective sensation. Several studies have promoted the testing of anywhere from 1
to 10 sites with varying criteria for determining protective sensation, with specificity
and sensitivity defined separately in each study [13–15].
Despite varying recommendations for testing and interpretation of results, SWM
remains a highly sensitive and specific method of predicting foot ulceration. Lee
et al. found sensitivity and specificity reached 93.1% and 100%, respectively, based
on testing of ten sites. The definition of insensate was defined as an inability to
sense four or more out of the ten sites tested [16]. Another study found that simplified
testing of four areas, namely the great toe, and base of the first, third and fifth meta-
tarsals has been shown to identify 90% of patients with loss of protective sensation
[17]. In addition to assessing for loss of protective sensation, the 10-g SWM has
also been demonstrated to independently predict the development of diabetic foot
ulcers and increased the risk of ulceration nearly ten-fold [15]. While the SWM test
has limitation in quantitative measurement of neuropathy, it remains a simple, cost-
effective, and practical test for detecting peripheral neuropathy.
Biothesiometry
The electronic measurement of vibration perception threshold (VPT) can be per-

formed with a biothesiometer and has successfully been used to screen patients for
peripheral neuropathy as well as to predict ulceration in those individuals with dia-
betes [18, 19]. The biothesiometer is a hand-held device with a probe that can be
178 T. Dinh et al.
made to vibrate at increasing intensity by turning a dial. The biothesiometer can

have a reading from 0 to 50 V. The reading is low in young, healthy individuals and
becomes progressively higher with age.
VPT is semiquantitative in nature and thus, potentially less prone to variability
compared to SWM. In a population based study, diabetic patients with past or present
history of foot ulceration were more likely to have a higher VPT than those without
history of ulceration [18]. Pham et al. found 12-fold risk of ulceration in diabetic
patients with high VPT compared to patients with low VPT [15]. Furthermore, a
VPT >25 V was strongly associated with the risk of foot ulceration.
Foot Ulcer and Pressure
In addition to the presence of neuropathy, high plantar foot pressures have been
implicated as an important predisposing risk factor for foot ulceration. In 1983,
Boulton et al. [20] used optical pedobarograph to examine the relationship between
high-foot pressures and foot ulceration in patients with diabetes. Their findings
showed that a significant number of patients with diabetic neuropathy had abnor-
mally high plantar foot pressures compared to the control subjects. Furthermore,
patients with a previous history of foot ulceration had higher plantar pressures at
these locations. Thus, it was inferred that reduction of pressures in this area may
reduce the incidence of foot ulceration.
In a prospective study, Veves et al. [15] followed 86 diabetic patients over the course
of 30 months. Over this period of time, 17% developed a foot ulcer, with the majority
of the ulcers occurring on the plantar surface of the foot. Of these patients, 93% had
neuropathy at baseline, and all had abnormally high foot pressures measured by pedo-
barography. The authors concluded that high plantar foot pressures in diabetic patients
were strongly predictive of subsequent plantar ulceration, particularly in the presence
of neuropathy. Furthermore, quantification of risk for high foot pressures have demon-
strated pressures in excess of 6 kg/cm2 twice as likely to ulcerate compared to those
patients with lower pressures [15]. However, it is important to note that elevated foot
pressure alone is not a reliable independent predictor or foot ulceration [20, 21].
The relationship between high foot pressures contributing to the development of
foot ulcer has been confirmed by several studies [20, 22]. Murray et al. [22] reported
a 57% ulcer incidence at high pressure locations. However, it was not clear if all
ulcers were observed at peak pressure points and more importantly, the pressure
threshold for foot ulceration has yet to be established by any study.
Foot Pressure Measurement
Biomechanical measurements of pressure distribution focus on the pressures exerted

on the plantar surface of the foot from the supporting ground surface. Various pres-
sure systems are available for the measurements of the pressures to the foot inside a
shoe, insole, or plantar surface of the foot during stance or gait. Such assessment has
proven to be useful in the diagnosis and management of pressure related foot
problems.
Foot pressure measurement can be measured in two manners: static and dynamic.
Static measurements are taken while the patient is standing and dynamic measure-
ments are taken during locomotion. With static measurement, it is not possible to
determine the form or the loading pattern of the foot during function. Dynamic
measurements are used to determine loading during the actual stance phase of gait,
quantified by parameters such as the length of stride, angle of gait, and speed of gait,
along with many other parameters.
Foot pressure measurement in the diabetic foot has historically been performed
in shoes as well as out of shoes. Out of shoe measurements tend to be easier to per-
form and more comprehensive, allowing for measurements of peak plantar pres-
sures throughout the entire plantar surface of the foot. However, since it is generally
inadvisable for diabetic patients to ambulate without shoes, out of shoe measure-
ments may be of poor value in determining the offloading capabilities of protective
shoes and inserts.
Peak Pressures
Foot pressure measurements have primarily focused on the peak plantar pressures,
measured by the vertical forces exerted by the ground. These vertical forces have
been correlated with the development of foot ulceration through numerous studies
as they are easily measured and quantifiable. Despite the ability to measure peak
pressures, there has been no agreement on the pressure threshold necessary for foot
ulceration. The absence of a pressure threshold may be attributed to a variety of
factors, including the variability of the plantar soft tissue, amount of vascular perfu-
sion, the calibration of the measuring devices used, the presence of shear forces, and
the duration of plantar pressures.
Measurement of peak plantar pressures has routinely been used as a surrogate
marker of trauma to the plantar foot. Due to the many variables affecting measure-
ment of peak plantar pressure and the resultant difficulty in the establishment of
plantar pressure threshold, the measurement of peak pressure gradient (PPG) has
been proposed. PPG has been defined as the spatial change in plantar pressure
around the peak plantar pressure (PPP) location. PPG was found to be substantially
higher in the forefoot compared to the rearfoot and may be a better indicator of skin
trauma since spatial changes in high plantar pressures may identify high stress con-
centrations within the soft tissues [23].
Another variable affecting peak plantar pressure measurement is the role of shear
pressures. Shear pressures are an important component of foot pressures and has been
implicated in the formation of callus tissue in the diabetic foot [24]. In the diabetic foot,
the skin responds to repeated shear stresses by buildup of callus tissue to protect the skin
from further damage. However, if this callus formation becomes excessive, it will con-
tribute to higher pressures, and thus ulceration develops. The association between peak
180 T. Dinh et al.
pressures and shear forces remains unclear, however, shear combined with pressure has
been associated with ulcer incidences [25, 26]. Conflicting data exists regarding the
overlap of peak pressures with shear stresses. One study showed peak pressure and peak
shear sites to overlap in only 50% [25] of cases while another study showed overlap in
20% [26]. Thus, further investigation into shear forces is warranted.
Shear Forces
Although the foot is exposed to three-dimensional force vectors during weight-

bearing activities, analysis is generally limited to the vertical component. Plantar
shear is an influential force behind callus formation and has previously been linked
with increased ulcer incidence [22]. Compared to peak pressure measurements,
peak maximum shear stress provides more information regarding plantar pressure
distribution and the potentially injurious internal stresses to the soft tissues, thus,
making it a more valuable variable. However, while peak plantar pressures are eas-
ily measured, shear forces are not. Thus, it has been recommended that plantar shear
should be investigated further from a broader perspective including the temporal
specifics and fatigue failure characteristics of the affected plantar tissue [27].
Out of Shoe Pressure Measurements
In an attempt to examine plantar pressure distribution in the diabetic foot, the Harris-
Beath Mat was described in 1947 [28]. This method employed a multilayered inked
rubber mat that allowed ink to escape when pressure was applied. The Harris-Beath
foot printing technique is inexpensive, easy to use, and has the ability of providing
a permanent record of the pressure distribution. While this method has the benefit of
observing high pressure points in the foot, the pressure load itself cannot be ade-
quately quantified. To address the failure of achieve quantifiable data, Silvino and
associates calibrated the Harris-Beath mat by using a contact area of known size and
weight, thereby producing both qualitative and semi-quantitative data [29].
Using a similar idea of footprint impression, the Podotrack system (Medical Gait
Technology, The Netherlands) employs a chemical reaction with carbon paper
instead of ink. The advantages of this system over the Harris-Beath mat is the ability
to calibrate the system with a range of colors corresponding to the amount of pres-
sures measured [30]. In one study comparing control subjects to diabetic subjects,
the Podotrack identified the majority of high pressure areas, suggesting that the
Podotrack could be a useful screening tool to identify areas at risk of ulceration in
diabetic patients. However, due to their inherent static design, the Podotrack and
Harris-Beath mat cannot measure dynamic pressure measurements, an essential
component of the diabetic foot.
The failure of early foot pressure measuring techniques to adequately measure

dynamic foot pressures was addressed with the use of the optical pedobarograph.
The optical pedobarograph is a well-established computerized pressure measure-
ment device that uses digital video capture technology to record the pressure varia-
tions on the sole of the foot [20]. The subject walks across a force plate fitted with
an illuminated glass plate. As the foot hits the device, the glass surface deflects due
to the force, causing the horizontal light beams to reflect downwards and be read by
the video camera. The amount of light reflected is proportional to the pressure
caused by the foot striking the plate. The optical pedobarograph has high spatial
resolution, allowing accurate measurement of high foot pressures under small areas
of the foot with satisfactory precision and has been used in many interventional
studies investigating the effectiveness of offloading high pressure areas [31–33].
However, the drawbacks to this system include its limited measurement of barefoot
pressures only and the substantial space required for use.
In Shoe Pressure Measurements
Recognizing the limitations of barefoot pressure measurements in the evaluation

shoes and inserts designed to decrease pressure in the insensate diabetic foot, meth-
ods of in-shoe pressure measurements were developed over the last half century.
Primarily based on computer technology using microprocessor recording devices,
these technologies are capable of measuring peak pressures as well as producing
images of the peak pressures. In addition to dynamic in-shoe pressure measure-
ments, they can also provide out of shoe measurements.
The Electrodynogram System (EDG System, Langer Biomechanics Group, Deer
Park, New York) uses seven small sensors that adhere to the plantar aspect of the
foot. The sensors are attached by a cable which is then attached to a computer pack
carried by the subject. The Electrodynogram can measure dynamic foot pressures,
but limitations of this system include the limited ability to measure pressures only
at the site of the sensors.
The EMED system is another computer based system that can also record both in
shoe and out of shoe dynamic pressure measurements. This device consists of a mat
wired with electrical capacitance, allowing foot pressures to be measured by record-
ing the electrical flow. The EMED system has enjoyed varied clinical applications in
the lower extremity, including evaluation of diabetic foot pressures [34], pressure
changes following surgery [35], and foot deformities such as clubfoot [36].
In our unit, the pressure measuring device commonly employed for study is the
F-scan System. This system is a high-resolution, computerized pressure, force and
gait analysis program. The hardware system is capable of collecting both static and
dynamic plantar pressures by using the mat apparatus or the F-scan in-shoe sensor.
The advantages of the F-scan system include its simplicity, ease of use and repro-
ducibility of data. While the F-scan cannot reach absolute accuracy, its peak plantar
182 T. Dinh et al.
measurements have been found to have a coefficient of variability of 7.8%, largely

due to calibration error [37].
The primary focus of the F-SCAN system is that of peak pressure distribution
over time. Vertical plantar pressure dispersion across the plantar surface of the foot
is recorded, processed, and graphically displayed in terms of sequential gait changes.
Its graphic display capabilities are color coded and easily understood. The system
further allows for the manipulation of the accumulated data to present it in a more
comprehensive manner [38].
The major benefit of the F-Scan system is the ability to measure in-shoe mea-
surement with an ultrathin, flexible in-shoe sensor which is of critical impor-
tance when evaluating the insensate diabetic foot. This unique sensor insert can
be trimmed to fit the subject’s shoes and does not interfere with foot function or
pressure load distribution. However, the thin nature of the sensor is also prone
to wrinkling and breakage, resulting in incorrect data. Additionally, the accu-
racy of the insoles decreased over time, with a sharp decline in sensitivity found
after 12 uses [39].
Foot Pressure Offloading
Foot pressure offloading is commonly performed with the use of shoes, insoles, and
customized devices with a twofold purpose: (1) protection and prevention of foot
ulceration and (2) treatment of neuropathic ulceration. Protection of the insensate
foot with off the shelf shoes, customized shoes, insoles, and socks has been widely
reported. When a neuropathic ulcer is present, offloading treatment changes to a
more aggressive regimen including total contact casts and removable and non-
removable offloading devices.
A recent review of the literature found that the evidence to support the use of
footwear for the prevention of ulceration is meager [40]. While plantar pressure
reduction can be achieved by several modalities including casts, walkers, and thera-
peutic footwear, the diversity in methods and materials used limits the comparison
of study results. However, there was sufficient evidence found to support the use of
total contact casts and other non-removable modalities for treatment of neuropathic
plantar ulcers.
In the clinical setting, health care providers commonly rely on information
obtained from a physical examination to identify the location of elevated plantar
pressures. For example, elevated plantar pressures are suggested in areas of boney
prominences or in an area where a callus is observed. Yet a level of agreement
among different providers on the specific location of peak pressures is varied and
can add to the confusion as to the best offloading device [41]. As a result of the limi-
tations of the current methodology of assessing peak pressures of the diabetic foot,
there exist a number of different socks, inserts, shoes and casting methods studied
with varying degrees of success.
Interventions for Prevention of Foot Ulceration
Socks
Pressure reduction can be simply performed with the used of something as simple
as socks. In their initial study, Veves et al. investigated the use of padded hosiery in
ten neuropathic, diabetic patients. They found a 31% reduction in peak plantar pres-
sures as measured with an optical pedobarograph when the socks were new [42]. In
order to examine the effect of sock wear after multiple uses, a subsequent study was
undertaken to assess the peak plantar pressures after 3 and 6 months. Results from
this follow-up study showed that although the socks continued to provide pressure
relief, the amount of pressure reduction had diminished by 15% at 3 months and
17% at 6 months [43].
While socks can provide cushioning to decrease plantar peak pressures, elements
of the sock itself may cause increased pressure and ulceration risk. In a laboratory
study, the influence of the sock seam on foot pressures was investigated. The inves-
tigators used commercially available socks with seams and evaluated pressures
from seamed areas and un-seamed areas with an F-scan pressure measuring device.
They found that the seamed areas had a tenfold increase in pressures compared to
un-seamed areas. As a direct result of this study, it is now commonly recommended
that diabetic patients at risk for foot ulceration wear socks without seams to limit
foot pressures to the toe areas.
In the real world, most diabetic patients are advised to wear shoes and socks on
a daily basis to protect their feet and prevent ulceration. Thus, further investigation
into pressure reduction with the use of both socks and shoes was warranted.
Donaghue et al. [44] prospectively examined the effect of using specially padded
hosiery in combination with custom fit shoewear on 50 patients at risk for foot
ulceration. Dynamic foot pressures were measured at baseline and at subsequent
visits over at 30-month period. Initial pressure relief was experienced by all subjects
and remained so at interim and final visits, indicating that the combination of socks
and shoes effectively reduces plantar foot pressures.
Insoles
Quantification of barefoot plantar pressures has greatly aided in the design of the
appropriate offloading device, especially in the creation of custom diabetic insoles.
The basic underlying philosophy of insole design is to uniformly redistribute the
pressure under non-risk areas of the foot in order to decrease the pressure under the
at risk areas of the foot.
Custom made insoles of both the soft and rigid variety have been described to
decrease foot pressures [45–47]. The most common type of insole recommended in
184 T. Dinh et al.
the diabetic foot involves the used of heat-pressed Plastizote. The use of Plastizote
addresses the shear stresses involved in the development of foot ulcerations and has
been found to decrease plantar peak pressures by 40–50% [46]. Additionally, con-
toured insoles were found to possess greater pressure capacity compared to flat
insoles [48].
One study investigated the combination of therapeutic footwear and custom-
made orthotic insoles on pressure and tissue strain along the second ray of the plan-
tar foot [49]. The results from this study demonstrated a reduction in pressure and
soft tissue strain at the second metatarsal head with the inclusion of the orthotic
devices during the simulated terminal stance of gait. This finding supports results of
other studies that have also found a reduction in pressure with the use of therapeutic
footwear.
Custom made insoles have been found to be significantly more successful at
reducing pressures compared to flat inserts [50]. However, insole customization was
based on subjective clinical assessment and failed to reduce pressures in one-third
of cases. Thus, the need for a more systematic, objective way of producing custom-
ized offloading devices still exists.
Owings et al. [34] compared the use of custom insoles fabricated from barefoot
plantar pressure measurements with conventional custom insoles in decreasing high
plantar foot pressures. The investigators found that custom insoles created based on
pressure measurements enhanced offloading of high-pressure areas under the fore-
foot. Furthermore, this offloading was achieved by a greater transfer of load to the
midfoot without additional loading of other forefoot structures.
The materials used in the fabrication of an insole is just as important as the man-
ner in which it is made. A material with an appropriate amount of resistance to shear
stress is a critical element in the specification of an insert. Although shear stress is
difficult to measure, approaches to reduce shear stresses with insoles have been pro-
posed. Lavery et al. lowered peak pressures and shear stresses with insoles fashioned
with a central area composed of material with low-friction characteristics [51].
Shoes
Shoes provide the important function of protection to the diabetic foot from the
threats of the environment including foreign bodies, thermal injuries, and hazardous
substances. They also serve the important function of decreasing plantar foot pres-
sures. Shoes for the diabetic foot have been recommended in both non-custom and
custom forms, depending on the presence of foot deformity. In a large randomized
study, Reiber et al. compared custom therapeutic footwear to off the shelf shoes to
evaluate for reulceration in patients with diabetes [52]. In those subjects with little
foot deformity, the reulceration was comparable between the two groups, suggest-
ing that custom shoes may not be necessary in all patients with diabetes.
Non-custom shoes have been shown to reduce plantar pressures in patients with
diabetes and neuropathy. Off the shelf running shoes may be an inexpensive, readily
Fig. 10.1 Running shoes can

reduce foot pressures. They
are readily available,
lightweight, and affordable.
The material of the shoe upper
is soft and padded on the
inside where it interfaces with
the foot
available option for the reduction of peak plantar pressure. (Fig. 10.1) Evaluation of
pressure reduction at the plantar forefoot with off the shelf running shoes versus
leather oxfords showed that running shoes were found to decrease mean plantar foot
pressures by 47% at the second and third metatarsal heads and 29% at the first meta-
tarsal head compared to the leather oxfords [53].
Customized diabetic shoes typically incorporate an extra depth feature to the
forefoot to accommodate forefoot deformities in the diabetic insensate foot.
Customized extra depth shoes have been shown to significantly decrease foot pres-
sures, particularly so when used in conjunction with padded socks and insoles [54, 55].
While customized extra depth shoes are considered the gold standard for prevention
of foot ulceration, their effectiveness in decreasing foot ulceration is experienced
when compliance exceeds 60% [56].
Modification of customized diabetic shoes to further reduce foot pressures and
reduce energy expenditure includes the addition of a rocker bottom sole. The rocker
bottom sole limits the need for sagittal plane motion in the joints of the foot and
alters the lower extremity gait kinematics. Reviews of rocker bottom insoles have
demonstrated that while effective in reduction of forefoot plantar pressures, the
reduction of sagittal plane motion appears to occur primarily at the ankle joint [57].
The effect of the rocker profile on other joints of the foot, muscle activity and gait
patterns is unclear and further investigation warranted.
Interventions for Pressure Reduction in Foot Ulceration
Total Contact Cast (TCC)
The TTC is a well molded, minimally padded cast that maintains contact along the
entire plantar aspect of the foot and lower leg. Pressure reduction to the ulceration
is achieved through the transmission of pressures to other areas of the foot and leg
186 T. Dinh et al.
through the wall of the cast. Considered the gold standard of offloading the neuro-
pathic ulceration, TCC has shown superior pressure reduction compared to other
pressure offloading devices such as custom-molded insole shoe, a cast shoe, and a
prefabricated pneumatic walking brace [58].
Advantages of the TTC include the reported reduction of pressure, immobiliza-
tion of tissues, edema reduction, and patient compliance. Disadvantages of its use
include secondary lesions, inability to observe the wounds daily for the presence of
infection, and the considerable time and cost associated with its application. In a
survey of specialty foot clinics in 2005, Wu et al. found that less than 2% of the
respondents used TCC for offloading of neuropathic ulcerations, despite acknowl-
edging its acceptance as the gold standard for offloading [59].
Removable Cast Walker
As an alternative to TCC, removable cast walkers have been proposed to be a sim-

ple, cost effective method of offloading the neuropathic ulcer. Reduction of plantar
pressures has been shown to be comparable between TCC and removable cast walk-
ers [60, 61]. However, TCC has been shown to heal a higher proportion of wounds
in a shorter time compared with a removable cast walker [62].
One criticism of removable cast walkers in the treatment of diabetic foot
ulceration stems from the removable nature of the device. There has been con-
cern that the ability to remove the device may limit adherence and subse-
quently effectiveness. To remedy this, Armstrong et al. has described using
plaster of Paris to wrap around the cast walker, coined an instant total contact
cast (iTCC), resulting in an offl oading device that cannot be removed by the
patient, thus ensuring compliance with treatment [63 ] . In a prospective, ran-
domized study of 50 patients, the iTCC treated ulcers were found to heal
significantly sooner compared to those treated with the traditional removable
cast [64].
Half Shoe
Half shoes and postoperative shoes have been used with some success in offloading
foot pressure reduction, but are generally recommended for forefoot ulcerations.
Fig. 10.2 Application of the

custom cut out felted foam
dressing with self adherent
gauze
Half shoes are a modification of the postoperative shoe that incorporates a large heel
wedge that extends just behind the forefoot. Pressure reduction has been reported to
be as high as 66% [54]. In comparison to postoperative shoes, the half shoe was
shown to relieve metatarsal head peak pressure to a significantly larger extent, esti-
mated at approximately 20% [65].
Felted Foam Dressings
The felted foam dressing involves the use of customized felted foam cut-outs glued
to the plantar surface of the foot with self adherent gauze. (Fig. 10.2) The felted
foam dressing possesses an aperture to accommodate the ulceration, resulting in
redistribution of plantar pressures to the adjacent skin. In a prospective cohort study
of 61 diabetic patients with neuropathic foot ulcer, use of the felted foam dressing
was found to be comparable to conventional offloading techniques with healing
times averaging 83 days in both groups [66].
Benefits of the felted foam dressing include its ease of application and the cus-
tomized nature of the dressing [67]. Additionally, the aperture portion of the dress-
ing allows for daily monitoring of the ulcer and application of wound therapies not
easily performed in offloading devices like the TCC. (Fig. 10.3) Finally, since the
offloading dressing is glued to the patient’s foot, patient adherence to the treatment
regimen can be assured.
188 T. Dinh et al.
Fig. 10.3 The open aperture

portion of the felted foam pad
allows for daily observation
of the wound along with
specialized dressing
applications
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Chapter 11
Skin and Vascular Assessments
Marco Romanelli, Valentina Dini, and Raj Mani
Keywords Non invasive assessment • Digital analysis • Blood flow • Ultrasound •

Oxygen tension
Introduction
This chapter is focused on assessing the skin and vascular systems with the objec-
tive of understanding the pathophysiology or monitoring clinical progress of heal-
ing. The majority of chronic wounds occur on the lower extremity and are the
result of hypertension (in the venous or arterial systems) or diabetes. Excess unre-
lieved pressure may also lead to cutaneous lesions that frequently become to
chronic wounds in patients who are frail and need help to change position (pressure
wounds) or are suffer from diabetes mellitus (neuropathic ulcers). Consequently
diagnostic knowledge and clinical experience have developed from working with
chronic wounds of venous aetiology or diabetic foot wounds. This chapter is
focused on techniques and the significance of techniques with established clinical
applications.
There may be several complications that occur during wound healing; common
ones are oedema and infection. In relevance to infection, microbiology for a tissue
M. Romanelli, M.D., Ph.D. • V. Dini, M.D., Ph.D.

Wound Healing Research Unit, Department of Dermatology,
University of Pisa, Pisa, Italy
R. Mani, D.Sc., FACA (*)
Department of Clinical Measurements,
Southampton University Hospital NHS Trust,
Southampton, SO16 6YD, UK
e-mail: rm1@soton.ac.uk

194 M. Romanelli et al.
diagnosis is essential to establish diagnosis; this is discussed in another chapter in

this book.
Skin Assessments
Chronic ulcerative skin lesions affect around 1.5% of the population and represent
a considerable medical and social problem. The population affected by this pathol-
ogy is generally elderly often suffering from concomitant illnesses. Chronic ulcers
have different causes and can be divided into the following main categories: vascu-
lar ulcers (venous, arterial, mixed aetiologies), diabetic foot ulcers, pressure ulcers,
and ulcers of different aetiology. The above pathologies refer to chronic and invali-
dating conditions that profoundly affect the patients’ quality of life and often lead to
psychological disturbances, such as depression.
New treatments for these pathologies have led to improvements in lesion manage-
ment and in the quality of assistance provided by medical and paramedical staff, but
lesion monitoring methodologies have not kept pace with this progress. The tech-
niques obtained a valid wound assessment currently based on the use of transparent
acetate sheets, which are positioned on the lesion so as to trace its perimeter manu-
ally, measuring the depth of the lesion by placing a q-tip inside it, or filling the lesion
cavity with hypoallergenic material to produce a cast, which is then measured to
obtain the volume of the lesion. However, most clinical diagnoses depend on visual
observation of the lesion. This is obviously an inaccurate, non-standardized, slow,
and above all subjective method, which depends on the experience of the physician.
An effective and accurate monitoring of skin lesions should be performed by
measuring in an objective, precise and reproducible way the complete status and
evolution of the skin lesion [1]. The main goal of current research projects is to
design a system that can monitor the qualitative and quantitative evolution of a skin
lesion, with an easy-to-use technological system.
Wound Size Analysis
The use of digital photography in wound management has amplified the possibili-
ties of diagnosis in case of cutaneous chronic lesions, through the acquisition of
optical characteristics which are not directly detectable with the naked eye. Such
methodology may be placed therefore half-way between pure clinical surveying and
microscopic examination.
Further innovation has been recently provided by the digital imaging, which pro-
vides the conversion of the optical medical report in a bi-dimensional series of
numerical values.
Digital imaging, currently employed almost exclusively on cutaneous lesions,
has achieved therefore new advantages, such as being non invasive, relative ease
of execution, and good reliability. Acquirements and records of detailed digital
11 Skin and Vascular Assessments 195
mappings of the lesions may provide an uniform object comparison, which allows
to detect several features, interpretable with mathematical models also in function
of their changes over time.
With reference to ulcerative cutaneous lesions, this methodology may reveal its
usefulness as an important tool in a field where variability of clinical findings often
does not favour effective appraisals.
This fact imposes the definition of standard, objective and uniformly acquirable
elements that may constitute the base for correct diagnostic guidelines as well as
therapeutic efficacy.
The morphologic features of an ulcerative cutaneous lesion can be substantially
analyzed according to two distinctive modalities: the quantification of the loss of
substance (extension and depth of the lesion, characteristics of the edges), and the
qualitative discrimination of the several areas of the wound bed (presence of necro-
sis, fibrin, fluid, extension of the surrounding phlogosis). All these characteristics
can be acquired through digital imaging, repeated at defined times. The comparison
of these findings would provide the object of a dynamic analysis of the healing pro-
cess, eventually in function of the type of performed therapy.
As regards the morphometric analysis of the loss of substance, it is possible to
obtain the geometric determination of the perimeter, area, and maximum vertical
and horizontal dimensions in a relatively simple way, and to study its variations by
repeating detections over time, thus obtaining an “a posteriori” evaluation of the
healing dynamics and its progressive or regressive tendency.
An additional survey may be carried out by observing the tendency to epithelial
proliferation, which is possible only when the wound is in optimal conditions
(absence of necrosis, absence of infection, right humidity, sufficient cleansing, and
sufficient perfusion).
In order to inquire effectively on such process, the study of the margins of the
wound (regular or not) and of their chromatic features (evocative of hyperaemia,
hyperkeratosis, necrosis,) is thought of remarkable importance, while the observa-
tions over time may focus on the epithelial edge progression.
From this last information, a perspective appraisal of dynamics of the healing
process is thought possible to be realized.
Previous attempts at monitoring the evolution of lesions in an objective manner
include systems based on the acquisition of 2D images or video streams [2, 3]. In
these studies, a characterization of the tissue status is reconstructed from the seg-
mentation of the red, green, blue (RGB) colouration of the image, and some 2D
measurements are inferred from this segmentation (e.g. the wound area computed in
the 2D image projection space). Calibration specimens are in general placed in
proximity of the lesion, to allow colour calibration and reconstruction of linear mea-
surements. The shape characteristics of a small skin section can be calculated by
using three-dimensional scanners in particular systems based on active optical
approaches [4, 5]. Some of these systems also support the integrated acquisition of
the colour of the scanned region, and colour plays a very important role in the analy-
sis of the status of a skin lesion. The quality of current 3D scanning devices allows
accurate geometric and chromatic characterizations of the skin lesion to be achieved.
Fig. 11.1 Portable laser

scanner for digital wound
image analysis
This objective characterization allows numerical measures to be assessed according

to a flexible set of parameters; the data may then be stored in a database and the skin
damage may be easily monitored over time. There are two different categories of
potential applications of such types of devices: medical treatment, to improve the
efficacy of therapeutic regimens, and pharmacological scientific research, to assess
the quality and effectiveness of new chemicals or clinical procedures (Fig. 11.1).
Once a 3D model of the lesion has been produced, the assessment of significant
parameters has to be performed either automatically or by a dermatologist. Two
related actions can be performed: firstly, the required shape-based measurements are
calculated, and secondly, the associated colour information is segmented to produce
an image where colour segmentation is integrated with the shape characterization.
Wound Bed Colour Assessment
Wound bed and surrounding skin colour evaluation is another instrumental analysis of
particular importance in wound assessment. Today the two main type of colour analy-
sis for skin conditions are the reflectance spectrophotometry and colorimetry. Most of
the portable instruments use the system in which a colour is numerically expressed as
coordinates in the standard colour space defined by CIE (Commission Internationale
de l’Eclairage) [6]. The range of colour of different tissues can be linked to different
Fig. 11.2 Non invasive

tristimulus colorimeter for
wound bed color assessment
conditions of the lesion such as in burn depth assessment [7]. In general, three main
colours are used by clinicians in wound assessment: black for necrotic eschar, yellow
for slough and fibrin, red for granulation tissue. Once we have segmented the lesion
region into a few classes, the system produces numerical data computed on the cor-
responding 3D mesh, such as: the perimeter, the surface area and the percentage of
each class with respect to the wound size. As usual, the segmented chromatic charac-
terization and the computed data can be saved in the database and can be used in the
comparison of the status of the lesion at different intervals [8].
In a recent study we monitored the efficacy of debridement through the use of tri-
stimulus colorimetric assessment in chronic wounds [9]. In this prospective trial we
were comparing a hydrogel and an enzymatic preparation, which were applied once a
day for 2 weeks. The colorimetric evaluation was more accurate than clinical scoring
in assessing granulation tissue and it has been shown to possess high reproducibility,
with the advantage of avoiding the bias involved in clinical scoring. Recently image
analysis of digital photography was used to develop several color indicators for granu-
lation tissue of pressure ulcers [10]. The authors were able to demonstrate the validity
and reliability in the clinical setting for the method investigated (Fig. 11.2).
High Frequency Ultrasound
High frequency ultrasound evaluation of wound healing process is relatively new.

This technology has shown consistent results in dermatology in the acquisition of
data on different dermatological diseases, such as skin cancer, psoriasis and sclero-
derma, as well as in wound healing [11, 12]. Non-invasive assessment of skin struc-
ture using this technique allows an accurate measurement and quantification of
oedema in venous insufficiency, providing greater information for the understand-
ing of this fundamental pathogenic factor [13]. The technique has been shown to be
an objective, valid and reproducible instrument for the evaluation of the healing
process up to the point of scar formation [14].
Using 20 MHz-B-mode high resolution ultrasonography, it is possible to obtain
a specific image of the skin and an identification of physiological and pathological
skin structures. Major changes observed in the skin involve epidermal atrophy and
dermal modifications, but high frequency ultrasound is used to identify the ultra
structure in chronic wounds, hypertrophic scars, keloids and normal surrounding
skin [15]. The use of an aqueous transmission gel over the wound bed allows for
optimal recording without any direct contact with the lesion.
The parameters analysed are the depth between skin surface and the inner limit
of the dermis and the tissue density (Fig. 11.3). The depth measurement, expressed
in millimetres, gives an estimate of wound and scar thickness. The values of echo-
genicity are an expression of tissue density and are characterized by a high echoge-
nicity of the dermis, compared to a relative hypoechogenicity of the subcutaneous
fat. Moreover, a significant correlation has been found between echogenicity and
the duration of scars (r = 0.91).
This technique also makes possible an accurate evaluation and quantification of
the amount of granulation, sloughy and necrotic tissue, together with a measure-
ment of the length and width of the wound [16]. The technique is also characterized
by the enormous advantages it holds over other methods such as callipers, xerora-
diography and punch biopsies [17].
High frequency ultrasound represents a safe, objective, non-invasive and painless
method for the evaluation of the wound healing process, allowing an accurate evalu-
ation of epithelialization, formation of granulation tissue and contraction of ulcers.
Laser Doppler Systems
Different techniques are available for measuring skin blood flow, such as skin tem-
perature measurement, dynamic capillaroscopy, isotope techniques, percutaneous
measurement of partial oxygen pressure, and assessment of capillary pressure,
fluorescence videomicroscopy, videodensitometry or photoplethysmography [18].
The different layers of local skin microcirculation can be directly detected by
Laser Doppler flowmetry and laser Doppler perfusion imaging. Laser Doppler
Fig. 11.3 High frequency ultrasound skin image
techniques are non-invasive medical devices based on the Doppler effect and laser
light source such as low power helium-neon laser (2.5 mW, l = 632.8 nm) or semi-
conductor laser diodes with near-infrared light of 780 nm. The main differences
between these two laser sources is that laser diodes are multi-channel instruments
and the light generated can penetrate more deeply into cutaneous tissue [19].
The photons of monochromatic light emitted by lasers penetrate into tissue and
are reflected by stationary and moving tissue components. Stationary tissue scatters
and reflects the incident photons at the same frequency, while red blood cells mov-
ing with a certain speed reflect the waves, which go through Doppler shifts in their
frequency. The radiation returning to the instrument is composed of two compo-
nents: the non-shifted waves reflected from non-moving tissue elements and the
frequency-modulated light. A proportion of shifted light is backscattered to the sys-
tem where the signal is detected by an optoelectronic device and processed. The
voltage output is proportional to the velocity and concentration of the moving red
cells and gives a measurement of changes in tissue perfusion as discussed in the
section “Vascular assessments”.
The development of laser-Doppler flowmetry dates back to 1972 and since 1975
has been used for the assessment of skin blood flow in humans [20].
Laser Doppler flowmetry is widely used because it is a non-invasive, simple,
objective and fast instrumental measurement which quantifies cutaneous blood flow
1–2 mm under the skin surface (including the upper papillary plexus) and provides
a continuous or near-continuous record. The monochromatic, coherent laser light is
conducted by glass fibres to a probe, attached to the skin by means of adhesive
discs. The movement of blood cells leads to a scattering of the laser light, generated
by a low-powered helium-neon source, inducing a Doppler shift.
The backscattered signal containing data on flux, cell concentration and cell
velocity is displayed on screens and the data may be recorded by a computer [21].
Capillaries and dermal vessels are usually present at a depth of 1 mm and can be
easily evaluated with this technique. This measurement is able to monitor perfusion
in the wound bed, adjacent normal skin and scars. Two parameters are monitored
with the patient supine and relaxed: resting flow and healing potential index (HPI)
(ml of blood/100 g of tissue/min). HPI represents the ratio of resting flow values for
the ulcer and the adjacent normal skin.
It has been shown that blood flow in all types of chronic ulcers is 170% higher
than in normal skin and that a potential healing index of less than 100% is not a
good prognosis. Blood flow in hypertrophic scars and keloids increases by 180%
when compared to normal skin [22]. However there are some limitations with this
technique, such as the necessity for contact with the organs evaluated, the potential
for pain or sepsis when applying the probe to skin surface, and poor accuracy in the
determination of tissue volume.
Since 1993, a development of the laser Doppler flowmetry called laser Doppler
imaging has been available, which combines laser Doppler and scanning techniques
and overcomes the above limitations [23]. This instrument is equipped with a mov-
ing mirror and light collection system, instead of optical fibres.
This technique displays on the screen of a computer a two-dimensional colour-coded
image of the local flux, in which each colour corresponds to a different level of perfu-
sion. It can therefore be used for evaluating tissue viability and ischaemic areas [24].
With regard to colours, blue-violet is an expression of poor flux, whereas green,
yellow and red correspond to areas with higher flux. Grey areas represent regions
where no flux can be detected.
Although some authors do not recommend these exams for routine use in wound
healing studies [25], laser Doppler flowmetry and laser Doppler perfusion imaging
(LDPI) have been used in the evaluation of wound healing and for definition of
ischaemia, inflammation and reperfusion. They could be useful in delimiting areas
that need debridement.
Laser Doppler flowmetry has been used to assess patch test reaction, providing
quantitative and more comparable data, as well as accurate statistical analysis
(although expensive and time-consuming) [26]. The advantage of this method is
that it can visualize subclinical reactions through blood flow changes, at a time
when clinical assessment cannot detect any erythema.
The use of laser Doppler flowmetry or the laser Doppler perfusion imager has
been reported in the evaluation of immediate wheal-and-flare reactions [27],
tuberculin reactions [28], and ultraviolet-induced erythema [25]. Vasodilating effects

of various systemic and topical drugs or blood flow changes in response to the effect
of chemical mediators can also be measured by laser Doppler flowmetry [29].
Laser Doppler flowmetry is useful in the evaluation of wound healing, microan-
giophathy in diabetic patients, and burn depth; it has also been used to monitor flaps
and replants [30].
The laser Doppler flowmeter-measured resting flux has been shown to be ele-
vated (and reversible with treatment) in venous disorders [31], while remaining
approximately at a normal value in peripheral arterial obliterative disease [32].
Laser Doppler flowmetry has been used in stage 2 and 3 pressure ulcers for the
continuous evaluation of local skin microcirculation and it has been shown that the
local blood flow increased at the ulcer edge at rest and after heat stress at 44°C,
when compared to surrounding skin [33].
Concomitant evaluation using laser Doppler perfusion imaging and capillary
microscopy within venous ulcers and ischaemic ulcers is capable of differentiating the
distribution of microcirculation in granulation tissue, non-granulation tissue, adjacent
normal skin and distant skin [34, 35]. In fact, up to 85% of the laser Doppler signal is
generated by deeper layers of skin microcirculation characterized by a thermoregula-
tory function, while the superficial nutritive layers are well detected by capillary
microscopy which detects the density and morphology of visible capillaries [36].
Laser Doppler imaging is also an accurate technique for the early and objective
assessment of burn depth, as well as being a useful instrument in assessing the tim-
ing of surgical treatment as discussed by Sarah Paper in another chapter in this
book. High flux indicates a favourable prognosis for wound healing, whereas low
flux is an expression of difficult or impossible spontaneous wound closure. With
this instrument, it is also possible to assess the whole burn without direct contact
with the burn surface [37].
There is evidences of local blood flow changes being assessed by laser Doppler
flowmetry in scleroderma in which the blood flow of sclerotic plaques is higher than
normal, and in rosacea [38], which is characterized by a lesional blood flow three to
four time higher than normal (control patients). Scanning laser Doppler flowmetry
allows the measurement of increased cutaneous blood flow in psoriatic plaques and
the assessment of its decrease with various treatment modalities, thanks to the rapid-
ity of evaluation and the ability to cover a large area of skin surface. This method also
seems very useful as an adjunct instrument in the differentiation between benign and
malignant pigmented skin lesion [39]. These days, this non-invasive instrument is
not directly applicable to clinical practice, but is reliable in several fields of dermato-
logical research, providing excellent monitoring of cutaneous microcirculation.
Transcutaneous Oximetry
Transcutaneous oximetry is the partial pressure of oxygen measured with an elec-

trode placed on the skin surface. The observation of the existence of an exchange of
O2 and CO2 between skin and ambient air was first made by Gerlach in 1851 [40].
Transcutaneous oximetry is an effective technique widely used in the evaluation

of local skin microcirculation, nutrition and tissue ischaemia. Several methods for
non-invasive measurements of tissue O2 are available [41]. One of these is the TcPO2
technique, based on the electrochemical reduction of oxygen, which is measured on
the skin surface with a calibrated Clark electrode. The TcPO2 measurement pro-
vides information about the content of tissue oxygenation in superficial skin layers.
The post-heating reactive hyperaemia responses of TcPO2 can be used as relative
measures of the vasodilatory capacity of skin micro-vessels. TcPO2 values depend
on cutaneous circulation, arterial PO2, oxygen consumption in skin tissue and oxy-
gen diffusiveness through the skin. The values measured represent the partial pres-
sure of oxygen diffusing from the capillaries and provide data on the oxygenation of
superficial skin layers. The TcPO2 values from undamaged skin can vary depending
on the body region and it has been noticed that low values are associated with failure
in wound healing [42]. In tissue with inflammatory reaction, the dramatic increase
in local metabolism and the infiltration of large amount of cells requiring oxygen
may increase the consumption of oxygen in the skin, leading to low TcPO2 values.
The TcPO2 and TcPCO2 values have been measured in a large number of skin condi-
tions, such as evaluation and management of leg ulcers [43], the assessment of skin
involvement in patients with morphea and scleroderma [44], investigation into
hypertrophic scars [45], and the understanding of the action and efficacy of vasoac-
tive drugs such as topical nitroglycerin [46]. These values can be influenced by
many local factors such as blood flow, thickness of the epidermis, metabolism of the
epidermis and glands, conductivity of the gases, and the production and consump-
tion of such gases in situ. The epidermal barrier has also to be considered in the
measurement of TcPO2, since the stratum corneum represents an important resis-
tance to oxygen diffusion; its removal (using a stripping technique or other meth-
ods) is recommended when studying the skin, for a better reproducibility of the
assessment [47].
Other perfusion analyses such as arteriography, capillaroscopy, plethysmogra-
phy and videomicroscopy are considered difficult to perform and too invasive.
pH Measurement
Measurement of cutaneous pH on wound surfaces is used to assess sudden changes

in pH following exposure to external factors, such as acid or alkaline products, and
in assessing the state of acute or chronic cutaneous diseases. pH is defined as the
negative logarithm of the activity of hydrogen ions in an aqueous solution, used to
express acidity and alkalinity on a scale of 0–14.
In 1892, Hesus first determined the acid nature of the skin surface [48] and since
then a large amount of studies have confirmed his observation and defined the rep-
resentative range of pH for the population as a whole. The first study on the scientific
assessment of skin surface pH was performed by Schade and Marchionni using an
electrometric technique [49]. More recently, the normal range of values in skin
surface pH [50] has been established. Normal values of pH ranges from 4.8 to 6.0
due to the presence of the acid mantle in intact skin by comparison interstitial fluid
is characterized by neutral values. The values are more alkaline for subjects over
80 years of age.
In recent years, a central role for the acid mantel is emerging as a regulating fac-
tor in stratum corneum homeostasis and this has been shown to be very relevant to
the integrity of the barrier function. Until now, no diseases have been associated
with an increase or decrease in skin surface pH, however alteration in the skin pH
(and the organic factors influencing it) seems to play a role in the pathogenesis,
prevention and healing of several cutaneous diseases, such as irritant contact derma-
titis, atopic dermatitis and ichthyosis; it also plays a role in wound healing.
Significant improvements in the methodology and instrumentation of skin sur-
face ph measurement took place during the last century. Two major methods are
currently used for measuring cutaneous pH: the colorimetric technique and the glass
electrode potentiometric measurement.
The most common pH instrument, in use since 1972, is a flat glass electrode con-
nected to a meter and applied to the skin, with one or two drops of bi-distilled water
interposed between the electrode and the skin [51]. The use of a flat electrode is
necessary to provide a good contact with the skin surface, providing high accuracy
and sensitivity for the assessment. The measurement is non-invasive and the electric
current is low, constant and causes no skin damage. In contrast, the colorimetric
procedure with dye pH indicators is less accurate, owing to the interference of many
factors.
The recommended regions for the measurement are the forehead in the midline,
3 cm above the glabella, and the cheek below the zygomatic bone, but if necessary
measurements can be performed on any area of the skin.
The electrode is attached to the skin for an interval of 10 s until stabilization of
the reading. Measurements are performed at a room temperature that is below 23°C
and a relative humidity of less than 65%, because sweat can influence the results.
Readings should be taken 12 h after the application of detergents or creams to the
skin.
One new instrument for pH reading is based on pH transistor technology, in
which the sensor is an ion-sensitive field effect transistor [52]. This non-invasive
technique for the measurement of skin surface pH has been used in the past to assess
the barrier properties of the stratum corneum and also to evaluate the relationship
between change in superficial skin microflora and the development of skin irrita-
tion. In fact, researchers have already noted that there is a relationship between the
acidity of the skin surface and its antimicrobial activity.
There are many reports on the relationship between skin pH and the incidence of
cutaneous diseases. In acute eczema (with erosions), the pH is alkaline due to extra-
cellular components. Some authors have found an increase in skin surface pH in
xeroderma, atopic dermatitis and seborrheic dermatitis [53].
Glibbery and Mani [54] used glass electrodes for the measurement of skin sur-
face pH on ulcers and control sites and showed a link between acid medium and
healing. Wound bed pH has been proven to be fundamental in the healing of chronic
wounds, since a prolonged acidification of the wound bed enhances the healing rate
of chronic leg ulcers; the pH of non-healing chronic venous leg ulcers and pressure
ulcers was shown to be alkaline or neutral when compared to normal perilesional
skin.
Sayeg [55] used reproducible wound pH measurements in experimental and
clinical studies to predict skin graft survival and found this assessment useful in the
determination of the percentage success or failure of the surgical treatment of burns
and chronic ulcers.
The use of occlusive dressings in wound healing provides the optimum environ-
ment for normal repair and regeneration, and the acidic wound fluid collected dur-
ing moist wound healing has been shown to inhibit the bacterial growth and to
promote fibroblast proliferation [56]. Using a foam dressing on venous leg ulcers,
we were able to change the wound bed pH from an alkaline to an acidic environment
and to maintain this acidic state until dressing removal, 72 h later [57].
These days, the use of skin pH measurements is limited to the assessment of the
effects of various materials and environmental factors on the skin surface, but we
believe that wound bed pH measurement can provide highly useful information
about the changes in bacterial burden in chronic wounds.
Infrared Thermal Imaging Systems
The skin represents a thermal interface between the body and the environment; it is
influenced by both internal and external factors. The link between temperature and
diseases has been observed for millennia.
The study of body temperature is based on the three fundamental methods of
heat transmission: conduction (fluid thermometers, thermistors and thermocouples),
convection, and radiation (radiation thermometers, infrared imaging system). Many
of these instruments alter the heat exchange between the skin and the environment,
but an ideal temperature measurement technique should in no way interfere with
this exchange.
Another essential parameter in the measurement of skin temperature is reproduc-
ibility and the thermal radiation system is thoroughly reliable in this sense. It pro-
vides a high speed, two dimensional temperature recordings, does not need direct
contact and does not interfere with the skin.
Thermal imaging is a highly efficient instrument for the assessment of skin tem-
perature, providing both thermal and spatial resolution. Thermography is a non-
invasive method and represents one of the most technically developed methods of
thermal imaging, which, when correctly used and under controlled conditions,
supersedes the data obtained from other thermometers. Through the use of thermog-
raphy, temperatures can be evaluated and recorded, allowing visualization of heat
flow.
There are three types of thermography currently in use: liquid crystal thermogra-
phy, infrared thermography and microwave thermography [58].
Infrared thermography has been found to be a fast and stable method, which is
relatively impervious to user technique. The technology used is based on electro-
optical systems for the detection of infrared radiation emitted by the skin. Cadmium-
mercury-telluride or indium-antimonide are the detectors most commonly used to
generate an electrical signal; at which point, a scanning optical arrangement allows
the creation of a two-dimensional image [59]. This real time imaging, in association
with efficient on-line processing, allows greater ease of use and higher quality of
information, while the use of digital infrared cameras has improved spatial and
thermal resolutions enormously.
Even though it is non-specific, thermal imaging can be used to monitor many
clinical conditions. Increased skin temperature has long been associated with infec-
tion, therefore thermography could provide a useful means to evaluate and monitor
the healing process in complicated chronic wounds. Inflammation with increased
local perfusion may be detected clearly and early, thanks to the precise relationship
between skin perfusion and temperature.
This technique is therefore reliable in monitoring local heat and quantifying the
degree, extent and response to therapy [60]. Reduction of body temperature assessed
by thermal index has been used to evaluate the efficacy of analgesic and anti-
inflammatory agents [61]. Increases in skin temperature have been measured during
erythema and urticarial eruptions [62]. Equally, a reduction in skin temperature can
be found in a large amount of conditions where there is a decrease in tissue perfu-
sion, such as venous ulceration. Low wound bed temperatures have been shown to
delay healing rate, mainly because there is a decrease in oxygen release [63].
Moreover, a region with poor vascularization, generating a cold area on the ther-
mography, will probably result in delayed or impaired healing, while a hot well-
perfused region can be expected to heal correctly.
Even though the cost for these techniques is currently high, new possibilities are
emerging through the use of low-cost portable thermal imaging cameras and high-
resolution thermal imaging will improve our knowledge of wound tissue thermal
regulation. The improved resolution and non-invasiveness of thermographic sys-
tems make them valuable options for the detection and diagnosis of several skin
diseases or abnormalities, such as infections, inflammation or malignancies charac-
terized by increased skin temperature.
Confocal Microscopy
Confocal microscopy (CM) has recently become quite commonly used in the der-
matological field. The basic principle uses a light source and a lens to focus on a
specific plane within the sample of tissues. The returning light from this focal point
is detected by the instrument and used to create an image that is a composite of a
large number of imaged points. The final image acquired is clear, because the sys-
tem is trained to detect mainly the light that is directly backscattered from the focal
point and to exclude any scattered and reflected light from out-of-focus planes, thus
minimizing image blur. The light source used can be either intense visible light or
near-infrared light, as used with the video-rate laser-scanning confocal
microscope.
The main advantage of CM is that it can allow the skin to be evaluated in its
native state either in vivo, or when freshly biopsied (ex vivo) without the fixing,
sectioning and staining that is necessary for routine histology.
Confocal microscope imaging of normal skin in vivo give clear images of the
cellular layers of the epidermis and upper dermal region. Beyond the dermoepider-
mal junction, at a depth of 100–150 mm, blood flow in the capillary loops within
each dermal papilla can be evaluated at the highest resolution; erythrocytes, leuco-
cytes and platelets can be distinguished in relation to their relative sizes and shapes.
Further imaging at depths of 100–350 mm below the stratum corneum can show a
network of fibres and bundles in the papillary dermis and superficial reticular net-
work that represent the collagen network. Skin appendages such as sebaceous
glands, hair shafts and sweat-gland ducts can also be seen [64].
The quality of images obtained by CM can depend on the type of confocal micro-
scope used and the ability of the operator. Traditional confocal microscopes require
the use of fluorescent dyes in order to achieve adequate tissue contrast in creating a
clear image. For in vivo skin examination, some commercially available confocal
microscopes can achieve image contrast entirely through the detection of reflected
light from within the skin, whereas others require an intradermal injection of a
fluorescent contrast agent before different skin-cell layers can be clearly visualized .
The completely non-invasive nature and high-resolution capability of CM has
made it a useful instrument in skin research. Some skin conditions that have been
studied in vivo with CM and have been reported in the literature include solar kera-
toses, psoriasis, amelanotic melanoma and allergic contact dermatitis, where den-
dritic cells resembling activated Langerhans cells have been directly visualized [65,
66]. Confocal microscopy has been shown to characterize the pattern of neovascu-
larization and reinnervation in a model of human skin equivalent grafted in a pig
[67], confirming that angiogenesis occurs first and act as an influencing and guiding
factor on innervation in experimental wound healing [68].
Currently, confocal microscopes are costly instruments and, although available
commercially, their use is mainly confined to research. However, as the technology
of CM improves, and smaller, more affordable instruments become available, the
technique could have immense potential as a diagnostic tool in wound healing,
enabling clinicians to characterize wound parameters, image skin lesions and diag-
nose them without the need for biopsy, and to define the margins of skin lesions
prior to any intended excision [69].
Vascular Assessments
Typical venous and arterial ulcers may be easily diagnosed clinically. Venous
ulcers occur mostly around the skin over the ankle, on the medial or lateral side or,
Fig. 11.4 Typical venous leg

ulcer
occasionally, circumferentially. Venous ulcers tend to be oval in shape with sloping

edges and a hard base as shown in Fig. 11.4. Patients with venous ulcers may com-
plain of pain though not all ulcers are painful. Ischaemic ulcers may occur any-
where on the legs including the toes, fingers or even the nose tip! These ulcers are
shallow, punched out looking and painful and occur in over 3% of the over 65 s in
Northern Europe, USA and Australia. However, a mixed arterio-venous ulcer such
as shown in Fig. 11.5 presents a diagnostic difficulty and confirmatory vascular
assessments are needed.
In the case of an uncomplicated venous ulcer, it is important to exclude periph-
eral arterial disease and to demonstrate the presence of venous disease in order to
assign the patient an accepted treatment pathway as discussed by Davies and Poskett
in this book. When treating an ischaemic leg ulcer, having baseline data of the arte-
rial haemodynamic and structure are essential for management be it surgical or
medical.
Patients with diabetes mellitus get chronic wounds, the well known risk factors
being diabetes, neuropathy and peripheral vascular disease. The assessment of
Fig. 11.5 The figure presents

a lower extremity chronic
wound of mixed arterio-
venous aetiology. Note the
extensive skin pigmentation
and the heavy ‘crinkles’ due
to bandaging over an
oedematous calf
neuropathy and its role in the pathogenesis of diabetic foot disease are well described
by Edward Jude in this book. Peripheral arterial disease is a known risk factor for the
development of foot ulcers in the patient with diabetes. In patients with critical
ischaemia, treatment is based on intervention and surgical bypass for adequate reper-
fusion of distal extremities of the legs. Faglia [70] studied 564 consecutive patients
with diabetes of whom 413 (74.5%) received angioplasty, 114 (20.6)% received
bypass grafts and 27 (4.8%) were unfit for surgery and received merely medical
management. Of the 440 patients who were treated with revascularization, 347
required some form of amputation, only 35 healed with dressings. Faglia measured
transcutaneous oxygen tension TcPO2 after treatment and observed that a high TcPO2
(OR 0.8 95% CI 0.74–0.87) in other words, a functional microcirculation, must offer
a protective effect. There is an increasing prevalence of neuroischaemic ulcers
reported in Europe which reinforces the argument for good, reliable early diagnosis
on the arterial, venous and microvascular function over the skin of the foot [71].
Involvement of the Microcirculation in the Pathophysiology

of Venous and Diabetic Foot Ulcers
High unrelieved venous pressures on standing resulting from valve damage due to
deep vein thrombosis or congenital aplasia lead to
• Impaired microvascular function through engorgement, increased permeability
and leucocyte trapping in the skin around the ankles [72].
• Hard, chronically dry, leathery skin over the lower legs
• Pericapillary cuffs capable of trapping substances including growth factors [73,
74] and impeding nutrient diffusion. This condition is described as lipodermato-
sclerosis (LDS) and these factors put the skin at risk of chronic wounds. Good
treatment starts with a clear diagnosis as described in the sub section to follow.
Figure 11.6 is a graphical representation of the microcirculation.
Flow in the microvascular network is driven by the pressure gradient between an
artery and a vein; it is controlled by sympathetic outflow as well as hydrostatic pres-
sure. In a person lying supine, the pressure in the foot veins is near zero. On standing,
the pressure in the foot veins immediately increases to around 115 mmHg instanta-
neously (assume this person is 1.8 m tall) reducing the pressure gradient between
artery and vein creating the need for control. In response, precapillary sphincter
Function is controlled by
• Pressures (hydrostatic and oncotic)
• Nervous system
Arterial inflow Venous outflow
Exchange from artery to

vein through a network of
microvascular vessels
The hierarchy of the microvascular network runs from the artery to arteriole to meta arteriole to capillary to
venule to vein. There is a pressure gradient along this network - high at the ardterial end low at the venous
end - this drives blood flow through the capillary in a slow ‘push-pull’ or vasomotion. Vasomotion speeds
vary at different skin sites typically between 5-12 cycles per minute.
Fig. 11.6 An arteriole concept of the microcirculation

resistance increases causing capillary pressure and hence flow to decrease and then
normalise. This decrease in capillary flow (with a parallel increase in pre-capillary
sphincter resistance) may be easily demonstrated, is known as the arterio-venous
(AV) response. Tooke [75] and colleagues used that the laser Doppler flowmeter to
monitor the A-V response. The AV response is local, mediated neurally and sympa-
thetically controlled. It is absent in patients with diabetic neuropathy [76] and dimin-
ished in patients with venous disease [77] who are therefore prone to oedema
formation. An intact AV response offers an oedema protective effect.
Other changes in the microcirculation elegantly demonstrated by clinical research
through the 1990s decade are
• Increase in foot skin capillary membrane thickness [78]

• Decrease in microvascular flow to such challenges as thermal [79], and chemical
[80] (acetylcholine and sodium nitroprusside). Challenging the microcirculation
with acetylcholine and sodium nitroprusside evokes endothelium and non
endothelium depended responses.
Schramm working with Veves demonstrated that microvascular reactivity is

impaired in patients with diabetic neuropathy with and without foot ulcer [81].
This finding, also reported by other workers, raises questions as to whether the
microvascular impairment precedes the ulcer and how widely this is distributed
over the foot skin. Impaired microvascular reactivity has been reported in skin over
the forearm in children and adults. Khan [82] reported impaired microvascular
function is associated with increased cardiovascular risk and fat content in chil-
dren. Gates and Shore [83] argued that dysfunction in the microvascular network
may precede dysfunction in the macrovascular system. In its Guidelines for
Diabetic Foot Management the National Institute for Clinical Excellence (NICE)
recommended that “Further research is required to identify the appropriate level
and combination of risk factors at which patients should be categorised as at high
risk for ulceration….” (www.nice.org.uk) [84]. These arguments favour the assess-
ment of the microcirculation in the skin over the foot in diabetic foot disease and
this is discussed in a sub-section to follow.
Assessment of the Arterial System of the Lower Limbs
(a) Palpation (of the tibial, dorsalis pedis vessels).

(b) Measurement of ankle to brachial pressure index (ABPI) or toe blood pressure
index (TBI).
(c) Duplex ultrasound imaging and flow measurements in the arterial segments of
the limb.
(d) Contrast Angiography.
(e) Magnetic Resonance Imaging with enhancement to detect flow.
Ankle Brachial Pressure Index (ABPI) and Toe Blood Pressure

Index (TBI)
ABPI (referred to as ABI in US literature) is a non invasive measurement of systolic

pressure at the ankle (in the posterior tibial/peroneal/the dorsalis pedis arteries). The
technique is validated and widely available [85] using portable ultrasound (5–8 MHz)
and sphygmomanometer cuffs. The normal range of ABPI is 0.8–1.2. Falsely high ABPI
values higher than 1.2 may be detected in patients with diabetes on account intimal
medial calcification that makes arteries incompressible. This is commonly found in
patients with diabetes suffering from Mënckerg’s media sclerosis. ABPI l < 0.5 is consis-
tent with the presence of significant disease and indicates the need for further work up
and assessments using Duplex ultrasonography prior to clinical management.
The technique has good intra and inter-observer reliability, inter observer variability
is of the order of 15–20% and a difference of 0.15 is considered the lower threshold
of clinical significance [86]. The technique is recommended especially for popula-
tion studies. It is also recommended that the width of the cuff used is about 75% of
the circumference of the upper arm. Large cuffs should be used for obese patients.
When ABPI is measured in an outpatient environment (or in a patient’s home while
a District Nurse is doing a domiciliary visit for example), the patient should be per-
mitted some 10 min to relax and the test done with the patient lying supine. Ideally
ipsilateral arm systolic pressures should be used to derive the value of ABPI.
The advantages of ABPI measurements are ease of use as wells as to train.
However, it is difficult to measure ABPI on some patients with lower extremity
wounds: Patel [87] reported that it in some 15% of patients from Southampton (UK)
area it was not possible to measure this ABPI. The reasons for this vary from pain
perceived to shape of the legs to the presence of widespread ulceration, the most
common cause being pain. Some patients may not be able to bear the pain associ-
ated with cuff compression while others report that their legs are painful to touch. In
some, leg arteries are incompressible at pressures exceeding 200 mmHg. In all cases
the pragmatic next step is either to measure Toe Blood Pressure Index (TBI) and or
use the Duplex Ultrasound technique that offers diagnostic information just as non-
invasively. It is important to first discuss the toe blood pressure index (TBI).
There is an increasing trend to measure toe blood pressure index (TBI) which may be
derived in much the same way as is ABPI. Toe blood pressure is measured with a detect-
ing probe positioned on the toe and using a blood pressure cuff in the same way as for
ABPI. Transducers used to detect toe pressures may be optical systems or electrical
resistance strain gauges though the former are used in currently available commercial
systems. Optical transducers detect changes in blood volume using infra red sensors to
pulse and detect reflection from tissues or flow, using lasers and the Doppler technique.
The detection of change in blood volume in response to applied increase in external
pressure, is called plethysmography; since changes in blood volume follow changes
in arterial pressure, calibrated systems can reliably detect pressure induced changes in
blood volume and, in principle, measure heart rate. Optical reflection from tissues is
conveyed to the optical detector by changes in arterial pressure waves or pulses from
Fig. 11.7 The Moor Laser

Doppler Flowmeter (MBF3) a
(a) and Laser Doppler
Imaging Systems (b) are
shown
which, after detection and electronic filtration, instantaneous volume (or pressure)
changes may be read out. Laser detection of blood velocity in dermal tissues relies on
detecting and measuring from the phase change in blood cells in the path of the laser
beam. This technique based on the Doppler shift principle was first described by Stern
[19]; Nilsson successfully developed the first laser Doppler flowmeter [20] as also
described in the section “Skin assessments” (Figs. 11.7 and 11.8).
Fig. 11.8 The Perimed PIM3

system is shown
Williams [88] measured TBI in a modest number of patients with vascular disease
or diabetes and reported these measurements correlate with Duplex ultrasound findings
consistent with the presence of peripheral arterial disease, ABPI and transcutaneous
oxygen tension levels (TcPO2). It ranges from 0.8 to 0.9 in normals; values less than
0.5 are usually considered as evidence of the presence of peripheral arterial occlusive
disease (PAOD). TBI is only measureable in patients with toes, a limitation of the
technique. Nonetheless TBI is simple to use and to train other users (Fig. 11.9).
TBI is less widely available though it is as equally appealing as ABPI. A high
value of toe blood pressure derived this way in a patient with leg ulcers treated with
compression bandaging will offer the attending Tissue Viability nurse confidence
that there a prescribed compression garment is doing no harm. ABPI and TBI are
good screening measurements that triage patients for further vascular work up start-
ing with Duplex ultrasound.
The Pole Test
The Pole Test [89] is a painless, simple way of measuring ankle systolic pressure in
patients who are unable to tolerate cuff compression. In principle, the height above
the heart at which ankle blood flow is not detectable is a measure of the systolic
pressure. In practise, the patient lies supine. Systolic pressure is located using a
portable ultrasound Doppler probe and contact gel. The patient’s ankle is then raised
against a vertically held graduated pole and the height at which the systolic sound
Degree of occlusion Approximate ABPI Waveform
Normal > 0.8
Triphasic
0-24% diameter reduction < 0.8
25-49% diameter reduction ∼ 0.6
Biphasic
> 50% diameter reduction < 0.5 Monophasic
Fig. 11.9 This figure shows changing Doppler frequency shift with corresponding ABPI (Courtesy
of Sage Publications, USA)
disappears is noted. The pole is graduated in divisions 13.6 cm wide since the den-
sity of mercury is 13 g/cm3 compared to that of blood which is 1 g/cm3. This is an
easy bedside test though not suitable for the very tall or those with very long legs.
In any case, a patient’s foot should not be raised over 50–60 cm.
Duplex Ultrasound
Duplex ultrasound, a technique that has been available for some 25 years permits blood
vessels to be imaged and haemodynamic data to be generated using the Doppler
technique. It is an incredible boon to the diagnosis and management of vascular dis-

eases. It is widely described in several chapters of this book – an indication of the
essential part played by this technique in wound management. Duplex imaging gives
accurate and reliable data of flow in the arteries and veins [90]. In patients with lower
extremity wounds, a full arterial scan should extend from the sapheno-femoral junction
to the tibial arteries with the patient lying supine, legs adducted and hips externally
rotated by 10°. Electronic images may be stored on computer hard drives and reprinted
on paper, film or viewed used the latest information transfer technology maintaining
patient confidentiality at all times. A full arterial scan takes up to 20 min.
Duplex angiograms together with images of the arteries permit visualisation and
localisation of obstructive or stenotic lesions in the arterial tree and have been dem-
onstrated to correlate very well with contrast angiograms.
The Duplex ultrasound technique uses the Doppler principle [91] to detect
change in the velocity of red cells given knowledge of the angle at which the
ultrasound beam interrogates blood cells within an artery, the frequency of
the transducer and velocity of ultrasound in the medium which is a constant. The
derived velocity data vary over time (since arterial blood flow is pulsatile) and
have been correlated with arteriograms. Figure 11.9 is the graphical representa-
tion of changes in blood flow and ABPI [92]. Duplex ultrasound imagers are now
available as lightweight portable machines that are also relatively inexpensive
making them attractive options for wound healing clinics as shown in Fig. 11.10.
Duplex Testing of Venous Function
Duplex ultrasound may also be used to detect deep venous thrombosis accurately
using the compression test [93] and to detect venous incompetence. Venous reflux
Fig. 11.10 This picture

shows a Sonosite™
(Sonosite, USA) portable
Duplex ultrasound imager.
Also shown in the picture is a
linear 5–8 MHz transducer
is measured using the Doppler facility to detect incompetence; reflux lasting longer
than 500 ms is usually consistent with the presence of incompetence [94]. Venous
assessment not only permits patients with lower extremity wounds to be treated
with confidence but also offers guidance in the treatment of the superficial veins.
Venous Haemodynamics
In order to measure calf pump function, haemodynamic assessments may be

achieved either using foot flexion exercises (to measure time taken for skin blood
flow to return to baseline a reliable reflection of the competence of connections
between the deep and superficial venous systems), or air displacement techniques to
measure maximum venous outflow. Both these techniques are correctly termed
plethysmographic.
Angiography and MR Imaging
Angiograms using contrast dyes yield images of the best quality permitting sur-
geons to make final decisions on clinical management. In the same way, magnetic
resonance imaging (MRI) using gadolinium to enhance the contrast has the poten-
tial to detect small and large vessels as well the ability of the perfused tissue to
extract oxygen. In wound healing, current use of MRI is limited to the area of imag-
ing foot deformities in the patient with diabetes mellitus.
Evaluating the Microcirculation
In homeostasis, the microcirculation supplies nutrition to the tissues. The principal

components of the microcirculation are blood flow and oxygen available at the right
time to sustain healing. In the wound healing laboratory, assessing functionality of
the microcirculation is important especially in patients with diabetic foot disease. In
current practice, the most widely used techniques are the laser Doppler technique to
measure changes in blood flow and the modified Clark sensor to measure transcuta-
neous oxygen previously described in the section “Skin assessments”.
Transcutaneous Oxygen Tension (TcPO2) is measured using small, flat surface
sensors that heat a sample volume of tissue to preset levels. The sensor is an elec-
trode system that is sealed in a solution of aqueous potassium hydroxide; the cath-
ode is held at a negative potential to the anode which leads to a production of
electrons so long as there is a supply of oxygen that diffuses from the tissue through
a membrane covering the probe. The electronic current generated is linearly related
Fig. 11.11 This is a typical duplex ultrasound image of venous reflux
to the partial pressure of oxygen driving the system when the appropriate biasing
conditions are maintained. This technique is reliable and reproducible with a
coefficient of variation of 7% in the skin over the lower legs and may be used to
study populations or to follow changes in the same patient [95]. TcPO2 may be
measured at 43oC or 44oC; the process may take up to 30 min. On no account should
the heated sensor be left on site for longer than 2 h. Figure 11.11 shows a transcuta-
neous oxygen measurement system.
TcPO2 should be measured in a ‘side’ room rather than an open ward, its use has
been well described. The system is calibrated in air. Sensors should be placed on
cleaned, dry skin and held in situ with double sided adhesive discs for 15 min for a
baseline level to be achieved. Ideally, control values should be derived on the chest
along the mid clavicular line in the intercostal space between the fourth and fifth
ribs. In practice this can be difficult as sick patients may be restive or connected to
other monitors. There is the matter of patient choice too. A pragmatic approach is to
derive control values from the skin over the right antecubittal fossa. Skin oxygen
tension may be recorded as a ratio of the control value or in absolute terms.
TcPO2 is well correlated to arterial PAO2 when measured on the chest wall or
forehead on neonates and young adults 1 and 0.79 respectively. In adults, this cor-
relation is less good since TcPO2 is not only a function of blood flow but also skin
thickness and oxygen consumed [99]. On adults therefore, TcPO2 measured at
Fig. 11.12 (a) This figure shows the TINA transcutaneous oxygen system with electrodes (b)
shows the TINA transcutaneous oxygen sensor. (c) This is a picture of a subject with transcutane-
ous oxygen sensors in situ
43/44°C, is an indication of the vasodilator status of the tissue under study and not
a measure of arterial oxygen supply. In patients with peripheral arterial occlusive
disease, there is a gradient of TcPO2 – high at the heart and low on the dorsum of the
foot. Hence in the protocol used in one of the authors’ group (RM), sensors are
positioned along the vascular plane as shown in Fig. 11.12c.
The best application of this technique is to determine levels of amputation [96]
especially in the diabetic foot. TcPO2 levels above 30 mmHg are considered safe;
levels below 20 mmHg are indicate that tissue is unable sustain nutrition. In
analysing these values, it is important to bear in mind that TcPO2 is affected by local
infection and oedema. TcPO2 measured at the edge of infected diabetic wounds has
increased as the wound infection responded to treatment suggesting a decreasing
level of oxygen consumption [97]. In critically ischaemic skin low TcPO2 (up to
10 mmHg) has been demonstrated at the edge of ischaemic ulcers too though
according to some reports, when this value has increased with inhaled oxygen, it
was associated with responsive ulcers. Low TcPO2 (2–10 mmHg) was demonstrated
at the edge of venous ulcers with poor positive predictive value though amputation
was never a treatment choice.
Efforts to combine LDF with TcPO2 measurements to predict amputation levels
did not significantly improve matters [98]. Spence [95] and colleagues, after detecting
the washout of 4-iodoantipyrine with a Gamma camera had significant levels of
detecting the success of below knee amputations. This technical success must be
hailed though for acceptance as a clinical application needs the team to have access to
high resolution imaging and facilities for isotope management. What is the future?
Optical techniques combining wavelengths to measure blood volume and oxy-
gen saturation are available and discussed in Chap. 12. Near infra red spectroscopy
with the potential to image oxygen available for healing, offer a beacon of promise
to measure and make reliable judgements of tissue viability.
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Chapter 12
Wound Tissue Oximetry: A Cornerstone
in Wound Care
Jaideep Banerjee and Chandan K. Sen
Keywords Hypoxia • Wound healing • TCOM • EPR • Laser Doppler flowmetry

microRNA • NIR spectroscopy
Introduction: The Biological Significance

of Hypoxia in Wound Healing
Wound healing represents an innate response to tissue injury. Broadly, wound heal-
ing comprises a wide range of mechanisms that occur in an orchestrated fashion.
Any disruption in the systems biology of cutaneous wound healing is accommo-
dated for by compensations which may compromise the quality of healing but none-
theless is effective in closing the defect caused by injury. Disruptions that cannot be
managed by such compensatory mechanisms result in a chronic wound, one which
loses its ability to close and therefore could be viewed as a fatal flaw. One such fatal
J. Banerjee
Department of Surgery, The Ohio State University Wexner Medical Center,
Columbus, OH 43210, USA
e-mail: jaideep.banerjee@osumc.edu
C.K. Sen, Ph.D. (*)
Department of Surgery, The Ohio State University Wexner Medical Center,
Columbus, OH 43210, USA
Department of Surgery, 512, Davis Heart and Lung Research Institute,
Ohio State University Medical Center,
473 W 12th Ave, Columbus, OH 43210, USA
e-mail: chandan.sen@osumc.edu

226 J. Banerjee and C.K. Sen
flaw, especially in lower limbs with peripheral vascular disease or wounds with
acute infection, is represented by inadequate supply of oxygen to the injured tissue.
In wounds, the partial pressure of tissue oxygen (pO2) may vary from almost zero to
100 mmHg; it is speculated that the central region of the wound is most hypoxic i.e.
a gradient exists from the periphery to the centre of a wound. Besides playing a role
in healing as a nutrient, O2 is converted to reactive oxygen species (ROS) in the
wound microenvironment which contribute as cellular messengers to promote pro-
cesses that support wound healing, such as cytokine action, angiogenesis, cell motil-
ity, and extracellular matrix formation. Nicotinamide adenine dinucleotide phosphate
oxidase (NADPH oxidase), the enzyme that generates reactive oxygen species
(ROS) has a Km (Michaelis constant) around 45–80 mmHg. If the wound pO2 drops
below 20 mmHg, the NADPH oxidase ceases to function. This prevents the essen-
tial signaling pathways from functioning and may at least in part be responsible for
the non-healing nature of the ischemic wounds.
Angiogenesis is a vital event in the wound healing response. Hypoxia results in
induction of various angiogenic factors like HIF (Hypoxia Inducible factor) and
VEGF. While hypoxia can and does initiate neovascularization, angiogenesis is
overtly observed only in the acute wounds. In the relatively hypoxic environment of
a chronic wounds cells are often deprived of a crucial source of energy which ren-
ders it difficult to sustain angiogenesis. Hypoxia has been reported to induce non-
coding genes like microRNAs along with the classically defined pathways involving
coding genes. HIF dependent hypoxia-inducible microRNA miR-210 has been
reported to impair wound re-epithelialization, a key aspect of overall wound closure
[1]. Hypoxia may also impair differentiation of fibroblasts to myofibroblasts, cells
responsible for wound contraction. Collagen deposition is another fundamental step
in wound healing which may be compromised under hypoxia. There are several
post-translational steps in collagen synthesis that are O2 dependent. The enzymes
prolyl hydroxylase, lysyl hydroxylase and lysyl oxidase all required during collagen
synthesis, require molecular O2 as a cofactor.
Hypoxic wounds are also highly susceptible to infection which impedes the heal-
ing of the wound. Therefore generally, moderate hypoxia can support adaptation
and survival, while chronic hypoxia leads to deterioration of the wound. Vascular
limitations are further complicated by coincident conditions like infection and
hypothermia and thus lead to poor healing outcomes. For a detailed review on the
oxygen cost of cutaneous healing the reader is referred to this reference [2].
Depending on the wound conditions, correction of tissue hypoxia alone may not be
sufficient to restore healing. However, it is understood and accepted inadequate sup-
plies of oxygen alone will stifle the healing process. Therefore, wound tissue oxim-
etry is a key parameter to manage a problem wound. While the science of tissue
oximetry has substantially advanced in recent years, the technology available to
wound clinicians represents the state of the art of the 1970s. Transcutaneous oxygen
measurement (TCOM or TcPO2) quantitates tissue oxygenation in the peripheral
skin surrounding the wound though not in the wound bed. Furthermore, TCOM
relies on electrochemical detection of oxygen that diffuses out of skin at rest and
when skin is warmed. In this form of measurement, the electrode consumes oxygen
12 Wound Tissue Oximetry: A Cornerstone in Wound Care 227
which limits the accuracy of the technique to detect very low levels of tissue oxy-
gen. Thus, in the absence of any other clinically applicable technique, TCOM or
TcPO2 has a use. However, there have been advances in tissue oximetry techniques.
In this chapter we outline the key significance of tissue oxygenation in wound heal-
ing and address the current technology platforms capable of addressing tissue oxy-
genation status.
The Oxygen Cost of Wound Healing
The major factors that may contribute to wound tissue hypoxia are [3]:
(i) Peripheral Vascular Diseases
(ii) Mitochondrial Respiration: Increased energy demand of the healing tissue
leads to a hypermetabolic state wherein additional energy is generated from
oxidative metabolism increasing the O2 demand of the healing tissue. ATP thus
generated powers tissue repair.
(iii) Respiratory Burst In Response to Infection: The open cutaneous wound bed is
often a site of infection. During phagocytosis of these microbial intruders,
phagocytes of the innate immune system respond to foreign products such as
bacterial lipopolysaccharides by increasing their O2 consumption through the
•-
inducible activity of NADPH-oxidase (NOX) that generates O 2 and H2O2.
These oxygen-derived metabolites give rise to yet other ROS (reactive oxygen
species) that are potently anti-microbial but which may also cause damage by
destroying surrounding tissue and cells. Approximately 98% of the O2 con-
sumed by wound neutrophils is utilized for respiratory burst. Thus, infection is
an “oxygen sink”: as the level of infection increases, so the tissue becomes
more hypoxic. Contrary wise, once infection is managed tissue oxygen is
spared and therefore available for other oxygen-driven vital functions.
(iv) Respiratory Burst in Response to Inflammation: Acute inflammation follow-
ing injury results in ROS production by phagocytic NADPH oxidases which
plays a key role in numerous processes such as cell death, phagocytosis and
autophagy.
(v) Redox Signaling: Numerous aspects of healing ranging from hemostasis, cell
proliferation, migration and tissue remodeling are supported by redox signaling
where low-level ROS act as messenger molecules. These redox signals are gener-
ated at the cost of tissue O2. Thus, extreme tissue hypoxia will limit redox signal-
ing and disable the function of several growth factors (e.g. PDGF, VEGF, KGF,
IGF, and TGFb) and numerous molecular mechanisms (e.g. leukocyte recruit-
ment, cell motility, integrin function) which rely on redox signaling [4–6].
(vi) Nitric Oxide (NO): Known for its effects on the vascular system, NO is
another major signaling messenger that is important in wound healing.
Generation of NO consumes oxygen. Thus, under extreme hypoxic condi-
tions as often noted in clinical wound tissue, NO cannot be generated by NO
synthases even when there is an abundance of such substrate as L-Arginine.

Under hypoxic conditions, NO inhibits mitochondrial respiration and pre-
vents mitochondria from depleting local oxygen, enabling the continued
hydroxylation and degradation of HIF-1a, thus leading to a situation in which
the cell may fail to register hypoxia [3, 7].
(vii) Systemic Limitations: Factors such as arterial hypoxia (e.g. pulmonary fibrosis
or pneumonia, sympathetic response to pain, hypothermia, anaemia caused by
major blood loss, cyanotic heart disease, high altitude) may also contribute to
wound hypoxia. Chronic intermittent hypoxia (CIH) may complicate wound
healing. CIH activates ROS dependent responses. In one study, 43% of patients
with chronic non-healing wound were deemed at high risk for obstructive
sleep apnea (OSA) which is characterized by CIH [3]. A direct relationship
between intermittent hypoxia associated with OSA and wound healing requires
further investigation.
As we discuss about how extreme hypoxia may compromise wound healing, it is
important to recognize that excessive oxygen could have adverse effects as well [3]
thus elevating the significance of wound tissue oximetry in clinical wound care. In a
wound with pockets of hypoxia, it is required to re-establish normoxia in the worst
affected hypoxic pockets without exposing other parts of the wound tissue to such
high levels of pO2 that would antagonize healing by hyperoxia-induced growth arrest
or simply overt oxygen toxicity. Among the factors that may oppose wound healing,
extreme hyperoxia causes growth arrest [8–11] and cell death by a mitochondria-
dependent apoptosis pathway [12–14] and may result in oxidative stress [15, 16].
Indirect Measurement of Hypoxia: Oxygen Saturation in Blood
Indicator Clearance Methods
Historically, measurements of perfusion were used to infer levels of tissue oxygen

saturation. Radio isotope clearance techniques or radioactive tracer methods were
used to measure blood flow and technetium labelled I131 or I125 4-iodoantipyrine were
commonly used for clearance measurements to measure the viability of skin prior to
amputation [17]. Holstein developed another technique which was dependent on
injecting a radio labelled dye into the site (or an organ when an estimate of regional
perfusion is sought) and detecting its clearance using a suitably sensitive transducer
that may be a portable scintillation counter or a gamma camera [18]. However, the
main disadvantages of these methods were that they were invasive, radioactive, cum-
bersome and not repeatable. Non invasive methods to estimate blood flow/perfusion
were also developed during this time using thermistors (detect heat clearance) and
photoplethysmography (PPG). There are optical and mechanical methods of PPG all
with uses as well as limitations. PPG measures changes in blood volume caused by
(i) arterial pulsatility and (ii) venous flow. Commercially available systems permit
either parameter to be selected. The simplicity and non invasiveness of this technique
led to its widespread availability though users were aware of its limitations which are
(a) difficult to calibrate (b) inability to identify the sample volume generating the
signal. With the advancement in technologies, indicator clearance methods have
gradually been phased out from clinical settings [19].
Laser Doppler Flowmetry
Laser Doppler Flowmetry (LDF) is a non-invasive, reliable and easy method for
measuring blood flow. The possibility of using LDF for clinical purposes of measur-
ing blood flow was first demonstrated by Riva in 1972 [20]. LDF allows for a real-
time and continuous monitoring suitable for cutaneous microcirculation
measurements and is based on the principle of detecting a change in the phase of
laser light incident on the microcirculation following the principle of Doppler shift.
As discussed in another chapter in this book, the LDF has a major role in the detec-
tion of depth of burns based on its reliability to detect the depth of dermal wounds
compared to tissue histopathology and clinical diagnosis [21]. The laser Doppler
imager consists of a low energy laser (<2 mW Helium Neon or a semiconductor
laser in the infra red region in more modern machines), a fiber optic probe, and a
photo detector with a signal processing unit. The fraction of back-scattered light
that is Doppler-shifted is signal processed to analyze the change in frequency, which
is related to blood cell velocity and power, and corresponds to the number of mov-
ing blood cells (CMBC) and thus, blood flow. Spatial resolution of the optical probe
is about 1 mm3 and thus it is important to recognize that LDF measurements address
only changes in skin microcirculation.
There have been several approaches to the development of laser blood flow
imagers but each have their own merits and specific limitations. The Laser
Doppler imaging (LDI) systems (scanning imagers) suffer from low imaging
speed. Full-field imagers: the laser speckle imagers (LSI) do not distinguish
between changes in blood concentration or velocity and cover only a small veloc-
ity range. To overcome these problems, a new imaging system using the CMOS
image sensor technology has been developed which permits the acquisition of
the Doppler signal at many spots simultaneously at a sub-frame rate of several
kHz. This minimizes the measurement time and also provides a better signal-to-
noise ratio [22]. A drawback of the Laser Doppler techniques is the sensitivity of
the probe to artifacts though this may be overcome by careful methodology and
data analysis. Transmission of the optical beam is impaired by particulate matter
and color (i.e., yellow coloured slough on a wound bed will transmit light differ-
ently than a healthy reddish coloured wound bed). However, since the introduc-
tion of high resolution glass optical fibres; this limitation has been greatly reduced
(Fig. 12.1a).
Near Infrared Spectroscopy and Hyperspectral Imaging
Near-infrared spectroscopy measures the percentage of hemoglobin oxygen satu-

ration in the microcirculation. Near infrared spectroscopy (NIRS) is based on the
differences in absorption in the spectra of oxygen carriers, such as hemoglobin,
myoglobin, and cytochrome a,a3 (cyta,a3) [21]. It is a noninvasive technique that
relies on the differences in absorption that occur at different wavelengths of light.
Fig. 12.1 (a) Laser

Doppler image of a mouse.
Left panel shows
photographic image and
right panel shows blood flow
imaged using a laser Doppler
machine. (b) Schematic of
probe used in Near-infra
Red spectroscopy. The
amplitude and phase shift of 0 500 1000 1500 2000 2500
light is measured as it
traverses through tissue. It
then uses a diffusion-based b
Optical
model to calculate the
fibres
optical absorption coefficient
and the reduced scattering
coefficient. A fiber optic Teflon
probe delivers light through probe
a single optical fiber and
collects light through four
optical fibers spaced at
distances of 4–16 mm from Average
the source fiber. The device penetration
generally uses laser diodes in depth =
the wavelength range of 3-5 mm
685–830 nm (Modified from
[23]). (c) Representative
absorption coefficient
spectra of oxygenated and
non-oxygenated hemoglobin Tissue
Fig. 12.1 (continued) c
Absorption coefficient Hb
Hbo2
400 500 600 700 800 900

Wavelength (nm)
As figuratively shown in Fig. 12.1 b, c at 805 nm the absorption coefficients (and

therefore the reflection) of oxygenated and reduced haemoglobin are equal. By
comparison at 633 nm, these coefficients are maximally different. NIRS devices
are based on the principle that near infrared light passes through the skin and the
subcutaneous tissue into the muscle and is absorbed by heme proteins, most of
which is hemoglobin. The average depth of penetration is about 3–4 cm below
the skin [23]. Light in the near-infrared range (600–1,000 nm) is capable of pen-
etrating through skin, soft tissue, and bone and is absorbed based on the relative
concentrations of oxygenated and deoxygenated hemoglobin in the microcircu-
lation. Since large vessels or hematomas absorb the light completely, the only
light collected by the sensors is light that is capable of passing through the micro-
circulation. By using two separate wavelengths of near infrared light (805 and
633 nm or more in sophisticated machines), and the Beer-Lambert law and the
concentration of both oxygenated and deoxygenated hemoglobin may be esti-
mated. NIRS systems may work in transmission or reflectance modes, the former
being best useful to study thin sample such as the ear lobe. Reflectance mode
systems by using relatively powerful incident beams of white light find a use in
studying tissue oxygen saturation in such sites as the brain and find use in neo-
natal monitoring.
Ischemia lowers the oxyhemoglobin/deoxyhemoglobin ratio which can be quan-
titatively and continuously measured by NIRS. The main advantage of NIRS over
Doppler blood pressure measurements and laser Doppler flowmetry is that NIRS
measurements enable direct and continuous assessment of tissue oxygen saturation.

Near-infrared spectroscopy has been studied, validated, and approved by the U.S.
Food and Drug Administration in the anesthesia setting to monitor cerebral oxygen-
ation (Fig. 12.1b, c).
Clinical Applications
NIRS provides a noninvasive continuous assessment of brain oxygenation which

can be made available at the bedside. Simultaneous measurements over multiple
areas give a global assessment. (NIR) spectroscopy has been demonstrated to
predict wound healing outcomes in human patients suffering from diabetic foot
ulcers [23]. Healed wounds showed a consistent reduction of hemoglobin con-
centration several weeks before closure that approached control site values. In
non-healing wounds, the hemoglobin concentration remained elevated. NIR
results correlated (n=16) well with wound size measurement and the degree of
wound contraction [23] and so NIR spectroscopy can determine wound healing
outcomes earlier than that visibly assessed by current clinical approaches. NIRS
has also been used to reliably differentiate superficial from full thickness burns
in human subjects. The full capacity of NIRS in the diagnosis of burn injury,
however, has not been fully elucidated.
Limitations of NIRS
The accuracy of NIRS can be reduced by tissue swelling and the presence of
extravascular blood collections in tissue compartments from theoretical consid-
erations. NIRS has also been reported to fail to detect some permanent ischemic
insults. With respect to neuro-critical care, NIRS signals may be adversely
affected by the skull thickness, cerebrospinal fluid, myelin sheaths or when there
is pre-existing brain injury. Also, skin pigmentation has an effect upon NIR spec-
troscopy values. Decrease in near-infrared spectroscopy values in darker pig-
mented subjects likely occur because skin pigments absorb more light. However,
advances in technology over time, is expected to overcome some of these techni-
cal problems.
Hyperspectral technology (HT) also characterizes tissue ischemia by moni-
toring the tissue oxyhemoglobin and deoxyhemoglobin ratio noninvasively
[1, 24]. It provides a spatial distribution of oxygen saturation where the multi-
dimensional (spatial & spectral) data is represented in what is called a “hyper-
cube”. The spectrum of reflected light is acquired for each pixel in a quadrant
and each such spectrum is subjected to standard analysis. From this an oxygen-
saturation map of the tissue is created based on the chemistry of the region of
interest.
Molecular Reporting of Tissue Hypoxia
The quantitative assessment of tissue oxygenation is objective but not a direct expres-
sion of its biological significance. For example what is the level of oxygenation that
may be sensed as hypoxia by wound tissue? To answer this question, a molecular
approach is advocated. Hypoxia is manifested by the stabilization of a protein called
hypoxia inducible factor (HIF-1). Under normoxic conditions, this protein is hydrox-
ylated by oxygen and then degraded. However under hypoxic conditions, the hydrox-
ylation process is limited disabling subsequent processes leading to degradation of
HIF-1. Thus in hypoxic conditions, HIF-1 is detected in tissues. It is very important
to recognize here that a hypoxic live tissue will show high HIF-1 levels. However, at
a later phase when the hypoxic tissue is dying, global protein expression is shutdown
and therefore HIF-1 levels will go down. This occurs not because HIF-1 was hydrox-
ylated and degraded but because it was simply produced in dying tissue. Stabilized
HIF-1 binds to a hypoxia regulatory elements (HRE) enhancing the expression of
hypoxia-inducible genes. This is an adaptive response and works well when hypoxia
is sub-extreme allowing for some oxygen to support the rescue process. In overtly
infected open wounds, however, tissue may be borderline anoxic. In such cases,
HIF-1 is unable to rescue as there is not enough oxygen to pay for the rescue. If
hypoxia-induced HIF-1 was sufficient to induce rescue, ischemic limbs should be
healing much faster. This simply does not happen, leading to the realization that
while HIF-driven adaptive processes may be helpful in other scenarios, in near-
anoxic wounds HIF-driven adaptations are not useful. This supported by observa-
tions demonstrating that loss of HIF in myeloid cells improve healing [25]. Also, we
have recently reported a novel mechanism by which HIF can induce the non-coding
gene miR-210 which impairs wound closure [1]. There is evidence in experimental
mouse model wounds where HIF apparently is helpful [26] but this is only evident in
experimental wounds that are not ischemic and have residual oxygen to benefit from
the adaptive signals elicited by HIF.
Now, let us get back to detecting HIF stabilization in vivo. A HRE-luciferase
reporter construct [1, 27] is delivered to open wounds using standard gene therapy
techniques. HIF-stabilization results in increased luciferase expression and can be
imaged using an in vivo imaging system (IVIS) imaging system. The system fea-
tures a 25 mm (1.0 in.) square back-thinned, back-illuminated CCD (charged couple
device) camera, which is cryogenically cooled to −105°C via a closed cycle refrig-
eration system (without liquid nitrogen) to minimize electronic background and
maximize sensitivity. The CCD camera is designed for high-efficiency photon
detection, particularly in the red region of the spectrum. It is sensitive to very small
signals i.e. numbers of photons and operates as if a common camera. The electronic
readout is optimized so that the data gathered to create the real-time in vivo images
have subdued noise. This approach not only enables visualization of the biological
response to hypoxia but it also allows for repetitive measurements (Fig. 12.2).
a b
HIF-1 transactivation
HRE up regulation
Inject intra-dermally
Ad-HRE-Iuciferase
More Luciferase
Luciferin
c
Luminescence
pAd-5HRE-Luc
generation of
5-HRE-Luc Non-ischemic ischemic
adenoviral particles
Fig. 12.2 (a) Principle underlying hypoxia imaging using the in-vivo imaging system. (b)
Xenogen IVIS instrumentation setup. (c) Representative image showing increased hypoxia mani-
fested by increased HIF transactivation in an ischemic wound [1]
Direct Measurements of Tissue Oxygen Tension
Transcutaneous Gas Measurements
Clinically, viability of the skin is assessed based on its color, capillary return, and
temperature. By raising or lower a limb, the effect of blood pressure may also be
assessed. There are many situations, however, where standard clinical examination
of the skin is inadequate and a more objective measurement such as transcutaneous
oxygen measurement is useful [28, 29]. Transcutaneous oxygen measurement
(TCOM) represents the current standard in clinical wound pO2 determination.
Surgeons use TCOM to guide their treatment planning and to determine limb ampu-
tation levels. The sensor system uses a modified Clark’s polarographic electrode, a
technology that originated in 1953 [30]. The current generated is linearly propor-
tional to the number of oxygen molecules reaching the electrode when the cathode
is biased negatively with respect to the anode in the band (−200 to 600 mV) [21].
Transcutaneous oxygen measurements have wide clinical applications. It has
been reported that amputation decisions can be made on the basis of measuring the
transcutaneous oxygen pressure and higher transcutaneous oxygen pressures were
associated with above-knee amputations and lower levels with below-knee and
more distal amputations [31]. High transcutaneous oxygen pressure (TcPO2) has
been shown to predict healing in diabetic patients. A peri wound TcPO2 above
35–40 mmHg is predictive of healing while a value lower than 20–25 mmHg pre-
dicts a high chance of amputation [32–40]. In non-diabetic patients, these critical
values are 30 mm and 10 mmHg respectively [41]. For critical limb ischemia, among
other tests, TcPO2 critical value lower than 30 mmHg is an important diagnostic
parameter [42].
However, the method has several limitations [21]
1. Differences in skin oxygen permeability (localization, thickness) and oxygen
consumption influence TcPO2 measurements. so not best suited for hypoxic
conditions
2. The site must be warmed which modifies skin properties by inducing vasodila-
tion and increasing oxygen permeability, which may vary from subject to sub-
ject, thus producing false differences in TcPO2 readings.
3. It estimates TcPO2 at wound perimeters it provides no information about the
wound bed.
4. Transcutaneous carbon dioxide pressure measurements (TcPCO2) have also been
used in clinical practice. However, due to the greater skin diffusion capability of
CO2 compared to O2, alterations in TcPCO2 appear later than in TcPO2 [43].
Typically, TcPCO2 values diffusing out of healthy skin are around 2–3 mmHg
which mitigates its sensitivity. TcPO2 is a reliable indicator of the vasodilatory
capacity of skin.
Optical Oximetry
This technique relies on a new generation fluorescence quenching O2 sensor

(OxyLite, Oxford Optronix) which use fibre optics to continuously and quantita-
tively monitor regional pO2 in tissue. In brief, short pulses of blue LED light are
transmitted along a fiber optic probe to excite a ruthenium-based fluorophore situ-
ated at the probe tip. The resulting emission of fluorescent light, quenched by the
presence of O2 molecules, travels back up the fiber and is detected by the instru-
ment. The lifetime of the emitted fluorescence is inversely proportional (non-lin-
ear) to the concentration of dissolved O2, and is accepted as an absolute value for
pO2 expressed in mmHg or kPa. Since the measurement is based on fluorescence
lifetime rather than fluorescent intensity it is much less prone to artifacts (e.g.
because of variation in the intensity of the light source, ambient lighting, or photo-
bleaching) [44].
Electron Paramagnetic Resonance (EPR) Spectroscopy

and Imaging
Over the last two decades the EPR imaging (EPRI) has evolved into an important
tool for imaging free radicals and paramagnetic species in many branches of science
[45] and was reported by Berliner in Science for biological applications [46, 47].
Oxygen is a paramagnetic molecule. It can be detected and quantified using the EPR
technique which enables visualization of the distribution of electron spins in tissues.
The development of low frequency EPR instrumentation at L-band, 1–2 GHz, or
lower frequencies, and lumped circuit resonators has made it possible to perform
EPR measurements on biological samples.
Basic Principles
EPR oximetry (measurement of oxygen concentration by EPR spectroscopy) is

based on the paramagnetic characteristics of molecular oxygen. Oxygen has two
unpaired electrons in its ground state and undergoes spin exchange interaction with
paramagnetic EPR spin probe. The presence of oxygen influences both, spin–lattice
and spin–spin relaxation times (T1 and T2) of the paramagnetic probe. Increased
spin-spin relaxation rate results in increased line-broadening. According to the
Smoluchowsky theory, the oxygen-broadened line-width is directly proportional to
oxygen concentration (Fig. 12.3). Therefore, an increase in oxygen concentration
increases the EPR spectra line-width.
In general, paniculate probes such as lithium phthalocyanine [48, 49] and syn-
thetic char [50] are suitable for measurements of oxygen partial pressure, while
soluble probes such as nitroxyls measure dissolved oxygen concentration. The
advantages in using the particulate probe are: (i) provides higher resolution in the
range of 0.1 Torr (mmHg), (ii) suitable for repeated measurements in vivo without
reintroduction of the probe into the tissue, (iii) enables non-invasive measurement,
(iv) localized measurements are possible because data are collected from a single
voxel/region containing the oxygen-sensing particle, (v) insolubility in aqueous sol-
vents anchoring at a specific location (can be mapped by MRI) in the tissue, (vi)
inert to biological oxido-reductants, pH, temperature etc., (vii) nontoxic, (viii) high
temporal resolution, usually less than a sec and (ix) responds to partial pressure of
oxygen (pO2), rather than concentration of oxygen, which may be quite heteroge-
neous in cellular/tissue environments and hence calibration can be difficult.
One of the major foci of EPR imaging has been to map the spatial distribution of
dissolved oxygen in tissue [51] and methods have been developed to produce image
contrast based on oxygen-induced line-broadening. Subtraction techniques involve
acquiring images with and without oxygen perfusion [52]. Another approach has
been to investigate the oxygen-dependent metabolism of nitroxyls that is indicative
of differences in oxygen concentration [52].
A conceptually identical technique termed as spectral-spatial imaging which
includes a spectral dimension in addition to the normal spatial information obtained
from the EPRI. The potential application of the spectral-spatial technique has been
recognized in performing EPRI oximetry on asymmetric biological samples. This
four-dimensional (4D) EPRI, however has a disadvantage in that it places severe
constraints on the design and power of field gradients, data acquisition time, and
sensitivity of the probe [53].
Fig. 12.3 (a) Basic block a

diagram of the EPRI
instrumentation [57]. (b) The
LiNc-BuO EPR spectra at Magnet power Field controller
room temperature. The supply
peak-to-peak width of the Signal channel
spectrum is highly dependent
Microwave Microwave counter
on the oxygen concentration
bridge
of environment. (c)
Relationship of line-width in
the EPR spectrum with pO2 Programmabel power
Modulation coil supplies
GIPB BUS
Resonator
Magnet
Magnet
computer
Personal
38 25 38
mm mm mm
Gradients Gradients
b
0% (0
mm
Hg)
20.9%
(159
mm
Hg)
c
Line width (mG)
pO2 (mmHg)
Table 12.1 TCOM versus EPR oximetry system

TCOM EPR-based magnetic oximetry
Clarke-electrode technology consumes O2 as Measurement does not consume O2; highly
it measures; not best suited for hypoxic sensitive for assay of low pO2
conditions
Estimates pO2 at wound perimeter on Provides information of the actual wound site;
vascularized skin; no wound-bed information; direct
indirect
Point measurement only Point measurement with imaging capability
Applicable to pO2 measurement only Platform may be extended to measure redox,
free radicals as well as NO
Has limited prognostic value, therefore Has prognostic, diagnostic and therapeutic
insurance reimbursed; long clinical history; value; bulky but can be designed to be portable
small size
Cannot be used to monitor or determine Can be used to monitor or determine response
response to treatment to treatment
End-user (nurse) friendly Capable of being programmed to be turn-key
Based on technology that is over five Based on current magnetic technology coupled
decades old with imaging capability
Differences Between TCOM and EPR Techniques
TCOM measures tissue pO2 but does not measure wound pO2. It overestimates pO2
in the intact tissue at the wound perimeter. Standard TCOM are conducted under
conditions where the skin is warmed to 42–44°C. From theoretical considerations
warming can lead to an overestimation of pO2, Electron paramagnetic resonance on
the other hand, enables reliable and accurate measurements of concentrations of
molecular oxygen [54]. Table 12.1 summarizes the comparison between the EPR
system and the TCOM measurement approach.
EPR from the Laboratory to Bedside
EPR oximetry of tumors is already in practice in the clinic to determine therapeutic

outcomes. These successes may lead to the use of this technique in wound healing clin-
ics. The main advantages of EPR which makes it suitable for clinical applications lie in
the fact that EPR enables direct non-invasive, continuous and repetitive measurement
of partial oxygen pressure in the tissue. EPR instruments have now been designed that
can comfortably accommodate patients including those with disabilities. The technique
lends itself to use over small areas such as the feet or teeth One of the area where EPR
finds clinical applications is [55, 56] in oximetry and for following the oxygenation in
tissues at risk in peripheral vascular disease, in tumors to optimize therapy, and in
wound healing. This facilitates following the course of disease and monitoring the
response to treatment. The other primary application is in measuring clinically
significant exposures to ionizing radiation, which is useful in dealing with unplanned

exposures due to acts of terrorism, nuclear wars and accidents [55, 56].
EPR oximetry technique has been used as a guide for individualizing radiother-
apy and chemotherapy. EPR technique also finds unique application in skin research.
Since the skin is largely diamagnetic, paramagnetic centers are easily detectable.
Currently this technique has been standardized in the clinic to measure local oxygen
tension in the skin and its change caused by muscle contraction. The present size of
magnets allows placing a lower extremity between the poles of an L-band spectrom-
eter. EPRI/MRI dual imaging technique has enabled a detailed observation of drug
delivery, resorption or imaging of nitric oxide. EPR with longitudinal detection
(LODEPR, LODESR) can directly map pO2 in the organ.
Wound tissue oxygen status is an inherent property of the wound which needs to
be understood and managed for favorable treatment outcomes. TCOM is a recom-
mended common clinical tool for measuring tissue partial pressure. It represents a
good start however given the advances in the technology of wound oximetry we
may soon see optical and EPR-based technology platforms to develop clinical tissue
oximetry tools for use in wound care.
Acknowledgements This work was supported by NIH grants GM069589 and GM077185 and
HL073087 to CK Sen.
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Chapter 13
Measurement of Biomarkers for Impaired
Healing in Fluids and Tissues
Gregory S. Schultz and Daniel J. Gibson
Keywords MMP • Proteinase • Chronic wound • Biofilm • Wound assessment

Molecular indicators
Introduction: Summary of Phases of Acute Wound Healing
Acute skin wounds normally heal in an orderly and efficient manner, and progress
smoothly through the four distinct, but overlapping, phases of wound healing: hemo-
stasis, inflammation, repair and remodeling (Fig. 13.1) [1–3]. Each stage of wound
healing has certain milestones that must occur in order for normal healing to progress.
• Hemostasis – establishes the fibrin provisional wound matrix that provides
hemostasis; creates the initial scaffolding for deposition of the scar; platelets
provide initial release of cytokines and growth factors in the wound that stimu-
late inflammatory cells to chemotactically move into the injured area.
• Inflammation – mediated primarily by neutrophils and macrophages that: phago-
cytize and kill bacteria; secrete proteases (including matrix metalloproteinases
(MMPs) and neutrophil elastase) that digest and remove denatured extracellular
G.S. Schultz, Ph.D. (*)

Department of Obstetrics and Gynecology,
Institute for Wound Research, University of Florida,
1600 SW Archer Rd. M337F, Gainesville, FL 32610-0294, USA
e-mail: schultzg@ufl.edu
D.J. Gibson, M.S.
Department of Biochemistry and Molecular Biology,
Institute for Wound Research, University of Florida,
1600 SW Archer Rd. M337F, Gainesville, FL 32610-0294, USA

244 G.S. Schultz and D.J. Gibson
12
Months 10
8
6
4
25
20
Days
15
Four Phases of Healing

1. Hemostasis
0 2. Inflammation
1 2 3. Repair
3 4 5
1 Clotting 6 7 4. Remodeling
2 Vascular response 4 Scar formation 6 Contraction
3 Inflammationa 5 Epithelial healing 7 Scar remodeling
1 2 3 4 5 6 7
Vascular Scar Epithelial Scar
Clotting response Inflammation formation healing Contraction remodeling
Fig. 13.1 Sequence of molecular and cellular events in skin wound healing
matrix (ECM) components; synthesize and secrete growth factors and cytokines
that stimulate help to initiate the third phase of healing, repair. Prolonged, elevated
inflammation stimulated by the presence of planktonic and biofilm based bacteria
retards healing due to excessively elevated levels of proteases and reactive oxygen
species (ROS) that destroy proteins and factors that are essential for healing.
• Repair – new capillaries sprout from undamaged veins adjacent to the injury
migrate into the fibrin provisional wound matrix and form granulation tissue;
fibroblasts supported by the new capillaries proliferate and rapidly synthesize a
relatively disorganized ECM that replaces the provisional fibrin wound matrix;
basal epithelial cells and stem cells in hair follicles proliferate and migrate over
the granulation tissue to re-epithelialize the wound surface.
• Remodeling – fibroblast and capillary density decreases, and initial scar tissue is
removed and replaced by ECM that is more similar to normal skin. ECM remod-
eling is the result of the balanced, regulated activity of proteases.
Dynamic Reciprocity and Regulation of Normal Wound Healing
The complex process of acute wound healing involves a variety of specialized cells,
including platelets, macrophages, fibroblasts, epithelial cells, and endothelial cells. In
addition to the various cellular interactions, healing is also influenced by the action of
proteins, such as cytokines, chemokines, growth factors and their receptors, proteases
and their inhibitors. Wound cells interact with each other and with the extracellular
13 Measurement of Biomarkers for Impaired Healing in Fluids and Tissues 245
matrix (ECM) in a process that has been termed dynamic reciprocity [4]. Specific
examples of dynamic reciprocity include the alteration in patterns of gene expression
that is induced in fibroblasts when the cells migrate from the ECM adjacent to the
injury that is dominated by collagen into the provisional wound matrix that is domi-
nated by fibrin. This transition in ECM components is recognized by integrin recep-
tors on the surface of fibroblasts, and binding of fibrin by the avB5 integrin receptor
induces an intracellular signaling cascade that changes the pattern of gene expression
in the fibroblasts, resulting in massive up-regulation of collagen and proteoglycan
synthesis. Another specific example of the ECM regulating cellular functions is the
requirement for the polysaccharide, heparin sulfate, to stabilize the binding of a key
growth factor, basic fibroblast growth factor (bFGF), to its cellular receptor. Thus,
components of the ECM play key roles in regulating the activity of fibroblasts. In a
reciprocal manner, wound cells synthesize key components of the ECM including the
glycosaminoglycans and proteoglycans that have extensive heparan sulfate polysac-
charides, and decorin, that binds and stabilizes transforming growth factor beta
(TGFb). Additionally, matrikines, or subcomponents of ECM molecules, can bind to
cell surface receptors in the cytokine, chemokine, or growth factor families and stimu-
late cellular activities (e.g., tenascin-C and laminin bind to epidermal growth factor
receptors, which enhances fibroblast migration). Thus, the interactions between
wound cells and ECM are bidirectional. As described below, these interactions are
altered in difficult to heal or chronic wounds, and treatments must re-establish this
dynamic reciprocity for chronic wounds to heal.
Measurements of Key Cytokines, Growth Factors and Proteases

During Healing of Acute Surgical Wounds
Analyses of key molecular regulators of wound healing during the phase of healing
have utilized various animal models and different types of human acute wounds.
These analyses have generally utilized two molecular approaches: analysis of wound
fluids or homogenates of biopsies for the proteins using selective antibody based
assays, like enzyme-linked immunosorbent assays (ELISA), or measurement of
enzyme activities using fluorogenic substrates.
Karl et al. [5] used ELISAs to analyze wound fluids collected in sealed wound
chambers placed on partial thickness porcine skin wounds. Levels of bFGF peaked on
day 1 (31 pg/ml), platelet-derived growth factor-AB (PDGF-AB) peaked on day 3
(45 pg/ml), TGFb levels peaked at day 7 (726 pg/ml), and epidermal growth factor
(EGF) levels remained fairly constant over the 10 days of sample collection at approx-
imately 1.5 ng/ml. Interestingly, addition of EGF and PDGF-AB in ng/ml concentra-
tions to the wound chambers accelerated epithelial healing of the partial thickness
wounds by about 1 day, whereas addition of bFGF and IGF-1 had no effect.
Using a similar approach, Vogt et al. [6] used specific ELISAs to analyze wound
fluid sampled collected daily for 10 days in sealed wound chambers placed on par-
tial thickness skin wounds of 16 patients undergoing reconstructive surgery with
split thickness skin grafts. They found two different patterns of growth factor con-
centrations that were higher than serum values as some point during healing: high
initial concentrations that decreased to baseline values or below serum levels by the
time of healing, which included bFGF, EGF, insulin-like growth factor binding pro-
tein-1 (IGFBP-1); a second pattern with low initial concentrations rising to a maxi-
mum at the time of epithelialization, which included interlekin-1 alpha (IL-1a) and
TGFb2. Interestingly, concentrations of three factors in wound fluid samples never
exceeded serum levels; PDGF-AB, TGFb1, and insulin-like growth factor-1 (IGF-1).
The different time course profiles of growth factors and cytokines in wound fluid
samples collected from split thickness skin wounds suggest that they regulate differ-
ent functions during the time course of healing.
Grayson et al. [7] also analyzed wound fluid samples collected daily for 4 days
under occlusive dressings placed on split thickness skin graft donor sites of 13
patients with moderate sized burn injuries. Using ELISAs, all patients had elevated
levels of EGF and TNFa in their donor site fluids compared to serum controls. Only
3 of the 13 patients had elevated levels of IL-1 compared to serum controls, and 5
of 13 patients had elevated levels of bFGF compared to serum controls. Similar to
the results of Vogt et al. [6], no PDGF was detected in the donor site fluid samples.
Other types of human wounds have also been used to study profiles of key
molecular regulators in wound fluids. Di Vita et al. [8] collected drainage fluid daily
for 4 days from ten female patients undergoing abdominal midline incisional hernia
repair and analyzed the samples for multiple cytokines and growth factors.
Concentrations of all the cytokines, IL-1a, IL-6, IL-10, IL-1 receptor antagonist
(IL-1ra) and interferon-gamma (IF-g) and the growth factor, bFGF, were all highest
on day 1 then deceased over the following 3 days. In contrast, concentrations of
vascular endothelial cell growth factor (VEGF) increased progressively during the
4 days post-surgery. Baker and Leaper [9] determined the profiles of MMPs (MMP-
1, -2, -3, -8, -9) and the tissue inhibitors of metalloproteinases (TIMPs-1 and −2) in
intraperitoneal drainage fluid samples collected daily for 8 days from 58 patients
after elective pelvic surgery. Although there was wide range of concentrations
between the different MMPs and TIMPS over the 8 days, the levels of total MMP-9
were highest in the first days after surgery then progressively decreased (Fig. 13.2).
Perhaps more importantly, there was a significant positive correlation with the
occurrence and severity of postoperative complications and high levels of MMP-2
and MMP-9 and a significant negative correlation with high levels of TIMPs.
In a separate study, Baker and Leaper [9] measured levels of MMPs, TIMPs,
plasminogen activator, pro-inflammatory cytokines, and growth factors in wound
fluid samples collected the first day following surgery from tube drains of 50 elec-
tive surgical patients (24 breast and 26 colorectal). Levels of EGF, PDGF, bFGF and
TGFb1 were significantly higher in breast surgery drain fluid samples than in col-
orectal surgery drain fluid samples. Colorectal wound fluid samples, however,
showed significantly higher levels for the MMPs, TIMPs, and proinflammatory
cytokines IL-1, IL-6 and TNFa than breast wound drain samples. These results sug-
gest that early wound fluid samples from different types of surgical wounds may
have different initial patterns of proteases, inhibitors and cytokines that may reflect
differences in the initial healing of different types of acute wounds.
200
150
Total MMP-9 levels (ng/ml)
100
50
0
N= 53 42 46 14 30 12 9 1
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
Post-operative day
Fig. 13.2 Levels of total MMP-9 protein in intraperitoneal drainage fluid from 58 patients under-
going elective colorectal surgery (From Baker and Leaper [10])
Molecular Biomarkers for Impaired Healing

of Acute Traumatic Wounds
In contrast to acute clean surgical wounds that go on to heal with minimal risk of
breakdown, acute traumatic wounds, especially modern combat wounds, are at higher
risk of breakdown and dehiscence. It is generally accepted that major factors that
contribute to delayed healing and/or dehiscence of acute surgical wounds are pro-
longed, elevated, levels of wound infection/inflammation, or excessive mechanical
tension across a closed incision. However, as reviewed recently by Hahm et al. [11],
the timing of wound closure historically has been based on the subjective assessment
of the surgeon, using the gross appearance of the wound and the general medical
condition of the patient as determinants in timing of wound closure. Thus, there is a
need for more objective assessments that predict successful wound closure.
A biomarker is defined as a substance that is objectively measured and evaluated
as an indicator of normal biologic processes, pathogenic processes, or pharmaco-
logic responses to a therapeutic intervention [11]. Biomarkers are currently in use
for some clinical conditions. Levels of procalcitonin have been used to guide anti-
biotic therapy in critically ill patients with sepsis [12]. C-reactive protein and eryth-
rocyte sedimentation rate have been widely used in orthopedics to globally assess a
patient’s inflammatory state as an indirect marker of infection. In a prospective
study by Greidanus et al. [13], erythrocyte sedimentation rate and C-reactive protein
were shown to be effective screening tests for the presence of infection in patients
presenting for revision total knee arthroplasty.
As summarized by Hahm et al. [11], Utz et al. [14] investigated the association
of matrix metalloproteinases and the timing of successful surgical closure of acute,
traumatic, extremity combat wounds. Thirty-eight extremity wounds in 25 combat
casualties were studied prospectively. Surgical debridement with negative-pressure
wound therapy (NPWT) was repeated every 48–72 h until wound closure. Wound
effluent collected from the NPWT canister and patient serum were collected at each
debridement and analyzed for MMPs, and levels were compared between normal
and impaired healing wounds, which was defined as delayed wound closure of
>21 days from injury or wound dehiscence. Serum proinflammatory MMP-2 and
MMP-7 levels were increased in the impaired healing group, while levels of MMP-3
in NPWT wound fluid, which is involved in degradation of extracellular matrix, re-
epithelialization, and contraction, was depressed. The state of excessive or pro-
longed inflammation appeared to delay progression through the wound healing
phases, resulting in impaired wound healing. The authors suggested that serum and
NPWT wound effluent levels of MMP-2, MMP-3, and MMP-7 may potentially
provide objective evidence for timing of wound closure.
In a pilot study of wartime extremity wounds, Forsberg et al. [15] evaluated the
inflammatory cytokine and chemokine profiles of severe extremity wounds in service
members at the time of wound closure to determine whether serum and NPWT effluent
markers could be used to predict wound healing. Fifty wounds were analyzed in 20
patients with multiple high-energy penetrating extremity wounds. Four of the 50 wounds
(8%) dehisced. Among the serum chemokines and cytokines, only serum procalcitonin
levels correlated with wound dehiscence. Effluent analysis of the patients with dehis-
cence showed increased levels of procalcitonin and decreased levels of chemokine
ligand-5 [i.e., regulated on activation, normal T-cell expressed, and secreted (RANTES)]
and interleukin-13, an anti-inflammatory cytokine. No wound with an effluent procalci-
tonin concentration of less than 220 pg/ml, interleukin-13 greater than 12 pg/ml, or
RANTES greater than 1,000 pg/ml failed to heal. Based on the preliminary results from
their study, Forsberg et al. posited that effluent biomarker analysis may provide an objec-
tive means of determining the timing of traumatic wound closure.
Hawksworth et al. [16] expanded on the work by Forsberg et al. [15]. Serum,
wound effluent, and wound bed tissue biopsy specimens were analyzed to look for
differences in levels of cytokines and chemokines at each debridement. Fifty-seven
extremity war wounds in 35 consecutive patients were studied prospectively.
Significant factors associated with wound failure included elevated Injury Severity
Score (ISS), larger wound surface area and volume, and associated vascular injury,
which corresponded to a more pronounced inflammatory response. In this cohort of
patients, nine wounds (17%) dehisced. on Serum levels of pro-inflammatory
cytokine IL-6, pro-inflammatory chemokine IL-8, and macrophage inflammatory
protein-1 (MIP-1) were all significantly elevated in patients with wound dehiscence.
Analysis NPWT effluent revealed statistically higher expression of IL-6 and lower
levels of IL-2 and inducible protein-10 in patients with wound dehiscence. Tissue
biopsy specimens taken at each debridement also demonstrated a proinflammatory
pattern with increased expression of IL-1, IL-8, monocyte chemoattractant protein-1

(MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF) and
decreased expression of IL-4, IL-5, and IL-3. In addition, several of these proteins
(serum and effluent) and RNA-based (tissue) cytokines and chemokines demon-
strated favorable characteristics as predictive biomarkers regardless of sampling
time. The authors concluded that cytokine and chemokine proteins and gene tran-
script expression patterns demonstrate a condition of inflammatory dysregulation
leading to poor wound healing and dehiscence. Having the ability to determine dif-
ferences in the cytokine and chemokine profile at the time of surgery would enable
surgeons to make decisions for wound closure based on objective parameters.
Molecular Biomarkers for Chronic Wounds
Since cytokines, growth factors, proteases, and ECM components play key roles in
regulating acute wound healing, it is reasonable to hypothesize that alterations in
the levels and/or actions of these molecules could contribute to the development of
chronic wounds. As with analysis of acute wounds, several methods have been
used to assess the molecular environments of chronic wounds, including ELISAs
to measure specific protein in wound fluids or homogenates of wound biopsies,
fluorogenic substrates that selectively measure protease activities and immunohis-
tochemical localization of proteins in histology sections of wound biopsies. From
these types of studies, several important concepts have emerged from the molecu-
lar analyses of acute and chronic wound environments.
The first major concept to emerge from analysis of wound fluids is that the
molecular environments of chronic wounds have reduced mitogenic activity com-
pared to the environments of acute wounds [17]. Fluids collected from acute mas-
tectomy wounds when added to cultures of normal human skin fibroblasts,
keratinocytes or vascular endothelial cells, consistently stimulated DNA synthesis
of the cultured cells. In contrast, addition of fluids collected from chronic leg ulcers
typically did not stimulate DNA synthesis of the cells in culture. Also, when acute
and chronic wound fluids were combined, the mitotic activity of acute wound fluids
was inhibited. Similar results were reported by several groups of investigators who
also found that acute wound fluids promoted DNA synthesis while chronic wound
fluids did not stimulate cell proliferation [18–20].
The second major concept that emerged from analyses of wound fluids was levels
of pro-inflammatory cytokines were highly elevated in chronic wounds as compared
to levels in fluids of acute healing wounds. The ratios of two key inflammatory cytok-
ines, TNFa and IL-1b, and their natural inhibitors, P55 and IL-1 receptor antagonist,
in mastectomy fluids were significantly higher in mastectomy wound fluids than in
chronic wound fluids. Trengove and colleagues [21] also reported high levels of the
inflammatory cytokines IL-1, IL-6 and TNFa in fluids collected from venous ulcers
of patients admitted to the hospital. More importantly, levels of the cytokines
significantly decreased in fluids collected 2 weeks after the chronic ulcers had begun
to heal. Harris and colleagues [20] also found cytokine levels were generally higher
in wounds fluids from non-healing ulcers than healing ulcers. These data suggest that
chronic wounds typically have elevated levels of pro-inflammatory cytokines, and
that the molecular environment changes to a less pro-inflammatory cytokine environ-
ment as chronic wounds begin to heal.
The third important concept that emerged from wound fluid analysis was the elevated
levels of protease activity in chronic wounds compared to acute wounds [17, 22, 23]. For
example, as shown in Fig. 13.3, the average level of protease activity in acute wound
fluids (mastectomy drain fluids) determined using the general MMP substrate, Azocoll,
was low (0.75 mg collagenase equivalents/ml, n = 20) with a range of 0.1–1.3 mg colla-
genase equivalents/ml [24]. This suggests that protease activity is tightly controlled dur-
ing the early phase of wound healing. In contrast, the average level of protease activity
in chronic wound fluids (87 mg collagenase equivalents/ml, n = 32) was approximately
116-fold higher (p < 0.05) than in mastectomy fluids. Also, the range of protease activity
in chronic wound fluids is rather large (from 1 to 584 mg collagenase equivalents/ml).
More importantly, as also shown in Fig. 13.3, the levels of protease activity decrease in
chronic venous ulcers 2 weeks after the ulcers begin to heal [24].
Yager and colleagues also found 10-fold higher levels of MMP-2 protein, 25-fold
higher levels of MMP-9 protein, and 10-fold higher collagenase activity in fluids from
pressure ulcers compared to surgical wound fluids using gelatin zymography and cleavage
of a radioactive collagen substrate [25]. Other studies using immunohistochemical local-
ization observed elevated levels of MMPs in granulation tissue of pressure ulcers along
with elevated levels of neutrophil elastase and cathepsin-G [26]. TIMP-1 levels were found
to be decreased while MMP-2 and MMP-9 levels were increased in fluids from chronic
venous ulcers compared to mastectomy wound fluids [27]. Recently, Ladwig and
colleagues reported that the ratio of active MMP-9/TIMP-1 was closely correlated with
healing outcome of pressure ulcers treated by a variety of protocols (Fig. 13.4) [28].
n = 15 n=5 n=3 n = 17 n = 22
250 150
Collagenase activity (μg/ml)
200
Protease eq (mg/mL)
100
150
100
50
50
0
Non healing
Healing
Venous Arterial Diabetic Decubiti Acute
Fig. 13.3 Levels of MMP activities are low in acute wound fluids (mastectomy drain fluids) com-
pared to fluids collected from chronic wounds (left panel). Furthermore, high levels of MMP activ-
ities decrease in chronic venous ulcer wound fluids as the ulcers begin to heal (right panel)
180
Good healing - >95% Area healed; n=12
160 Intermediate healing - <95% But >65% Area healed; n=36
Ratio of MMP-9 (Pro + Active) :TIMP-1
Poor healing - <65% Area healed; n= 8

140
120
100
80
60
40
20
0
Day 0 Day 10 Day 36
Time course
Fig. 13.4 The MMP/TIMP ratio is high in fluids of chronic wounds that do not heal well and low
in wound fluids of wounds that heal in the period of treatment. Thus, low MMP/TIMP is a good
biomarker for healing of chronic wounds
It is interesting to note that the major collagenase found in non-healing chronic

pressure ulcers was MMP-8, the neutrophil-derived collagenase [2]. Thus, the per-
sistent influx of neutrophils releasing MMP-8 and elastase appears to be a major
underlying mechanism resulting in tissue and growth factor destruction and thus
impaired healing [3]. This suggests that chronic inflammation must be decreased if
pressure ulcers are to heal.
Other classes of proteases also appear to be elevated in chronic wound fluids. It
has been reported that fluids from skin graft donor sites or breast surgery patients
contained intact a1-antitrypsin, a potent inhibitor of serine proteases, very low lev-
els of neutrophil elastase activity, and intact fibronectin [29]. In contrast, fluids from
the chronic venous ulcers contained debrided a1-antitrypsin and 10- to 40-fold
higher levels of neutrophil elastase activity, and degraded fibronectin. Chronic leg
ulcers were also found to contain elevated MMP-2 and MMP-9, and that fibronectin
degradation in chronic wounds was dependent on the relative levels of elastase,
a1-proteinase inhibitor, and a2-macroglobulin [30, 31].
Besides being implicated in degrading essential extracellular matrix compo-
nents like fibronectin, proteases in chronic wound fluids also have been reported to
degrade exogenous growth factors in vitro such as EGF, TGF-a, or PDGF [1, 24,
32, 33]. In contrast, exogenous growth factors were stable in acute surgical wound
fluids in vitro. Supporting this general concept of increased degradation of endog-
enous growth factors by proteases in chronic wounds, the average immunoreactive
levels of some growth factors such as EGF, TGF-b, PDGF were found to be lower
in chronic wound fluids than in acute wound fluids while PDGF-AB, TGF-a and
IGF-1 were not lower [9, 32].
In general, these results suggest that many chronic wounds contain elevated
MMP and neutrophil elastase activities. The physiological implications of these
data are that elevated protease activities in some chronic wounds may directly con-
tribute to the failure of wounds to heal by degrading proteins which are essential for
wound healing such as extracellular matrix proteins, growth factors, their receptors
and protease inhibitors. Interestingly, Steed and colleagues [34] reported that exten-
sive debridement of diabetic foot ulcers improved healing in patients treated with
placebo or with recombinant human PDGF. It is likely that frequent sharp debride-
ment of diabetic ulcers helps to convert the detrimental molecular environment of a
chronic wound into a pseudo-acute wound molecular environment.
Rapid Point-of-Care Detectors for Protease Biomarkers
As described above, one of the first biomarkers for impaired healing that can be mea-
sured in wound fluids is the activity of MMPs, especially MMP-9. Thus, a prototype
device to provide rapid, point-of-care measurement for MMP-9 activity has been
developed. As shown in Fig. 13.5, this device consists of a standard swab on a hollow
plastic shaft that can be used to collect a sample of wound fluid by standard tech-
niques of gently pressing the swab into the surface of a wound bed at several places
(or at a specific area of interest) and rolling the swab between the thumb and first
finger until the swab becomes saturated with wound fluid. The swab is then placed
back into the “straw”, the snap valve broken by pressing the reservoir sideways then
the top reservoir is squeezed which forces the substrate solution through the hollow
shaft and washes the wound fluid out of the swab while mixing it with the substrate
solution. The substrate is a small peptide that contains a fluorescent dye molecule at
one end and a quencher group at the other end of the peptide. If the substrate peptide
is not cleaved, the quencher group absorbs photons released by the adjacent
fluorescent group when it is exposed to UV light, but if the MMPs cleave the peptide,
the fragments containing the quencher group and the fluorescent group diffuse away
from each other and a fluorescent signal is generated when the solution is exposed to
UV light. This arrangement of a fluorescent group combined next to a quencher
group on a cleavable substrate is called fluorescent resonance energy transfer (FRET).
The cleavage reaction of the FRET substrate is stopped after 10 min by pushing the
cap down to a second ‘stop position’, which punctures a foil barrier and allows the
reaction solution to flow into the bottom chamber where a chemical immediately
stops the reaction. The fluorescence signal is read by placing the unit into a simple
hand help fluorometer that measures the fluorescence intensity.
Initial analyses of wound samples collected from three acute wounds and seven
chronic wounds using the MMP indicator showed low levels of MMP activities in
the acute wound fluid samples and elevated levels of MMP activities in the chronic
wound fluid samples. Clinical studies to fully validate the MMP indicator are
underway.
a
Snap valve: releases buffer and
substrate solution to the swabbed sample.
Foil seal, separates

reaction chamber
Wound swab
Proteinase inhibitor to stop

reaction after specified time.
Full lengh mmp−9 standard curve and clinical samples

b 12
Standard curve
10
Concentration (μg/ml FL-MMP-9 Equivalent)
∗ acute wounds
8
∗
2
∗ ∗
0 .
. . . td
td td td ls
ls ls ls 01 02 04 05 06 07 08 09 10
/m /m /m g/m wc wc wc wc wc wc wc wc wc
μg μg μg μ
0 2 5 10
Acute and chronic wound patients
Fig. 13.5 A rapid point-of-care MMP detector that can accurately measure levels of active MMPs in
wound fluid samples collected with the swab (a). Measurement of MMP activities in three acute wound
fluid samples and six chronic wound fluid samples (b) show substantial differences in MMP activities
Assessment of Biofilms and Bacterial

and Fungal Species by PCR
Bacterial biofilms are well known in other medical specialities to cause a variety of
chronic pathologies including periodontal disease, cystic fibrosis, chronic otitis
media and osteomyelitis [35]. Biofilms are characterized by an exopolymeric matrix
of polysaccharides, proteins, and DNA synthesized by the multiple bacterial species
(polymicrobial) comprising the biofilm community. Bacteria (and fungi) contained
within the biofilm matrix are highly tolerant to killing phagocytic inflammatory
cells (neutrophils and macrophages), antibodies, and exogenous antibiotics, anti-
septics and disinfectants. Several factors contribute to the increased tolerance of
bacteria in biofilms to these agents, including reduced penetration of large proteins
(antibodies) into the dense exopolymeric matrix, binding of oppositely charged
molecules like antibiotics or cationic heavy metal ions (silver ion) by negatively
charged components of the exopolymeric matrix, or neutralization of high reactive
chemicals like hypochlorous acid (bleach) by reaction with molecules comprising
the exopolymeric matrix. Also, some bacteria in mature biofilms become metaboli-
cally quiescent and these “persister cells” are therefore high resistant to antibiotics
that disrupt bacterial metabolism. The factors contribute to make biofilms extremely
difficult to kill and clear from chronic wounds. Furthermore, components of the
biofilm matrix and products produced by bacteria in the biofilm stimulate chronic
inflammation, which leads to persistently elevated levels of molecules like proteases
and reactive oxygen species that kill wound cells and damage proteins that are
essential for healing.
Assessment of the “bioburden” of wounds has traditionally relied upon rela-
tively simple microbiology laboratory techniques that typically provide informa-
tion on major bacterial or fungal species in swabs or biopsies that can grow under
the nutritional and environmental conditions provided in the lab. These assess-
ments of bacteria and fungi in wound samples have unquestionably generated
important data that have been used for decades to help select therapeutic regi-
mens for patients and their wounds. However, multiple publications have pointed
out that, in many patients, measurements of total bacterial bioburden (expressed
as colony forming units per gram of tissue biopsy or 0–4+ levels of bacterial
growth) alone do not correlate well with the failure of wounds to heal. As shown
in Fig. 13.6 this led to the concept of “critical colonization” or “occult infection”
to explain the discrepancy, because there was an apparent link between microbial
bioburden in these wounds and the impaired healing in the wounds. However, it
was not clear what aspect of the relatively low total bioburden was ‘critical’ to
impairing healing. More thorough evaluation of these ‘standard’ clinical micro-
biology assays led to the realization that these assays are inherently limited by
the rather poor ability to culture or identify most of the bacterial and fungal spe-
cies that are actually present in an individual chronic wound. In other words,
standard clinical microbiology assays only culture planktonic bacterial and fun-
gal species that are able (capable) of growing on agar media plates supplemented
Increasing clinical problems
BIOFILM
Contamination Colonization Infection
critical colonization
Monitoring required Intervention required
Fig. 13.6 Spectrum of Bacterial Bioburden in Wounds. Contamination and colonization of bacte-
ria usually do not substantially retard healing whereas infection clearly impairs healing. The con-
cept of critical colonization evolved to describe a condition where levels of planktonic bacteria
were not above 106 cfu/g, but healing was impaired. Since biofilm bacteria are not detected by
standard clinical microbiology assays, critical colonization probably represents a condition when
biofilm bacteria are present in wounds and stimulate chronic inflammation that retards healing
with general nutrients in air at 37°C. Thus, it is reasonable to assume that a more
complete picture of different bacterial species (aerobes, facultative anaerobes,
and obligate anaerobes) and fungal species in a particular wound should improve
the ability to assess the microbial bioburden on individual wounds and to indi-
cate what therapeutic strategies would be optimal for each wound. Fortunately,
in the last few years sophisticated laboratory research techniques have been
developed that allowed more complete assessment of bacterial bioburden.
Specifically, these techniques demonstrated that a high percentage (~60%) of
chronic skin wounds have extensive bacterial biofilms [36]. Using sophisticated
polymerase chain reaction (PCR) techniques Dowd et al. [37] reported that the
bacterial and fungal complexity of chronic wound samples was much greater
than previously thought. In fact, on average, ~60% of the bacterial species pres-
ent in chronic pressure ulcers and ~30% present in diabetic ulcers were strict
anaerobic bacteria, and many bacterial species were present that had never been
reported in cultures of chronic wounds. These data suggest that many of the bac-
teria present in biofilms in a chronic wound may never be successfully cultured
in the standard clinical microbiology laboratory due to obligate cooperation with
other bacteria that create unique environmental conditions in a polymicrobial
community of bacteria in biofilms. A second major concept recently reported by
Wolcott and colleagues [38] showed that mature biofilms are rapidly re-estab-
lished in chronic wounds following surgical debridement, on the time frame of
24–72 h. This indicates that sharp debridement opens a time-dependent thera-
peutic window to prevent the re-establishment of mature biofilms that are highly
tolerant to host inflammatory response or to exogenous antimicrobial
The clinical principle that should guide “biofilm-based wound care” is to reduce
planktonic and biofilm bacterial burdens by the most appropriate and effective
means (surgical debridement, curettage, irrigation, etc.), then follow the debride-
ment by covering the wound with an effective bacterial barrier dressing, of which
there are many types, including dressings with microbicidal metal ions (silver), qua-
ternary amines, or occlusive films [39].
Conclusions
The molecular environment of chronic wounds contains elevated levels of

inflammatory cytokines and proteases, low levels of mitogenic activity, and cells
that often respond poorly to growth factors compared to acute healing wounds. As
chronic wounds begin to heal, this molecular pattern shifts to one that resembles a
healing wound. As more information is learned about the molecular and cellular
profiles of healing and chronic wounds, new therapies will be developed that selec-
tively correct the abnormal aspects of chronic wounds and promote healing of these
costly clinical problems.
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Care. 2008;17(11):502–8.
Chapter 14
Measurements in Burns
Tom McKinnell and Sarah A. Pape
Keywords Burn • Depth • Extent • Severity score • Monitoring • Microbiology

Nutrition • Healing • Outcome
Introduction
Measurement is fundamental to the assessment of patients with burns throughout

their care. Burns are common injuries, with potentially serious sequelae [1, 2].
Burn injuries produce a major drain on health service resources and are a source of
national economic loss [3, 4]. Survival following a burn injury is directly related to
the extent and depth of the burn as well as being influenced by additional risk fac-
tors such as inhalation injury, pre-existing illness and age. There is considerable
evidence that doctors working in Emergency Departments may lack sufficient
experience to make an accurate assessment of the seriousness of a burn and may
overlook other risk factors. This may have a significant effect upon the final out-
come [5–7]. Throughout the burn patient’s hospital stay it is necessary to measure
the response to treatment in order to tailor the healthcare to individual need. Once
treatment is complete there are further opportunities to measure outcomes.
In this chapter, three questions will be considered (Table 14.1):
• “What can be measured?”,
• “Why do we need to measure it?”
• “Does measurement influence outcome?”
We will discuss the main evidence for each.
T. McKinnell, MB, ChB, M.Sc., MRCS (Ed)

Department of Plastic Surgery, James Cook University Hospital, Middlesbrough, UK
S.A. Pape, MB, ChB, FRCSEd (Plast), MClinEd (*)
Department of Plastic Surgery, Royal Victoria Infirmary,
Queen Victoria Rd., NE1 4LP, Newcastle-upon-Tyne, UK
e-mail: sarah.pape@nuth.nhs.uk

260 T. McKinnell and S.A. Pape
Table 14.1 Three questions to ask

What can be measured? Burn extent
Burn depth
Severity scores
Adequacy of resuscitation
Microbiological assessment of wound
Nutritional status
Healing
Outcomes
Why do we need to measure it? To aid clinical decision making
To predict prognosis
To plan and finance burn services
To measure individual outcomes
To conduct clinical audit
To enable comparative research
Does measurement influence Fluid requirement is proportional to burn extent
outcome? Inadequate fluid replacement causes complications
Severity of burn and co-morbidity affects mortality
Burn depth is related to healing time and scarring
Complex burns require intensive and specialist care
Inadequate nutrition in children and young adults
interferes with growth
Poor outcomes cause disfigurement and functional
disability
Patients with poor outcomes may have ongoing needs for
social and medical care
Burn Extent
It has been recognised since eighteenth century CE that the extent of a burn is the
main predictor of its outcome [8] (Richter 1788, cited by Irwin [5]. The volume of
fluid lost from the circulation following a burn is directly proportional to the extent
of the burn. This knowledge has enabled standardised formulae to be devised that
can predict the volume and rate of fluid administration required to avoid dehydra-
tion [9, 10]. Burn extent is also a useful predictor of the need for admission to an
intensive care unit. Thus, accurate measurement of burn extent is crucial for plan-
ning treatment, predicting outcome and ensuring that patients are managed in the
most appropriate environment for their needs.
The extent of a burn may be measured in terms of either its absolute or its relative
size. An absolute measure of width, length and area is useful for small burns though
the significance of burns of equal area differs greatly between a small child and an
adult. In larger burns it is more appropriate to use a relative measure of burn extent,
expressed as a percentage of the total body surface area (TBSA). Relative burn
extent can be measured in a number of ways.
14 Measurements in Burns 261
The One Percent Rule
The simplest measurement uses an approximation of the area of the palm of the
patient’s hand to 1% of their TBSA. Some authors use the palm alone and others the
palm plus the palmar surface of the fingers. Rossiter et al. [11] showed that the palm
plus fingers represents only 0.8% TBSA in men and 0.7% in women. Jose et al. [12]
found less variation between the sexes if the palm alone was used but they found
that this represented only 0.5% TBSA. Measurement of burn extent using the
patient’s hand is only appropriate for very small burns or for very large burns (where
the unburned skin is measured and the figure subtracted from 100%).
The Lund & Browder Chart
Since 1944, it has been common practice to depict burns on a body chart. These charts
were devised by Lund and Browder [13], using data collected by Dubois and
Dubois [14] in 1916 (Fig. 14.1). Children have proportionally larger heads and smaller
A A
1
1
2 2 2
2
13 13
1 12 1 12 1 12
1 12
1 12 1 12 1 12 2 12 2 12 1 12
1
B B B B
C C C C
13
4 13
4 13 13
4 4
Fig. 14.1 Lund & Browder chart

lower limbs than adults so a separate chart is included to indicate their relative contri-
butions according to age. Lund & Browder charts are used throughout the world for
documenting and measuring burn surface area. However, one must question whether
the charts are still valid given the changes in body shape since 1916. In addition, these
charts make no allowance for variation in body shape according to sex, race, muscu-
larity, obesity, pregnancy or disproportionate body size (e.g. variation in breast size
[15]). New body surface area charts have been prepared in Taiwan, based upon a large
sample of their population [16]. This research needs to be extended to other popula-
tion groups.
The Wallace Rule of Nines
There will be times when a Lund & Browder chart is not readily to hand. In this situ-
ation, the Wallace Rule of Nines [17] may be used. This rule assumes that the adult
body can be divided into areas each representing approximately 9% of TBSA or
multiples of 9%. The head and neck and each upper limb represent 9%. The front of
the trunk from clavicles to groins equals 2 ×9%, as does the back of the torso from
the base of neck to the buttock crease. Each lower limb is another 18%. The perineum
and external genitalia account for the final 1%.
The rule of nines must be modified for children to allow for their relatively larger
heads and smaller legs. A neonate’s head represents 18% TBSA rather than 9% and
each lower limb represents 14%. As children grow the relative proportions change.
For each year from age 1–9 years subtract 1% from the head and add 0.5% to each
lower limb. It is assumed that children over 9 years of age have normal adult propor-
tions. The Wallace rule of nines has been shown to have a higher inter-rater vari-
ability than a Lund and Browder chart [18] but it remains useful in situations where
a chart is not readily available.
Serial Halving
Another technique for measurement of burn extent is ‘serial halving’. This may be
used on its own or in conjunction with the rule of nines. The observer makes a series
of judgements as to whether the burn represents more or less than one half of the
TBSA. If it is less than 50% TBSA then is it more or less than half of a half? If it is
less than 25% TBSA then is it more or less than half of a half of a half? This has
been found to be helpful in the field in military burns because the latter measure-
ment (12.5%) approximates to the level at which intravenous fluid resuscitation
should commence in an adult [19].
Sources of Error
Burn charts are useful tools, their use increases the accuracy of measurement of
burn extent [18]. However, the effectiveness of a burns chart is only as good as the
extent to which they are used [5]. When charts are not used, estimations of burn
extent can be wildly inaccurate.
Any method of recording burn extent is affected by the differences between a
three-dimensional body shape and a two-dimensional burn chart. This results in a
foreshortening and makes it difficult to record burns on the lateral aspect of the
body. This can affect the perceived size of burn. Computer systems exist that are
used to measure body shape and size in the fashion industry and for the assess-
ment of facial asymmetry [20]. The use of 3-D surface scanning would make it
possible to adjust the calculation for individuals with non-average body propor-
tions (e.g. large pendulous breasts or distended abdomen). It remains to be seen
whether computer-aided measurement of burn extent becomes a practical alterna-
tive to the burn chart.
Studies have shown that Emergency room doctors are more likely to overesti-
mate the size of a small burn and under estimate the extent of a large burn [6, 7, 18].
In part, this is due to inexperience. However, it is also important to remember that
burns may change during the first few hours after injury. Redness (erythema) may
subside without producing any blisters. These areas of first degree burn should not
be included when measuring the %TBSA. On the other hand, erythema may develop
into a superficial dermal burn with blistering and fluid loss, in which case these
areas should be included. Therefore, when burns are seen in the emergency depart-
ment one has to make a judgement whether or not to include the erythema.
Importance of Burn Extent
Patients with extensive burns should be identified early, given appropriate fluid
therapy to prevent dehydration and transferred to a properly equipped burn service.
When using the burn extent to predict fluid requirements both surface area and
volume (mass) must be taken into account. The most universally used formula for
the calculation of fluid requirements is the Parkland formula [10]. This is expressed
as: Fluid required for first 24 h = 3 to 4 ml Hartmann’s solution × %TBSA × body
mass (kg). Because the rate of fluid loss is greater in the first 8 h compared with the
next 16 h after injury, half of this volume should be infused by 8 h after injury and
the rest by 24 h after injury. Small children (<30 kg body mass) have a greater sur-
face area to volume ratio than older children and adults, so they require additional
fluid. This is roughly equivalent to the maintenance fluid requirement of a healthy
fasting child.
Inaccurate measurement of burn extent leads to inaccurate fluid administration.

In the healthy individual with a small burn, over-resuscitation for a few hours will
create additional tissue oedema and be partially compensated by a diuresis. On the
other hand, if a patient with a large burn is under-resuscitated, they will become
hypovolaemic. If this persists for more than a few hours the patient may develop
irreversible shock and multi-organ dysfunction and may even die from a potentially
survivable burn.
Patients with extensive burns have a longer hospital stay, make greater use of
specialised services and are more likely to have ongoing health needs than patients
with smaller burns. Data about the incidence of extensive burns within a population
or geographical area can be used as a guide to planning burn services. Services that
treat large numbers of patients with extensive burns can be appropriately funded and
staffed.
Burn Depth
The measurement of burn depth is of paramount importance in making decisions

about the management of the skin injury. This assessment is usually relatively easy
in very superficial and very deep burns but it is particularly difficult in burns of
intermediate depth. It is rare for any burn to be of uniform depth, so it is important
to make a detailed analysis of all areas of the burn. A further problem is that the
level of cell death may progress to a deeper plane in the first few days after injury.
Thus, an early evaluation may be accurate at the time but become invalid later.
Burn Wound Pathology
In order to assess burn depth it is important to understand what happens in the first
few days after injury and how healing occurs. Injured but viable tissues in and
around a superficial burn are able to mount an inflammatory response, which maxi-
mise within a few hours and then subside over a few days. The vascular component
of the inflammation will make the burned skin appear bright pink. Light pressure
over these areas will cause blood to move out of patent blood vessels in the dermis
and the skin will blanch. The bright pink colour will return within 2 s on release of
the pressure (capillary refill test). By contrast, in a deeper burn the dermal capillar-
ies will be damaged and therefore partially or completely occluded and the vascular
component of the inflammatory response will be reduced. The burned skin will
appear pale and there may be evidence of congealed blood in the vessels, appearing
as red dots that do not blanch on pressure.
In a superficial burn there will be viable remnants of epidermal cells in the base
of hair follicles, sweat glands and sebaceous glands. Given ideal circumstances,
these cells will multiply and grow across the surface of the burn until it has been
completely repopulated by epidermal cells. The normal layers of the epidermis will
re-form. Once the outermost layers have keratinised the skin surface will appear to
be matte and dry. At this point the integrity of the epidermal barrier has been restored
and it is said to be “healed”. For burns where the damage is confined to the superficial
layers of the dermis this process is complete within 3 weeks and there will not be
any scarring. In a deeper burn there may still be some remnants of epidermal cells
in the deepest hair follicles but these will be fewer and further apart. There may be
some attempt at regeneration but this is likely to be patchy and incomplete. In the
deepest burns there will be no nests of epidermal cells and healing will only occur
through secondary intention. This will be associated with the growth of granulation
tissue (buds of endothelial cells from capillaries in the wound bed), contraction,
epidermal migration from the margins of the wound and scar formation. Deep burns
take longer than 3 weeks to heal and always leave scars Unless the burn wound is
very small, the outcome would be improved by replacing the missing skin surgically
(e.g. with a skin graft or local skin flap).
The methods currently used to measure burn depth may be divided into three
groups:
• Measurement of surface appearance
• Measurement of structural integrity
• Measurement of blood flow
Measurement of Surface Appearance of a Burn
The traditional (and still the commonest) method of assessing burn depth is by
inspection of the surface of the burn. This can be done in the clinical setting and
does not require any special equipment. The clinician observes and records the
colour of the wound, presence or absence of blistering (hallmark of a superficial
dermal burn), pliability of the skin, the presence or absence of capillary blanching
and the sensitivity of the skin [21–24].
Burns involving only the epidermis are described as erythema (first-degree
burns). They are characterised by redness, absence of blistering, soft and pliable
skin, capillary blanching on light pressure, rapid refill on release of pressure and
intact sensation. These signs of inflammation subside over a few days, when the
skin will look normal again, apart from some surface peeling. Sunburn is an exam-
ple of an epidermal burn. Superficial dermal (superficial second-degree) burns are
bright pink, swollen, painful and demonstrate capillary blanching and refill. Their
characteristic feature is the presence of blisters, although the blisters may separate
at an early stage leaving behind a moist wound bed with remnants of blisters only
around the margin. Deep dermal (deep second-degree) burns are dark pink. Close
inspection will reveal that this colour is due to the presence of multiple tiny red dots
within a white wound bed. Pressure does not cause blanching. Sub-dermal or full-
thickness (third-degree) burns are white and leathery or charred. Although not pain-
less they are not as exquisitely painful as superficial burns.
The accuracy of assessment of burn depth by inspection alone is highly depen-

dent upon the experience of the observer and varies between 65 and 83% [25–29].
Accuracy can be improved by delaying the assessment until 48 h after injury, when
the inflammatory response has stabilised.
With the availability of digital photography, it is easy to photograph burns.
However, this method suffers from the same limitations as clinical assessment of
burn depth by observation: the burn may progress later and the surface appearance
may not reveal what is taking place at a deeper level. One potential role of digital
photography is to transmit images to a more experienced burn clinician for an opin-
ion. However, no formal study has yet been conducted to see whether this improves
the accuracy of burn depth assessment. Potential limitations of this method include
low image resolution, colour variation due to differences in lighting conditions and
reproduction on different screens and printers, and the inability to assess capillary
refill in a still image.
Because of the limitations of clinical assessment of burn depth, researchers have
been seeking technological solutions [30]. The surface colour of a burn can be mea-
sured in a more objective fashion by reflectance spectrophotometry [31, 32]. This
technology is extremely accurate and is used, for instance, to colour-match paint.
Light reflected from the surface of an object is analysed to assess its range of wave-
length (colour). In order to avoid artefact caused by variations in the colour of ambi-
ent light, the incident beam should be of a standard and constant wavelength (e.g.
green, red, infra-red). Research has shown that reflectance spectrophotometry is
highly accurate in the assessment of the depth of very superficial and very deep
burns. However, its accuracy in the assessment of intermediate depth burns is only
75% [32]. This is the same as the results of clinical assessment alone.
Measurement of Structural Integrity
Instead of evaluating the surface appearance of a burn and using that information to
form an opinion about what is happening at a deeper level, one could make an
assessment of the structural integrity of the skin. This can be done, albeit by using
an invasive method, by taking a small biopsy, which is processed and analysed by a
Histopathologist. The report can include information about the structure and cellu-
lar integrity of the excised skin. The staining properties of cells and fibrous proteins
(e.g. collagen and elastin) can be used to identify the deepest level of damage [33].
This can be supported by information about the deepest level of thrombosis within
the dermal capillaries [34]. A limitation of the method is the fact that it samples
relatively small portions of the burn. Full histological preparation also takes a num-
ber of days, delaying the decision making process, and requires the opinion of a
histopathologist with particular experience in burn biopsy assessment. In addition,
the technique is painful, may require a general anaesthetic and inevitably increases
the risk of scarring. A number of studies have looked at the use of biopsy techniques
for the assessment of burn depth and it is often said to be the “gold standard” against
which other methods are compared [35–37]. However, to date, no universal method
has been adopted and histology is rarely used in the assessment of burn depth out-
side the research field.
High-resolution ultrasound can be used to analyse the structure of skin and the
extent of denatured collagen [38–41]. Ultrasound will also demonstrate the pres-
ence or absence of dermal oedema but cannot assess cellular integrity. Although the
technique is relatively easy to learn, it does require specialist training. The main
disadvantage of this technique is the fact that direct and close contact is required
between the transducer and the skin surface. This is painful and there is the risk of
cross contamination.
Optical coherence tomography (0CT) takes an ‘optical biopsy’ of the skin [42].
It works by analysing the properties of light reflected from the dermis. Visible light
cannot penetrate deeply enough but infra-red, which has a longer wavelength and
penetration, can. The data can be processed to create a cross-sectional or three-
dimensional image. This can be used to detect and quantify denatured collagen up
to 2 mm below the surface. The technique is non-invasive and has been shown to be
accurate in both animal and human studies [43]. However, to date there are no pub-
lished reports that compare it with other technologies. Like traditional biopsy tech-
niques, it provides only a single-point analysis.
Measurement of Blood Flow
The third group of techniques used to assess burn depth measure capillary blood
flow in the dermis. This can be used to quantify the vascular component of the
inflammatory response within a burn. Because of the time-scale of the inflammatory
response, these methods are only accurate between 48 h and 5 days following injury.
Injection techniques can be used to demonstrate patency of the dermal capillaries
[44–46]. The agents that have been used include tetracycline [47], fluorescein [48,
49], indocyanine green [50] and radioactive isotopes [51, 52]. Following intrave-
nous injection these agents can be detected in viable skin. Where a deeper burn has
caused destruction or loss of function of the dermal capillary bed the quantity of
agent detected will be reduced or even absent. In the past these injection techniques
have been used clinically to establish the viability of skin flaps and injured skin.
Although used experimentally in burn depth assessment they have not become
established in clinical practice. In addition, they cannot be used in women of child-
bearing age and their use carries a risk of allergy.
Thermography is a non-invasive and non-contact technique that can also be used to
examine skin blood flow by measuring the rate of heat loss at the surface. This requires
standardised environmental conditions including air temperature, humidity and air
movement. Thermography has been shown to be more accurate than clinical assess-
ment alone up to 72 h following burn injury [53–61]. The equipment required is por-
table but relatively expensive. Although there are a number of published studies,
thermography has not gained widespread acceptance in routine clinical practice.
Video microscopy can be used to investigate the integrity of dermal blood vessels
by detecting red blood cell movement. The equipment is able to assess only a fairly
restricted field at any one time. Visualisation of larger areas is a time-consuming
process, during which the patient must keep still. The technique can be supple-
mented by intravenous injection of a vital dye. In experienced hands video micros-
copy is highly accurate and correlates well with observed healing rates. However, it
requires that a trained assessor to use the microscopy system and to judge the areas
of interest to scan. Video microscopy has been shown to be more accurate than
clinical examination alone and similar to laser Doppler imaging [62] but has yet to
gain widespread acceptance beyond the centre where it was originally introduced
Laser Doppler flowmetry (LDF) and laser Doppler imaging (LDI) are both based
on the same principle. When lights bounces off a moving object (e.g. red blood
cells flowing in a capillary) the frequency of the incident beam of light is altered.
The magnitude of this alteration is called the Doppler shift. The Doppler shift is
directly proportional to the speed of blood movement. LDF requires close contact
between a laser emitting probe and the skin surface but LDI uses a scanning head
that is placed away from the patient. Thus, there is no contact between the LDI
equipment and the patient. Provided that there is no other source of movement (e.g.
restless or shivering patient) the only source of movement in the skin is the red
blood cells. LDF has been shown to be 92% accurate in the assessment of burn
depth [27, 63]. This is a considerable improvement on clinical assessment alone.
However, it can be difficult to maintain adequate contact between the LDF probe
and the wet burn without exerting undue pressure. This is not an issue for LDI.
Another drawback with LDF is that it only assesses a small area at a time. LDI, on
the other hand, can assess the whole of the burn surface in just a few seconds and
produce a two-dimensional, colour-coded image of the burn (Figs. 14.1, 14.2, 14.3
and 14.4) that gives an accurate prediction of healing times [28, 29, 64–70].
To date, LDI and LDF are the most accurate tools for the measurement of burn
depth in regular clinical use. LDI has some advantages over LDI because it does not
require contact with the burn and provides a picture of the whole burn.
Sources of Error
Irrespective of the method used, making an assessment too early is a common source
of inaccuracy [28, 71]. This is because of the dynamic nature of the burn wound,
which can progressively deepen due to death of severely damaged cells, oedema,
tissue hypoxia, changes in tissue perfusion, additional local trauma and infection. It
is not due to any inherent inaccuracy in any of the individual methods. Even when
the assessment is delayed to between 2 and 5 days after injury, the clinician who
inspects the surface of a mid to deep dermal burn frequently over-estimates the depth
[65]. Thus, if inspection of the burn surface is the only basis upon which treatment
decisions are made, some patients will receive unnecessary operations and, hence,
scarring [72]. Jackson acknowledged this problem in 1950 when he noted how
Fig. 14.2 LDI scan of mixed depth burn. Fig. 14.3 Colour photograph of mixed
Red and pink areas have extremely high depth burn on leg, taken with DCD camera
flow, indicating intact dermal circulation on Laser Doppler Imager at time of scan.
and a brisk inflammatory response in areas Only by comparison with the photograph is
of superficial burn. Note that the unburned it possible to distinguish between normal
skin and areas of deep burn do not show an skin and deep burn
inflammatory response and appear as blue
areas. Areas with intermediate blood flow
are shown in yellow and green
Low blood flow High blood flow

0 500 1000 1500 2000 2500
Fig. 14.4 Palette thresholds for LDI. Note that the numerical steps between the colours are not
uniform. These thresholds have been chosen because they correspond with healing at the important
steps of 14 and 21 days . Red and pink correlate with areas have a healing potential of less than
14 days (HP < 14). Blue correlates with a healing potential of longer than 21 days (HP > 21).
Yellow and green correlate with a healing potential of 14–21 days (HP14-21)
difficult it was to see below the surface of a burn to assess what was happening in the
deeper layers. Even LDI, the most accurate tool in regular clinical use, cannot be
relied upon if the assessment is made before the burn has stabilised. This is frustrat-
ing for the clinician who wishes to perform very early surgery, before 48 h have
elapsed. It is then down to the surgeon to make a judgement, recognising that even
the most experienced surgeon will have an error rate of 20–30% in intermediate
depth burns.
Local sources of error include un-debrided blisters, foreign material (e.g. molten
plastic or bitumen) and topical creams. Silver sulfadiazine is commonly used as a
burn dressing but has been noted to discolour the surface of the burn, making it
appear white and opaque. This makes clinical assessment of burn depth very difficult
but, because it does not affect the dermal blood flow, silver sulfadiazine does not
interfere with assessment by LDI. Cerium nitrate is another topical wound cream
that converts the burned skin into a dry, leathery eschar. LDI has been shown to be
effective even in the presence of cerium nitrate [73]. Evaporation from the surface
of a wet burn causes cooling and is a source of error in thermography [74].
General sources of error include hypothermia and hypovolaemia, both of which
reduce skin blood flow. This interferes with burn depth assessment by clinical
inspection or by measurement of blood flow. Unwanted movement (shivering or
restless patient) is a problem for a number of methods (e.g. LDI, OCT, thermogra-
phy, videomicroscopy). Patients should, therefore, be warm, well-perfused and
comfortable during measurement.
Importance of Burn Depth Assessment
In order to distinguish between superficial and deep burns one could just wait to see
whether or not the skin heals as was the practice However, it has been shown that
the incidence of hypertrophic scarring increases significantly if healing takes longer
than 14 days in a child [75] or 21 days in an adult [76]. It is, therefore, desirable to
minimise scarring by making an early and accurate assessment of the potential for
a burn to heal and to plan early surgery for those predicted to have prolonged heal-
ing. In recent years there has been a trend to early, total excision of deep burns, even
on the day of injury. This has been shown to improve survival, but it must be recog-
nised that reliance on clinical assessment by the surgeon in the first 24 h almost
certainly leads to excision of viable skin. The benefits of reduction in morbidity and
mortality therefore have to be balanced against the risk of increased scarring.
Severity Scores
Trauma surgeons and intensivists have developed systems for the classification of
severity of disease and non-burn trauma, one example being the Apache II system [77].
Apache II is not burns specific but the physiological elements of it are as useful for
burns as for any other injury. However, [78] concluded that existing, general trauma
scores did not predict outcomes for patients with burns accurately and that specific
burn severity scores should be developed.
Richter (1788 cited by Irwin [5]) recognised that the risk of death from a burn
was proportional to its extent. In Bull and Fisher [79] reported survival statistics for
patients with burns admitted to the Massachusetts General Hospital, USA. They
noted that age and burn extent played significant roles in the prediction of burn
mortality and that these two factors were interlinked. As a result of this work, for
many years it was said that if the age of the patient in years plus the %TBSA
exceeded 100, the patient was unlikely to survive. Bull revisited this work in 1971,
adjusting his predictions to factor for the increased risk of mortality if the burn was
associated with inhalation injury [80]. In 1981 Roi et al. [81] developed a burn
specific severity scoring system. They developed a quick and simple graphical
method for the assessment of burn severity on arrival in the emergency department,
using age, %TBSA and the presence or absence of a perineal burn but they did not
take inhalation injury into account. The work was based on the outcome of over
11,000 burn patients in 12 major burn centres in the USA. In 1983 Bowser et al. [82]
created the concept of LA50 – the %TBSA at which 50% of patients die. They used
this to study trends in burn survival between 1979 and 1983.
The presence of an inhalation injury has an enormous effect upon the mortality
risk of a burn [83]. Indeed, patients may die from simple hypoxaemia and carbon
monoxide poisoning in the absence of any cutaneous burns. In addition, an inhala-
tion injury increases the early post-burn fluid requirements, and oxygen demand
[84, 85]. Thus, the presence and severity of inhalation injury should be measured in
any burn severity score. In concept an inhalation injury is either present or absent.
This can be assessed by clinical examination, looking for burns in the upper airway,
soot in the nose, singeing of the nasal hairs and evidence of stridor or respiratory
distress [83]. The anatomical distribution and severity can be confirmed by flexible
bronchoscopy but chest x-ray (at least in the early stages after injury) is rarely diag-
nostic. Arterial blood gas analysis is a useful tool for assessment of the adequacy of
gaseous exchange. Carboxyhaemoglobin levels in the blood within the first few
hours after burn can be used to calculate the patient’s likely exposure to carbon
monoxide at the time of injury [86]. Due to the rapid washout effect of therapeutic
oxygen, carboxyhaemoglobin levels usually return to normal within a few hours.
However, whilst it is relatively to identify that an inhalation injury has occurred, it
is much more difficult to measure its severity. Inhalation injury is usually arbitrarily
classified as either absent, mild, moderate or severe.
It must be kept in mind that severity scores, reflecting as they do the general out-
comes of a group of patients, cannot be used to make specific predictions about an
individual patient. Whilst they can be used as part of a general discussion about the
likely outcome and to identify high risk patients they should not be used alone or by
inexperienced individuals to make decisions about starting or continuing intensive
therapy. Thus, burn severity scores are useful for the study of populations and trends but
they have not been (and probably should not be) used on an individual patient basis.
Measurement of the Adequacy of Resuscitation
Patients with extensive burns will rapidly become dehydrated due to the loss of fluid
from the circulation. In part this is due to leakage of protein rich fluid into the blis-
ters and evaporation from the surface of the burned skin. However, the inflammatory
response to a large burn also causes net loss of fluid from the circulation through
leaky capillary walls throughout the body. This leads to oedema in the subcutaneous
tissues, lungs, peritoneal cavity and most of the vital organs. When the burn extent
exceeds 30% there is oedema in virtually the whole of the body.
In order to prevent hypovolaemia, patients are resuscitated with oral or intrave-
nous fluids. As stated above, the volume of fluid required can be predicted, for
example by using the Parkland formula [10]. It is worth remembering that any for-
mula is simply an estimation based on population studies. The clinical effect in any
individual burn patient must be monitored and the fluid input adjusted according to
their physiological state. The simplest measure of the adequacy of fluid resuscita-
tion is the quantity and quality of urine output. This is an indirect measure of the
perfusion of the kidneys and hence an indicator of the adequacy of the circulating
blood volume. An accurate fluid balance chart, recording all sources of fluid input
and output, together with trends in the blood pressure and heart rate, will be a
sufficient monitor of fluid therapy for many burn patients.
In previous years invasive monitoring was avoided because of fears of introduc-
ing organisms into the bloodstream. Patients with major burns are already at
increased risk of infection through loss of integrity of the skin surface and the
immunosuppression caused by their injury. However, it became recognised that
urine output and monitoring of the vital signs may not be sufficient for those patients
with the most extensive burns or with additional factors, such as co-morbidities or
other injuries [87, 88]. Intra-arterial monitoring and serial measurement of central
venous pressure are now used routinely to monitor the adequacy of fluid resuscita-
tion in patients with major burns.
Alternative measurement techniques have been described. The original work by
Baxter and Shires [10] used radioisotope dilution methods to study post burn fluid
volume changes in terms of cardiac output. However, this invasive technique has
never been used in clinical practice. Thermodilution of arterial blood, either via the
pulmonary artery [89] or by a transpulmonary route [90–92] can be used to measure
cardiac output and cardiac index in patients with burns and this information can be
used to monitor and adjust fluid replacement therapy. Concern has been expressed
that the presence of hypothermia or pyrexia (both common in patients with major
burns) might cause unacceptable inaccuracy in the use of thermodilution. However,
this has not been shown to be a significant problem [91].
Transthoracic echocardiography (TTE) can be used to monitor cardiac output in
critically ill patients but this is not feasible in patients with major burns involving
the chest wall. An alternative method is transesophageal echocardiography (TEE).
This method has been evaluated in patients with burns [93–95]. In addition to mea-
suring cardiac output, TEE can also provide valuable information about pulmonary
hypertension, pericardial effusion, fluid overload and heart failure and has identified
vegetations on cardiac valves in cases of unexplained hypotension [95]. TEE can be
used to make decisions about therapeutic interventions such as fluid resuscitation,
the need for inotropes and introduction of antibiotics. It can be used during burn
surgery, where there may be massive volume shifts due to blood loss. TEE is highly
sensitive to small changes in cardiac output and can predict the need for fluid chal-
lenge or inotrope administration before these otherwise become apparent
No single measurement of the adequacy of fluid resuscitation should be taken in
isolation. What is more important is the overall picture and trends followed over a
period of time. Clinical records of fluid balance, intra-arterial and central venous
pressure are sufficient measures for most patients. Other more technological meth-
ods have not gained widespread clinical acceptance. What is not in doubt is that the
adequacy of resuscitation must be carefully monitored by some means or other. It is
vital to guard against under-resuscitation on the one hand, leading to inadequate
perfusion of the vital organs, and over-resuscitation on the other hand, with the risks
of excessive oedema, increased lung water, abdominal compartment syndrome and
hyponatraemia.
Microbiological Assessment of Burns
There are two aspects to the microbiological assessment of a burn: qualitative data
(which organisms are present?) and quantitative data (how many organisms are
present?) [96].
No skin, burned or otherwise, is sterile. There will be a range of contamination
from normal skin flora, colonisation, wound infection, spreading infection (celluli-
tis) and systemic infection. In USA the Center for Disease Control and Prevention
(CDC) gives a broad definition of burn infection [97] (Table 14.2). The authors note
that the presence of purulence alone is not adequate for the diagnosis of burn infec-
tion because this may simply reflect inadequate wound care and fever alone is
insufficient because of the systemic effect of the burn or the possibility of infection
in another site.
There are two main methods of obtaining burn wound samples for the measure-
ment of infection: surface sampling and biopsy. Surface samples may be harvested
by swabbing or by mechanical abrasion (dermabrasion) of the surface [98]. Some
authors favour swabbing small areas [99] but others suggest that the swab should be
twirled and swept over a large area. Any organisms that are grown can be identified
and quantified and their sensitivities to a range of commonly used antibiotics can be
assessed. Levine indicated that a value of 106 organisms per swab was associated
with a significant risk of sepsis. In practice many clinical laboratories simply pres-
ent the qualitative results in relative terms of no growth, normal skin flora, scanty,
moderate or heavy growth [100]. The CDC guidance makes no attempt to quantify
the number of organisms. Herruzo-Caberra et al. [101] used semi-quantitative cul-
tures of the surface of burn eschar to devise a threshold value between colonisation
Table 14.2 CDC/NHSN definition of burn infection

Must meet at least one of the 1. Patient has a change in burn wound appearance or character,
following criteria: such as rapid eschar separation, or dark brown, black, or
violaceous discolouration of the eschar, or oedema at wound
margin
and
Histologic examination of burn biopsy shows invasion of
organisms into adjacent viable tissue
2. Patient has a change in burn wound appearance or character,
such as rapid eschar separation, or dark brown, black, or
violaceous discolouration of the eschar, or oedema at wound
margin
and at least one of the following
a. Organisms cultured from blood in the absence of other
identifiable infection
b. Isolation of herpes simplex virus, histologic identification
of inclusions by light or electron microscopy, or visualization of
viral particles by electron microscopy in biopsies or lesion
scrapings
3. Patient with a burn has at least two of the following signs or
symptoms with no other recognized cause: fever (.388°C) or
hypothermia (.368°C), hypotension, oliguria (.20 cc/h),
hyperglycemia at previously tolerated level of dietary carbohy-
drate, or mental confusion
and at least one of the following:
a. Histologic examination of burn biopsy shows invasion of
organisms into adjacent viable tissue
b. Organisms cultured from blood
c. Isolation of herpes simplex virus, histologic identification
of inclusions by light or electron microscopy, or visualization of
viral particles by electron microscopy in biopsies or lesion
scrapings
From Horan et al. [97]
and infection of 105 organisms/g of tissue. This level of contamination is associated

with a higher rate of sepsis [102, 103] and reduced take of skin grafts [104].
Biopsy of the burn wound allows sampling at a deeper layer. The sample can be
homogenised and cultured or examined by a histopathologist for evidence of infec-
tion. Pruitt and Foley [105] stated that histological invasion of viable tissue, together
with at least 105 organisms per gram of tissue had a significant correlation with
increased mortality. Volenec et al. [106] reported similar findings. McManus et al.
[107] showed that histological invasion occurred in only 36% of specimens with
counts greater than 105 organisms per gram. Steer et al. [108] did not find any cor-
relation between sepsis and bacterial count, or between bacterial count and graft
take (although they attributed the latter fact to their technique of burn excision).
Overall, there may be no additional value of harvesting biopsy specimens.
There are a number of comparative studies that assess whether surface sampling
accurately represents the situation at a deeper level [109–115]. Herruzo-Cabrera
et al. [101] used semi-quantitative methods for the analysis of surface swabs and the
results had a high correlation with those obtained from punch biopsies. Uppal et al.
[116] showed good agreement for identification of the causative organism, but poor
correlation with quantification of infective load. Sj berg et al. [117] agreed that
biopsy was more informative than surface sampling but required additional time
and resources.
All of the above methods of sampling inevitably involve a time delay before
results can be reported. A possible means of avoiding this delay is to prepare a Gram
stain of a homogenised biopsy specimen. Taddonio et al. [118] showed a good cor-
relation between the identification of organisms on gram stain and the subsequent
culture results, but they admit that its sensitivity is questionable.
Murray et al. [119] showed that pyrexia and increased white blood cell (or neu-
trophil) count were poor predictors of a bloodstream infection in burns patients. If
systemic sepsis is suspected, venous blood should be drawn for culture using an
aseptic technique. Any intra-vascular catheters that are removed should also be sent
for culture. CDC has defined catheter-related blood stream infection as the presence
on a catheter tip of > 15 colony-forming units of the same organism as the blood
culture. Fungal septicaemia is a rare but potentially fatal complication of burns.
Blood needs to be cultured for up to 14 days to detect fungal growth and even then
it may be missed, leading to a significant delay in starting treatment [120].
Importance of Microbiological Assessment of Burns
Sepsis has always been a major cause of mortality in patients with burns [121]. Infection
of the burn itself can delay wound healing; indeed a superficial burn may progress to
such an extent that surgery is required. Once micro-organisms have breached the burned
skin they may access the circulation causing bacteraemia, septicaemia and toxaemia.
Clusters of micro-organisms can cause infective emboli and may lodge on heart valves
causing bacterial endocarditis. Following skin grafting some organisms will compete
with the cells in the graft, preventing take. Knowledge of the microbiological status of
a wound is clearly vital in the management of all burn wounds. It is cost-effective to
undertake regular qualitative surface swabs from the surface of burn wounds for the
identification of colonising micro-organisms. If a patient subsequently develops inva-
sive sepsis, the clinician has information about the most likely causative information.
However, the additional value of a biopsy has not been proven.
Measurement of Nutritional Status
Extensive burns place a huge catabolic demand on the patient. In order for them to
heal adequately, the patient’s nutritional status must be optimised. In the past, the
calorie requirement for patients with burns has been grossly overestimated because
the relative inactivity of the patients was not taken into consideration. While the
burn causes catabolism this is counterbalanced by a greatly reduced energy expen-

diture (especially in a ventilated and sedated patient who is not even making mus-
cular effort to breathe). Estimates for the nutritional requirements of a patient with
a burn are based on gender, weight, age, height and burn extent. Patients with major
burns are difficult to weigh because they are usually unable to stand or sit on scales.
Special beds that weight the patient can be used but these fail to take into account
variations in weight caused by dressings and fluid retention. Likewise, height is
rarely measured in critically ill patients.
There are standardised charts that can be used to calculate the patient’s caloric and
protein requirements but these should be considered to be no more than a guide.
Sufficient calories should be supplied to provide energy for basic body functions,
given as carbohydrate. Protein should be added to reduce the nitrogen depletion asso-
ciated with catabolism (although some loss of muscle bulk seems to be inevitable and
unavoidable). A small quantity of lipid, with its higher caloric content, is useful to
reduce the overall volume of feed required. No adjustment should be made for any
pre-existing nutritional depletion in order to avoid the re-feeding syndrome, which is
associated with instability of nitrogen balance (often with increased ammonia levels
in the blood) and fatty liver infiltration. These patients need careful monitoring with
serial liver function tests and blood ammonia levels (not a standard assay).
Calories and protein alone are insufficient for wound healing, and specific micro-
nutrients are also required, including vitamins and trace elements such as vitamins
A and C, magnesium, zinc and selenium [122]. Many of these agents act as cofac-
tors for enzymatic processes involved in metabolism. During the stress response to
a burn their absorption is decreased and utilisation is increased. Deficiency of any
of these nutrients will interfere with wound healing and it is important to ensure
adequate supplementation with micronutrients for all patients with major burns.
The response to feeding can be monitored by weighing the patient on a regular
basis. Most patients with major burns will suffer a significant weight loss, which
will take weeks or months to recover. However, when they regain weight this will
largely be adipose tissue rather than muscle. Additional measurements of the meta-
bolic effects of the burn include subcutaneous fat measurement
Serum albumin levels should also be monitored. However, these are likely to be
extremely low (even below the detectable range of the assay) in the early stages of a
major burn due to protein leakage from the circulation and dilution by resuscitation
fluid. Albumin has a half-life of 30 days in the blood so is not a sensitive marker of
nutritional status. Pre-albumin, with a half life of a few days is a better indicator. Serial
measurements of serum transferrin and white blood cell counts are also useful.
Why Measure Nutritional Status?
The incidence of burns is higher in socially deprived individuals, those with sub-
stance abuse (including alcohol dependency) and psychiatric disorders (including
anorexia nervosa). Some of these patients will have suffered from chronic malnutri-
tion before being burned, which will exacerbate the inevitable catabolism and may
precipitate metabolic derangement on re-feeding. Known or suspected weight loss
of >10% in the previous 6 months or >5% in the previous 30 days are indicators of
an at-risk patient. It is, therefore, essential that an initial measurement is made of the
patient’s nutritional status and that this is updated at least weekly. Blood levels of
micronutrients should be measured as soon as possible after admission and moni-
tored on a weekly basis. However, in reality, many of these are specialised assays,
the results may take days or weeks to arrive and blood levels have not been shown
to correlate with nutritional status [123].
Measurement of Healing
The ultimate goal for burn healing is to re-establish an intact skin surface as early as
possible in order to prevent or minimise scarring. Identification of the exact moment
of healing is not as simple as one might think. In clinical practice the day of healing
is often identified as the day on which a dressing is no longer required. However,
unless the dressing is being changed daily, one cannot be sure that the burn had not
actually healed a day or two beforehand.
The endpoint of healing differs between superficial burns, deep burns left to heal
by secondary intention and burns that have been skin grafted. Superficial burns heal
by re-epithelialisation by surface migration of epidermal cells that have survived in
the depths of the hair follicles and sweat glands. When the cells meet with others,
contact inhibition takes place and cell migration ceases. The epidermal cells then
undergo a period of maturation during which they form into multiple layers, fill with
keratin and die. The surface of an unhealed superficial burn is wet and shiny. The
surface of keratinised epithelium is dry and matte. At this point the clinician recog-
nises the skin to be healed. Whilst this may seem rather imprecise, there is surpris-
ingly good inter-observer agreement.
Deep burns left to heal by secondary intention undergo contraction of the wound
bed and migration of epidermal cells from the margin of healthy skin. A deep burn
is said to be healed when the advancing edges of epidermis meet and the whole
wound is covered with epithelium. The unhealed portion is often covered with a dry
scab, beneath which the epidermal cells are migrating. While the scab remains
adherent the wound appears to be unhealed. However, gentle lifting of the scab
often reveals that the burn is already healed.
Deep burns that have been covered with a skin graft heal through “take” of the
grafted skin. The surface of a sheet of split-thickness skin graft has very similar
qualities to those of intact and uninjured skin. When applied over an excised, deep
burn the surface of the graft is dry and matte and remains so unless it suffers some
insult (e.g. infection) that causes it to lose its keratinised surface. Take of a skin graft
involves the ingrowth of blood vessels from the wound bed through the cut ends of
empty blood vessels on the under-surface of the skin graft. When first applied a skin
graft will not have a circulation and appears pale. It will be held in place by a fragile
fibrin mesh that has formed via the normal blood clotting mechanisms. For the first
few days the graft can easily be detached. As blood starts to enter the skin graft, but
struggles to get out again, the graft will take on a blueish discolouration due to slug-
gish circulation and maximum deoxygenation of the stagnant blood. At this point
ingrowth of blood vessels into the skin graft secures it more firmly to the wound bed
but it could still be detached if sufficient force were to be applied. After around
5 days blood is better able to flow into and out of the vessels. The skin graft will then
look redder than the surrounding, unburned skin and one would need to use consid-
erable force to detach the graft.
In many situations a split-thickness skin graft will be “meshed” before it is
applied to a burn. This requires the insertion of a grid of perforations, either by hand
or by machine. Meshing allows diamond-shaped spaces to open up between the lat-
tice of skin, permitting fluid to escape, encouraging the skin graft to conform to a
complex three-dimension shape and stretching the skin graft out over a wider sur-
face area. A meshed skin graft can be left unexpanded or expanded by a ratio of up
to 6 to 1. A meshed skin graft creates a much more complex situation because the
interstices will heal by migration of epidermal cells from the edges of the skin graft
(like a deep burn) and the graft itself will heal by “take”.
In a mixed depth burn where all of the above is taking place it is very difficult to
measure healing. Is the burn healed when epidermis has reformed over the superficial
areas? Or is it the day on which the epidermis has reached full maturity and acquired
a dry, matte surface? Is it the point at which the circulation in the skin graft is estab-
lished or when the interstices in the mesh are filled? Or is it when all of the scabs
have separated from the deeper, ungrafted areas?
As with the measurement of burn depth, it would be possible to biopsy the wound
to assess the integrity of the surface epithelium. It would also be possible to use the
biopsy to quantify the level of expression of chemicals called integrins, which play
an important function in the epidermal barrier [124]. However, an invasive process
is rarely justified outside of the research field unless there is a suspicion of serious
pathology such as malignancy.
In superficial burns the clinical consensus is that it is healed when the skin
surface is dry and matte, at which point the normal barrier function has been
restored. It is therefore reasonable to say that the most appropriate measurement
of healing of a burn is one that quantifies this process. Where precise measure-
ment is required (e.g. comparison of rate of healing of standardised wounds with
different dressings) this can be done by quantifying trans-epidermal water loss
(TEWL) [125], surface conductance [126, 127] or surface impedance [128]. A
healed burn with normal barrier function will have near normal TEWL and elec-
trical conductance/impedance, as measured on a control, adjacent, undamaged
skin site.
In small, deep burns allowed to heal by secondary intention photoplanimetry
may be used to measure the process [129]. The size of the unhealed portion is
calculated from standardised digital photographs of the burn using planimetry
software. The rate of decrease in the surface area is expressed as the Healing
Index (HI):
HI = ( Area of ulceron day 0 - Area of ulceron day x) / Area of ulceron day 0
This method is not applicable to superficial burns, whose surface area changes
little during healing [130].
Importance of Measurement of Burn Healing
Given that slow healing is related to the development of scarring, it is important that
clinicians are able to identify wounds that have not healed in order that an alternative
treatment plan can be devised. In the clinical setting, observation by an experienced
burn clinician is sufficient and has been shown to be reliable. If measurement of
healing is required for research (e.g. comparative data on two wound management
protocols) quantification of TEWL is more precise than clinical opinion but still suf-
fers from the drawback that dressings would need to be removed on a daily basis.
Measurement of Burn Outcomes
Apart from healing there are a number of other useful outcome measures for patients
recovering from burns. Survival and mortality rates are fairly crude outcome mea-
sures. The mortality rate has been found to be proportional to the age of the patient,
total extent of burn (%TBSA), extent of deep burn, presence and severity of inhala-
tion injury, the number of co-morbidities and the length of any delay in commenc-
ing resuscitation. For those who survive, the outcome can also be measured in terms
of length of hospital stay, number of days in the intensive care unit, time to healing,
time off work/education, and the cost of treatment. Some of these factors are clearly
influenced by the severity of burn and are therefore not reliable indicators of the
quality of care. As many of these measurements vary according to burn extent they
can also be related to %TBSA (e.g. length of stay per %TBSA).
When considering the outcome for an individual patient, who should make the
assessment?. Should it be the doctor, the therapist or the patient? This is not an
academic question. Studies have shown that the factors considered important by
patients are different from those identified by clinicians [131–134]. Clinicians use
items such as pliability, vascularity, height, appearance, skin instability, surface tex-
ture, physical limitations and colour [131, 134–136]. Patients live with their scars
24 h a day. Their awareness of the scars is related to the tightness and thickness of
the scar, together with sensory disturbance (itch, pain, hypersensitivity, numbness),
interference with activities of daily living and psychological factors, as well as their
visibility.
Standardised measurements of scars have been suggested by a number of authors.

The system most commonly used is the Vancouver Scar Scale (VSS) [137]. It was
designed to be a reliable, objective and universal scar assessment tool. A trained
observer uses the VSS to assign individual numerical scores for: pigmentation, vas-
cularity, pliability and scar height. The authors suggested that VSS could be used to
compare scar outcome for individual patients and the changes over time. They also
envisaged that it could be used to compare outcomes both within an institution and
between different burn services. The most significant drawback of VSS is that it is
very difficult to represent the heterogeneity of scarring even within a small burn
scar. Other authors have attempted to refine VSS or to introduce an alternative scar
assessment tool [131, 135, 136, 138–144]. None has proved to be significantly bet-
ter than the VSS, which remains in use today both in clinical practice and in
research.
Burns are prone to hypertrophic scarring, particularly when healing has been
prolonged [78], in children and individuals with darker skin types. The characteris-
tics of hypertrophic scars include thickening, erythema and itching or pain. Surface
elevation can be measured by taking impressions of the outer surface of the skin
[144] or by surface scanning electron-microscopy [145]. However, neither of these
methods makes an assessment of thickening that extends into the deeper tissues.
Ultrasound can measure the total thickness of a scar, elevation above the surround-
ing skin surface and the level of involvement of the dermis [146–151]. Scar thicken-
ing decreases as the scar matures. Indeed, some burn scars are atrophic 1 year
following injury [144]. Thus, any scale of scar thickness must include the possibil-
ity of a flat, raised or depressed scar.
Pigmentation changes are a common complication of burn scars. Skin colour is
determined by the concentration of melanin pigment in the skin and its vascularity.
Normal melanin is confined to the epidermis; skin capillaries lie within the dermis.
External measurement of skin colour will be influenced by the relative proportions
of melanin and haemoglobin present in the skin at the time of assessment [152].
Inter-rater variability for the visual assessment of the scar colour is low. While the
vascularity can be assessed reliably by a single observer, inter-rater variability for
melanin pigmentation is much higher and at least three observers are required for a
reliable assessment [134]. A number of studies have attempted to quantify colour
disturbances in burn scars. One method is reflectance spectroscopy. A tri-stimulus
colorimeter can be used to characterise colour according to an industrial standard
known as the CIE L*a*b* colour system [134, 153–156]. Alternatively a narrow
band of light can be used to measure simple reflectance and produce an erythema:
melanin index [134, 154, 157, 158]. Both methods have been shown to have high
inter-rater reliability in burn scars [134, 155, 157, 158].
Laser Doppler flowmetry and imaging have been used to quantify perfusion in
burn scars and to monitor changes over time [151, 159–167] (Wang 2010). Both
LDF and LDI have been shown to be valid and reliable. Early and hypertrophic
scars consistently demonstrate increased blood flow, which can be used to monitor
the response to treatment. For example, it has been shown that skin perfusion
remains constantly low in the face while wearing a transparent plastic face-mask,
despite movements and changes of facial expression but is followed by rebound

hyperaemia for up to 30 min after removal of the mask [167].
Other studies have been conducted to measure the biomechanical properties of
burn scars. Pliability is quantified using a tonometer, which is be simple to use in
burn scars and has a high degree of reliability [168–171]. Elasticity can be measured
by using an elastometer to measure skin stretch and deformability by applying gen-
tle suction to the scar [142, 147, 172]. Skin stiffness can be quantified by measuring
the velocity of propagation of surface (shear) waves within the scar. Skin stiffness is
higher both in burn scars and in skin that had healed following a burn without any
visible scarring [173].
Surprisingly few studies have been carried out on the histology of burn scars,
despite the frequency with which scars are excised during surgery to improve their
appearance and function. It is possible to examine a full-thickness biopsy for degrees
of hyperkeratosis, epidermal hyperplasia, presence and depth of scar (defined as
abnormally orientated collagen seen under polarised light), fibroplasia, vascular
proliferation and absence of skin adnexae and to assign each factor a numerical
score [174]. Intra-observer correlation for individual histological features is highly
variable but the total histologic score shows good intra-observer correlation.
However, it has been shown that correlation between the histomorphologic scale
and a visual analogue scale carried out by an experienced burn clinician has low
correlation [174]. This suggests that histology and visual inspection measure differ-
ent features of burn scars
There are many methods for measuring function following a burn. The active and
passive range of movement of individual joints can be measured using a goniometer.
Any limitation in movement due to skin or joint tightness can be measured in degrees
and compared with charts for the normal range of movement. Three-dimensional
kinematic analysis can also be used to quantify active joint movement during activi-
ties of daily living [175] e.g. high reach and hand to back pocket movement. Complex
movements, such as those of facial expression are more difficult to assess. The
Faciometer® can be used to quantify the movements of facial expression in patients
with facial burns [176]. This equipment, consisting of callipers and an electronic dis-
play, was introduced for the measurement of facial movement in patients with facial
palsy. Patients can be asked to copy movements (e.g. raising the eyebrows, showing
the teeth, pursing the lips) to measure asymmetry at rest and functional deficits.
From the patient’s point of view there are also a number of factors that
influence their psychosocial adjustment to burn scars (e.g. visible scarring, body
esteem, perceived stigmatisation, social adjustment and depression) [177].
These can be measured by validated self-administered questionnaires and moni-
tored over time.
Why Measure Outcomes in Burns?
Patients and those who fund healthcare want to be assured that burns services pro-
vide the best possible outcomes. In an ideal world, data would be collected on a
routine basis for validated outcome measures. Individual clinicians and teams would
be able to compare their results with peers and identify areas in which improve-
ments can be made. In reality, robust data and reliable outcome measures cannot be
assured.
For individual patients, outcome measures could be used to identify aspects of
their recovery that could be improved and to measure the result of treatment.
Measurement of the range of movement of individual joints is routinely done by
physical therapists but few other outcome measures are used to guide treatment in
clinical practice.
Many patients acquire their burns through accident, negligence or criminal acts.
Medical experts are often called upon to assess victims to facilitate decisions regard-
ing sentencing and compensation for functional and aesthetic impairment [178]. In
the absence of any standardised, reliable and valid scar assessment tool, the judge
must rely upon the expert witness statements. It is therefore imperative that experts
include a full narrative description of the aesthetic, physical and psychosocial con-
sequences of the burn, the expected time-scale of any natural improvements and the
feasibility of future treatment to modify the scars.
Conclusion
There are multiple factors that can be used to quantify the magnitude of a burn
injury. Throughout a patient’s clinical course there are also many aspects of their
care and their response to therapy that can be measured and monitored. Some are
measured as a matter of routine clinical practice. Others are only measured in the
research setting. Increasingly, those who fund health care systems are seeking evi-
dence of outcomes that can be quantified and compared. We hope that we have
provided useful information that may help those who take on this role.
Acknowledgements Thanks are due to Miss Christina C Mackenzie and Dr Rodrigo Figueiredo
for logistical support.
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Epub 2009 Jan 23.
Chapter 15
Managing Scars: Measurements to Improve
Scar Management
Luc Téot, Claude Roques, Sami Otman, Antonio Brancati,

and Rainer Mittermayr
Keywords Scar • Scar Assessment Scales • Vancouver Scar Scale • Seattle Scar
Scale • Manchester Scar Scale • POSAS • Stony Brook Scar Scale • Tristimulus
System • Narrow band simple reflectance meter • Laser Doppler • Planimetry
TUPS
Introduction
Cutaneous scars are the normal and inevitable response to wounding in mamma-
lian tissue repair. Scar formation is considered as an integrative part of the complex
and dynamic process of normal physiological wound healing to restore skin integ-
rity following injury. Scarring represents the final stage of tissue repair and is
mainly characterized by a balanced extracellular matrix production, deposition,
L. Téot, M.D., Ph.D. (*)

Wound Healing Unit, Department of Surgery, Montpellier University Hospital,
Avenue Doyen Giraud, Montpellier, Hérault 34295, France
e-mail: lteot@aol.com
C. Roques, M.D.
Département of Physical Therapy, Pediatric Rehabilitation Centre,
CSRE Lamalou le Haut, Lamalou les Bains, France
S. Otman, M.D. • A. Brancati, M.D.
Wound Healing Medico-Surgical Unit Burns Unit, Montpellier University Hospital (MUH),
Montpellier, France
R. Mittermayr, M.D.
Department of Wound Healing, AUVA Research Center, Ludwig Boltzmann Institute for
Experimental and Clinical Traumatology, Vienna, Austria

292 L. Téot et al.
Fig. 15.1 Extensive burn

scars
degradation, and remodeling. In the optimal case, a minor scar results with limited
functional and aesthetic outcome. However, in some individual and in particular
burn victims, an abnormal scar formation is observed (Fig. 15.1). Causative factors
which are implicated in pathological scar formation are a prolonged and subse-
quently dysregulated inflammatory phase, a misbalance between pro- and
antifibrogenic factors, the involvement of specific cell subpopulations, wound
depth and localization as well as a genetic predisposition. Abnormal scar forma-
tion can end up in long-term problems such as aesthetical disfiguring often causing
a certain psychological burden and functional limitations (e.g. decreased range of
motion of joints due to scar contraction – contractures).
It is therefore mandatory to tightly assess the process of scar formation in order
to early recognize abnormal scarring. The early diagnosis of a developing patho-
logic scar can have a considerable impact on the final outcome, due to the fact that
preventing evoluting pathologies are easier to treat than a manifest pathology.
Scars normally develop in 6–8 weeks after complete reepithelialization and the
maturation phase is considered to take up to 2 years depending on the wound etiol-
ogy. During this period different features of the maturing scar are predominant like
redness, hypertrophy, indurations, retractions, pigmentation disorders, pain or itch-
ing. However, it remains difficult to determine in an early stage if a scar will become
pathologic. A gradual assessment can be proposed from a month to month basis,
beginning with the initial clinical evaluation at the end of the first month after com-
plete healing [1]:
1. First month evaluation:
(a) General features of the scar are determined focusing on color and elevation.
At this stage, color and vascularity seem to be the most reliable parameters.
If a scar is red, hypervascularized, the risk of hypertrophy is high. Preventive
measures as silicone gel sheets will be useful.
(b) Laser Doppler evaluation can be proposed as a complementary technique to
assess hypervascularization.
15 Managing Scars: Measurements to Improve Scar Management 293
2. Second month evaluation:

(a) Clinically, changes in width, height, color are more evident. Local treatments
can be indicated in the presence of hypertrophy, redness and increase in
width.
(b) Laser Doppler evaluation can confirm the scar hypervascularization.
3. Third month evaluation:
(a) Signs of hypertrophy are generally evident and visible and troubles in pli-
ability and texture are patent.
An appropriate clinical assessment strategy can be considered as a good measure
to prevent pathologic evolution of scars, independent of scar origin and intensity of
the pathologic process.
Clinical evaluation has to be standardized in order to obtain the maximum of
useful information. This is important in determination of the progression profile and
to anticipate therapeutic consequences. Clinically, some tools can be used in order
to help physicians to characterize and categorize the developing abnormal scar.
Semi-quantitative methods to assess scars have been developed by using scales to
make subjective methods more objective. Principles of these health measurement
scales [2, 3] should be considered when applying and interpreting them. Key attri-
butes include validity, reliability, responsiveness, and feasibility [4]. Reliability
refers to the reproducibility of the results obtained by a certain measurements or
ratings assessed by observers. Validity declares the question if we really measure
what we want to measure (=accuracy of measurements or ratings).
Validity
This describes the accuracy of the measures assessed by a certain scale to what it is
supposed to be measured. Evidence is accumulated longitudinally over time.
Comprehensiveness and clarity of the scale items with relevance for the target popu-
lation/scars are assessed by the content validity. The degree of correlation to a gold
standard measure is given by the criterion validity. For scars, instead of criterion
validity often the construct validity is used due to the lack of gold standard mea-
sures. It proposes logical relations between the scale to be evaluated and other mea-
sures. Additionally, patterns of scale scores for groups known to differ on relevant
variables are evaluated. Methods in this context are (1) convergent validity; the
scale correlates with instruments measuring the same attribute (2) divergent valid-
ity; no correlation between the scale and relevant instruments.
Instruments measuring similar attributes have a correlation index ranging from
0.4 to 0.8, with a correlation coefficient of 0.6 between two measures representing
a strong association.
Discriminative validity assesses whether the scale scores will reflect known dif-
ferences of a certain attribute amongst different groups. ANOVA can be used to
294 L. Téot et al.
detect significant difference in mean scores between evaluated groups, thus provid-
ing evidence of construct validity.
Reliability
A measurement should obtain same results repeatedly to be considered as reliable

or consistent. It can be differentiated between multiple assessors using a scale (Inter-
rater reliability; degree of same results between different raters) and intra-rater reli-
ability (degree of results consistency over time assessed by one rater). Coefficients
to assess this both parameter include Pearson’s correlation, intraclass correlation
coefficient (continuous data), and the Kappa coefficient (categorical data). Minimum
levels for reproducibility are 0.7 for group comparisons and 0.9 for individual mea-
surements over time.
The internal consistency evaluates if each scale item is measuring a distinct but
related aspect of the scar attribute being assessed. An indicator of internal consis-
tency is Cronbach’s alpha and a value >0.7 is considered acceptable.
The scale intended to be used should be additionally suitable to detect differ-
ences or changes in outcome (responsiveness) of the specific scar entity. The semi-
quantitative results obtained by the specific scale should be easily interpretable with
immediate translation into clinic (result with consequences in further treatment?).
Furthermore, the scale should be realizable with a minimum of administrative as
well as patient effort.
Subjective Assessment by Scales
To apply scales in scar evaluation was already introduced in 1978 [5] with assess-
ment of various parameter of scar appearance (color, consistency, and thickness) but
lacking appropriateness in others than hypertrophic scars.
In 1988, Smith et al. [6] proposed to use colored photographs in scar assessment
and included the assessment of aesthetic/cosmetic aspect. The inter-rater reliability
with 4 investigators was up to 0.94, however, with poor single rater reliability. A
similar scale was developed in 1989 by Leung et al. [7] who used laser doppler
flowmetry as an additional (objective) tool in assessing scars.
Vancouver Scar Scale
The first validated and widely used assessment scale was described in 1990 by
Sullivan et al. [8]. The Vancouver Scar Scale (VSS) is used to assess scars following
burn injury. Variables covered by this scale include: vascularity, analyzed by scar
Table 15.1 Vancouver Scar Scale

Parameter Score Description
Vascularity 0 Normal
1 Pink
2 Red
3 Purple
Pliability 0 Normal
1 Supple (flexible with minimal resistance)
2 Yielding (gives way to pressure)
3 Firm (inflexible; resistant to manual pressure)
4 Banding (rope-like tissue that blanches with scar extension)
5 Contracture (persistent shortening, causing deformity or
distortion)
Height 0 Normal
1 >0 mm; <2 mm
2 >2 mm; <5 mm
3 >5 mm
Pigmentation 0 Normal
1 Hypopigmentation
2 Hyperpigmentation
Total Score 0 (normal) to 13 (maximum alterations)
redness; degree of hypertrophy, quantified by scar height; pliability, related to con-

tracture and elastic texture of the scar; and pigmentation (Table 15.1). Initially vali-
dated on a defined area of 4 m2, it was shown that this scale is not applicable in large
and irregular scars where hypertrophy, pliability and color are not homogeneous.
Moreover, visual distinction of dark pink to light red or others colors linked to a
vascularity subscale is difficult. It is also particularly difficult in assessing the scar
color to judge the two different color parameters of pigmentation and vascularity
separately. Moreover, the parameter of pigmentation is not an ordinal variable and
therefore cannot be added equally to the three other parameters. In the item pliabil-
ity the VSS associates elasticity and scar contraction. These items, however, are in
fact complementary and cannot be considered as an identical phenomenon.
Clinically, measurement of hypertrophy in millimeters is also easily subjected to
errors, dependant on the assessor. Similarly, the assessment of pliability as well as
pigmentation has also limitations due to high inter-rater variability (moderate inter-
rater reliability for each item; Kappa 0.4–0.56). No evidence of intra-rater reliabil-
ity, internal consistency and validity was provided. Perception of the scar by the
patient is not recognized in the overall score.
Even the authors propose to include further parameter (pain and itching) to the
scale and recommend a 3–4 month follow-up in order to improve score reliability.
Therefore, several modifications were proposed to the original VSS described by
Sullivan et al. [8]. Baryza et al. [9] supplemented the pigmentation item with a
“mixed pigmentation” category. The scar height was changed to a ordinal 5-point
scale. These modifications demonstrated a higher inter-rater reliability (intra class
296 L. Téot et al.
coefficient 0.81). In order to increase staff compliance and to aid in scoring a pocket
size VSS was designed.
Nedelec et al. [10] proposed several modifications to increase reliability and
validity of VSS, an increase in the awareness by training evaluators to the use of the
scale, an improvement of the quality of subscales and the documentation of addi-
tional pertinent information (e.g. visual analogue scales to rate pain and itching
subjectively by the patient). However, inter-rater reliability was poor for the sepa-
rate variable (0.2–0.42).
Seattle Scar Scale
Yeong et al. [11] offered a rating scale to assess scar surface, thickness, border
height, and color differences between a scar and the adjacent normal skin. Raters
were trained with the use of a standardized set of photographs that provide examples
of the scores to be assigned to each level of severity of each scar characteristics. The
intrarater reliability was 0.94, 0.95, 0.90, and 0.85 for scar surface, border height,
thickness, and color, respectively (weighted kappa <0.81). First, negative values
were included in the assessment scale to describe the opposite range of the hyper-
trophic score (hypo-pigmentation, atrophy). However, major criticism was that even
rating parameters negatively, an “improved” total score is yielded.
Manchester Scar Scale
In 1998, Beausang et al. proposed [12] several modifications to the VSS by realiza-
tion of separate color assessment and introduction of a visual analog scale (VAS) to
rate global aspect of the scar. Parameters included in this scar scale are: color (per-
fect, slight, obvious, or gross mismatch to surrounding tissue); mate versus shiny;
contour (range from flush to keloid); distorsion (range from none to severe); and
texture (range from normal to hard). The scores from the two scales (VAS and cat-
egorical scale) are summed to give an overall score for the evaluated scar, with
higher scores representing clinically poorer scars. The authors found a good inter-
rater reliability (0.87), although at least three observers are required to obtain such
reliability.
To the original Manchester Scale the size of the scar (<1 cm; 1–5 cm; >5 cm)
as well as the number of scars (single vs. multiple) were included by Bayat et al.
[13, 14] and these complete dataset represent the physical scar examination in the
Manchester Scar Proforma (Table 15.2). Furthermore, other informations such as
the ethnic background, scar history, location, scar symptoms (pruritus, pain, psy-
chological, etc.), and already applied treatment modalities with treatment response
are assessed. A standardized color photograph is required at each consultation as
a reference to evaluate effectiveness of treatment since changes occur slowly.
Table 15.2 Manchester scar proforma – physical examination

Visual analogue scale
Excellent Poor
A Colour
Lighter Perfect 1
Slight mismatch 2
Obvious mismatch 3
Darker Gross mismatch 4
B Mate (1) Shiny (2)
C Contour
Flush with surrounding skin 1
Slightly proud/indented 2
Hypertrophic 3
Keloid 4
D Texture
Normal 1
Just palpable 2
Firm 3
Hard 4
E Margins
Distinct 1
Indistinct 2
F Size
<1 cm 1
1–5 cm 2
>5 cm 3
G Number
Single 1
Multiple 2
The image of the scar is captured under standard light conditions, using a digital
color video camera linked to a computer. The Manchester Scar Proforma is a useful
means in the assessment of localized burn scars, especially for hypertrophic or kel-
oid scars, however, the vascularity is not being evaluated.
Martin et al. [15] investigated the correlation between VSS and patient subjective
opinion of their scars; patients were asked to rate they own scar (question 1) and
how the scar was visually perceived by other people (question 2) using a visual
analog scale. A significant correlation existed between the VSS score and the VAS
score for question 1 (r = 0.646, P = 0.003) but not between the VSS score and VAS
score for question 2 (r = 0.099). “The VSS measurement of the scar bears no rela-
tionship to the patient’s opinion of their scar early after a burn injury. As the scar
improves over time, the patient’s opinion of their scar appears to improve and shows
better correlation with the VSS rating. Conversely, the patient’s impression of what
people around can think of their scar still has no relationship to the VSS rating; sug-
gesting that scar acceptance by the patient is incomplete despite objective improve-
ment in the quality of the scar. Although the VSS was never intended to measure a
298 L. Téot et al.
patient’s opinion of their scar, these preliminary findings emphasize the necessity of
including a patient-centered subjective component to routine scar monitoring and
assessment”.
POSAS
The Patient and Observers Scars Assessment Scale (POSAS) introduced the opin-
ion of the patient [16]. Initially this scale was tested on burn scars. The observer
scale assesses vascularity, pigmentation, thickness, relief, pliability and surface area
(Table 15.3) and the patient scale contains six parameters: pain, itching, color, stiff-
ness, thickness and relief (Table 15.4).
The consistency of POSAS seems to be superior to the VSS. Additionally, the
intra class variation coefficient values of the observer scale are better than those of
VSS (Table 15.5).
Linear regression of the general opinions on scars of the observer and the patient
showed that “the observer’s opinion is influenced by vascularization, thickness, pig-
mentation and relief, whereas the patient’s opinion is mainly influenced by itching and
the thickness of the scar”. In POSAS the observer scale shows less variability than
VSS between repeated assessments. Van de Kar et al. [17] evaluated the POSAS on
linear scars with an item add in the observer scale, the surface area. The authors con-
clude that Patient and Observer Scar Assessment Scale is an appropriate subjective
tool for the evaluation of linear scars and offer a suitable, reliable, and complete scar
evaluation tool. The opinion of the patient should not be neglected in order to evaluate
properly the quality of life of the patients; POSAS takes into account their experience.
But, as the VSS, each scar characteristic must be assessed in POSAS. Yet this subjec-
tive scale is very interesting because it associates clinical and human parameters.
Stony Brook Scar Evaluation Scale
In 2007 Singer et al. [18] developed a six item (width, height, color, hatch or suture
marks, and overall appearance) ordinal scale to measure short term outcome. A
binary response (1 or 0) to each individual attribute as well as overall appearance is
assessed thus yielding to scar score ranging from 0 (worst) to 5 (best). However, due
to the short term evaluation it has limited applicability in pathological scar assess-
ment as is therefore used primarily in research. The inter-rater reliability was shown
to range between 0.75 and 0.92.
One of the problems of this scales with which an observer is confronted is to
discriminate both pigmentation and vascularization, which finally gives the color of
the scar. A useful mean to more objectively evaluate pigmentation, which can be
masked by the scar vasculature, is to use a Plexiglas tool. In the same time when the
scar is compress by this tool the scar vessels are compressed and the pigmentation
15
Table 15.3 Observer scar assessment scale (OSAS)

Normal Scoring Worst imaginable
Parameter Skin 1 2 3 4 5 6 7 8 9 10 Scar
Vascularity ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Normal skin (1–2)
Pale-pink (3–4)
Pale-red (5–6)
Red (7–8)
Dark red (9–10)
Pigmentation ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Normal skin (1–2)
Slight hypopigmentation
(3–4)
Hypopigmentation (5–6)
Mix (7–8)
Hyperpigmentation (9–10)
Thickness ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Normal skin (1–2)
Slight hypertrophic (3–4)
Hypertrophic pale-red (5–6)
Managing Scars: Measurements to Improve Scar Management
Hypertrophic red (7–8)

Hypertrophic dark red
(9–10)
Relief ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Smooth (1–2)
Slightly uneven (3–4)
Uneven (5–6)
Very uneven (7–8)
(continued)
299
300
Normal Scoring Worst imaginable

Parameter Skin 1 2 3 4 5 6 7 8 9 10 Scar
Disfiguring (9–10)
Pliability ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Normal elasticity (1–2)
Almost normal (3–4)
Moderate (5–6)
Poor (7–8)
Contracture (9–10)
Size ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Non-existent (1–2)
Very small (3–4)
Small (5–6)
Medium (7–8)
Large (9–10)
Total score
L. Téot et al.
Table 15.4 Patient scar assessment scale (PSAS)

Scoring
Question 1 2 3 4 5 6 7 8 9 10
No, no Yes, worst
complaint imaginable
Is the scar painful? ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Is the scar itching? ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
No, as Yes, very different
normal skin
Is the color of the scar different? ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Is the scar stiffer? ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Is the thickness of the scar ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
different?
Is the scar irregular? ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪
Total score
Table 15.5 Intraclass correlation coefficient observer vs. Vancouver scales dependent on number
of observers
No. of observers ICCa Observer scale VSS
4 Mean (95% CI) 0.92 (0.87–0.95) 0.90 (0.84–0.94)
3 Range 0.88–0.90 0.86–0.88
2 Range 0.81–0.89 0.78–0.84
1 Range (0.95% CI) 0.73 (0.62–0.82) 0.69 (0.57–0.79)
ICC Intraclass correlation coefficient, CI confidence interval
a
The ICC together with the 95% CI is given for the scores of the observer scale and the Vancouver
Scar Scale completed by a single observer and completed by four observers. For two and three
observers, separate ICC calculations were required for each possible observer combination. The
minimum and the maximum ICC of these combinations are given (From Draaijers et al. [16])
can be judged via the transparent Plexiglas [19]. Evaluation of vascularity was
described by Gavroy et al. in 1995 [20] using the vitro pressure test. A transparent
plate is applied on the scar and the time of recoloration (=recapilarization) is mea-
sured. This time is reversely proportional to the current intensity of inflammation.
However, test conditions have to be very strict and where is no validation yet.
Various other scales or modifications on already existing scales were developed
in order to describe scars more appropriately with increasing reliable results.
However, all these scales are long and difficult to use on extended scars. They are
subjective and each one rates different items useful for scar assessment (inflammation,
pain, itching, around of the scar, history of the patient and his family, patient opin-
ion); they are useful in the daily practice. POSAS seems to be the more interesting
scale to rate burn scars and also linear scars because the inter class variation
coefficient values of the observer scale are better than those of VSS and it takes on
amount the patient opinion. Moreover for clinician, it takes into account the patient
opinion and the objective of the treatment is the patient’s quality of life. Manchester
scale is adapted for hypertrophic scars and keloids as it associate the history of the
patient and the scar to the physical examination and photograph.
302 L. Téot et al.
Technical Assessment
Many factors can distort technical assessment and interpretation of these results
remains subjective. This renders it difficult to consider precisely a scar evolution
and the effectiveness of various applied therapies. Scars can be assessed by non
invasive objective means studying morphological as well as physiological charac-
teristics. These are primarily color as a function of vascularity (difficulties to dif-
ferentiate to pigmentation as outlined above), scar volume and area, and elasticity.
Color – Vascularization
Scar coloration is determined by pigmentation and perfusion. Color mismatch can

be often stigmatizing for patients suffering from extended scars especially in
exposed body areas. Inflammation induces an increase of the vascularity. Scars
prone to abnormal evolution show increased and prolonged inflammation.
Assessment by Photographs
The authors proposed in a preliminary study [21] to assess the color of scars by
artificial vision. Assessment required precise experimental conditions to limit the
sources of errors (e.g. temperature, ambient light, and position of the patient). The
use of black and white photographs appeared more reliable than evaluating colored
images because of their complex composition. Therefore, the photographs were first
captured in color, further processed in black and white and the evolution was evalu-
ated by substraction of these two images. The quantification was done in the level
of grey scale from 0 for black to 255 for the white (Fig. 15.2); the use of artificial
vision appeared to be an interesting means for evaluating of scar color. However,
limitations of the study were a low patient number and a difficult set-up of condi-
tions to reproduce reliable results in a daily clinical practice.
Davey et al. [22] proposed to capture an image from a video on a computer to
carry out quantitative scar color analysis using a custom-written computer program.
In conclusion, they found a theoretical possibility to measure the progress of a scar
and to compare effects of different treatment modalities.
Although color assessment is a part of most of the subjective assessment scales,
perception of the observer is limited in reliably quantifying color and/or intensity.
Even more, it is difficult for the human brain to memorize colors and to compare
adequately when assessing a scar over time.
The Tristiumulus System or narrow band scanning reflectance instruments can be
used to assess the cutaneous (scar) color using the reflectance spectroscopy methodology.
Fig. 15.2 Subtraction of two images – the white area shows the evolution of the scar
Tristimulus Systems
The Tristimulus System is implemented in several devices and is based on the

human color perception model [23]. Currently, the Minolta Chromameter CR-200;
CR-221-R and CR300 (Minolta, Osaka, Japan), the Labscan (Hunter Associates
Laboratory, Reston, Va.), and MicroColor (Dr. Bruno Lange GmbH, Düsseldorf,
Germany) are available.
The Chromameter emits light from a xenon lamp and detects the reflected light
by photodetectors at different wavelengths. Brightness (black/white) as well as
color values can immediately be obtained.
The MicroColor device is based on a similar technology as the Chromameter
[24] while the Labscan (full-spectrocolorimeter) uses a similar method to that of the
human eye to perceive color details [25].
Narrow Band Simple Reflectance Meters
The narrow band spectrophotometric devices uses the known light absorption curves
of melanin and hemoglobin to estimate the constituent element content values [26].
In the DermaSpectrometer (Cortex Technology, Hadshund, Denmark), diodes are
304 L. Téot et al.
used as the light source emitting green light (568 nm) and red light (655 nm). The
reflected light is measured by photodetectors, and due to the absorption of green
light by hemoglobin and absorption of red light by melanin indices for erythema
(hemoglobin) and melanin can be calculated [27].
Draaijers et al. [19] compared a tristimulus colorimeter (Chromameter), and a
narrow band simple reflectance meter (DermaSpectrometer) to assess the color of
the scars. While the results obtained by the DermaSpectrometer are immediately
displayed and available, the Chromameter necessitates a computer connection, and
after calibration, results can be accessed via the computers monitor. They conclude
that the reliability of measurements obtained by the DermaSpectrometer and the
Chromameter is good for both devices. These tools were more reliable to assess scar
color and vascularization with a single observer than subjective scales. However,
the single measure ICC (r = 0.76) of the vascularization assessment was acceptable,
whereas the single measure ICC of pigmentation was moderate (r = 0.59). But for a
reliable assessment of pigmentation three observers were necessary (r ³0.77).
Laser Doppler
Laser Doppler Systems are used to assess local superficial blood flow thus reflecting
the vascularity [20]. Red or near-infrared wavelengths are used to detect moving
blood cells which shift the frequency of the laser light according to the Doppler
principle.
Laser Doppler Flowmeter measure local cutaneous perfusion and hence scar color
can be evaluated. It correlates positively with various colorimeters such as the
Chromameter and MicroMeter [24]. However, this device has less sensitivity in com-
parison to visual erythema assessment, can only measure a limited area, and is there-
fore not recommended in determining scar color [28]. In assessing scar color, the blood
volume rather than the velocity of erythrocytes determines the skin redness [29].
The Laser Doppler Imaging technology uses a laser light beam which scans a
surface and generates a two-dimensional image of perfusion. This technology is
applied in burn depth assessment [30] but is also suitable for scar evaluation [31].
Scar Area (Planimetry)
Using the continuous measurement of scar surface area which is referred as planim-
etry various parameters can be calculated. Percentage of hypo- or hyperpigmented
areas as well as changes in hypertrophic scar extension can be expressed in com-
parison to the original scar. Furthermore, contraction over time can be assessed
which is clinical interest in terms of recognizing future contractures. The most com-
mon techniques to perform surface area measurement are planimetry by photogra-
phy or tracing the area of interest on a transparent foil which is when measured.
However, attention has to be drawn on taking images with standard conditions (e.g.
object distance, ambient light, camera settings). Photographic planimetry is reliable
on flat surface areas whereas in curved body parts the tracing technique is more reli-
able and should be therefore applied. A drawback of this mean is that during time
the borders of a scar can be difficult to discriminate thus making it more difficult to
measure.
Scar Height and Volume
Evolution of the proliferative process and is also used to differentiate an hypertro-

phic scar, a process generally limited in extension but also in time, from a keloid, a
permanently promitotic stage with cell proliferation, covering the edges and tres-
passing the scar itself.
To evaluate the proliferative process of a scar and to determine an eventually
abnormal scarring the extent of hypertrophy can be checked. A scale analyzing
especially hypertrophy was developed by Crowe et al. [32] . This scale has a “sub-
stantial” reliability when used by expert or novice observers. However, evaluating
scar height subjectively it neglects the proportion below the surface and is therefore
only of limited accuracy. An objective evaluation of the scar thickness is the ultra-
sound technique. This methodology acquires information due to the different ultra
sound wave propagation at the interface of different tissues using high frequency
(5–20 MHz). It allows assessing the entire scar volume and the thickness under the
skin surface (Fig. 15.3). To obtain reliable and useful information a professional
training is mandatory. Already many different devices are available such as
DermaScan (Cortex Technology) and studies on validation as well as in clinical
scenarios were performed. They have demonstrated to be reliable and accurate [33,
34]. Lau et al. [35] used an ultra sound palpation system (TUPS – tissue ultrasound
palpation system) which is portable. TUPS showed a good correlation with the ultra
sound skin scanner and facilitated clinical application.
The system demonstrated also a good inter-rater reliability (p <0.01), but with
the drawback of only fair correlations with the VSS score (0.42, p <0.01).The
authors explained this discrepancy with the argument that TUPS measures the entire
scar thickness.
Three dimensional (3D) systems allow studying the scar surface with high
definition and reproducibility [36], and are available for volume assessment and
planimetry. Current disadvantages of these highly sophisticated devices are the high
costs and the availability of only few (scientific) studies. An optical 3D in vivo
device uses the principle of digital stripe projection technique, whereby a parallel
stripe pattern is projected on the skin. This scene is captured by a CCD camera and
deflection of the parallel stripes on skin surface irregularities can be measured quali-
tatively and quantitatively. The thickness of the scar is represented by colors (yellow
– most elevated areas, blue – lowest areas) varying according to the grade of hyper-
trophy (Fig. 15.4). The hypertrophy can be measured by first defining a transversal
306 L. Téot et al.
Fig. 15.3 Thickness

Skin
measured by TUPS (Lau
surface
et al. [15])
Skin/fat
interface
Tissue
bottom
Fig. 15.4 Defining a

transversal section. The
differences in height will be
measured between these two
points
line within the image. The difference within this transversal line are when depicted
in a curve by a reconstruction process (Fig. 15.5) demonstrating the thickness varia-
tions of the scar. The exact evaluation of the scar changes in height issues to cartog-
raphy of the surface variations with a less than 1 mm precision, the volume of
selected zone can also be obtained and measured.
The volume is calculated from selected surface areas and varies according to
the degree of hypertrophy in this area. This 3D system has the following advan-
tages: non-contact measurement, fast assessment, direct measurement of the skin
surface, macro- and micro-topometry, high resolution, high precision and its easy
handling. It allows objective and precise measurements in evaluating hypertro-
phy during its evolution and the opportunity to visualize treatment efficacy.
Fig. 15.5 Transversal image reconstruction in the PRIMOS software can define the height of
hypertrophy
Additionally, surface irregularities such roughness can also be measured.

Requirements for accurate measurement are: strict perpendicular position of the
area of interest and the CCD camera and for repeated measurements same condi-
tions to compare the data.
Ben Taylor et al. [37] used a 3D digitizer to assess keloids and concluded that
“this technique could allow monitoring of patients on treatment or comparisons of
treatments in research setting”.
Measurement of the Biomechanical Properties of a Scar
Normal skin is elastic, extensable, and resist to a certain degree of tensile strength.
A scar can substantially affect these properties which are also often subsummized
under the term of pliability. Therefore, testing pliability can comprise important
information concerning the correct diagnosis of a scar and whose advancement.
Pliability can be tested by using different types of loading mechanism which can be
applied in a vertical direction (suction and pressure) and in a horizontal direction
(torsion and extension) [38].
308 L. Téot et al.
Pressure (Tonometer)
Originating from the ophtalmometry (pneumatotonometer) to measure the intraocu-

lar pressure these devices were adapted to measure scar elasticity. Several different
apparatus were developed and tested in evaluating scars including durometer,
tonometer, pneumotonometer, and cicatrometer.
The Tonometer evaluates the effects of therapies on burn scars [39]. Tonometry
affords a precise evaluation of burn scar diagnosis and prognosis; it is possible to
avoid arbitrary clinical evaluation of the burn scarring process. The tissue
Tonometer provides a repeatable, objective index of burn scar pliability. Using the
methods described, it is a simple clinically useful technique for monitoring an
individual scar. Hence, it provides to clinicians a reliable, transportable and objec-
tive tool to document scar outcomes. The modified tissue Tonometer, [40, 41] pro-
vides a standardized method to longitudinally measure scar tissue pliability after
burn injury and a repeatable, objective index of burn scar pliability. Using the
methods described, it is a simple, clinically useful technique for monitoring an
individual’s scar.
A pneumatonometer (Medtronic Xomed, Jacksonville, Fl) showed a good cor-
relation with the Vancouver Scar scale in burn scar assessment and demonstrated a
sensitivity of 87% [27]. However, in thicker and non-compliant scars a ceiling effect
was demonstrated making the method in these cases not reliable [42] .
The use of Rex Durometer Max Hand to measure the degree of skin indura-
tions in lipodermatosclerosis was described by Romanelli et al. [43]. This instru-
ment is the international standard for measuring the hardness of plastic and
nonmetallic material. It was when Magliaro et al. [44] who tested the Durometer
on hypertrophic burn scars. A direct linear relation (r = 0.769) between the durom-
eter and a skin severity score was observed (p <0.01). The authors categorize the
device as easy to use, and as a simple method with a very large inter- and intrao-
bservers variability. This technique is used to assess tissue hardness in sclero-
derma [45] and in skin induration in chronic venous diseases [46]. However, it is
noteworthy that in specific anatomic locations with the bone directly under the
skin the system is not applicable. In this case the hardness of the bone influences
the measurements [45].
Suction
Within a defined scar area controlled negative pressure is applied causing skin
deformation which can be analyzed by a software program giving a typical defor-
mation versus time curve. The Cutometer skin elasticity meter [47] was compared
to a subjective evaluation of scar pliability (from 1 for normal skin to 10 for stiffest
scar imaginable). Only one observer can reliably use the Cutometer for measure-
ment of scar elasticity (r = 0.76) which is in contrast to the subjective evaluation
scale where at least two observers are needed to make evaluation reliable (r = 0.79).
An alternative device with a handheld larger in suction chamber diameter (10 mm
vs. 6 mm) is the dermaflex, although currently without any scar assessment trials
published.
Torsion
The principle of this technique is that a rotating disk is placed in contact with the
skin surface and the rotational force is applied by a motor with adjustable torsion
power. The Dermal Torque Meter (Dia-stron) was used to evaluate burn scars [48]
with resemblance of Cutometer readings. However, parameters of both techniques
which corresponds to each other show only poor correlation [28].
Extension
Two certain loci of skin are distracted and this specific strip is stretched measuring
extensibility or stiffness. The method has been described for scar evaluation,
although only few and anecdotal reports are available.
All this techniques can help to rate the evolution of scars by the assessment of
one of the scarring parameters; Ultra sound Palpation System, Tristimulus
Colorimeter, Durometer, Cutometer, Tonometer are easy to use and reliable. They
allow assessing color, height, induration or pliability objectively. Ultra sounds skin
scanner, laser Doppler color, 3D analysis are more complicated and expensive; their
use are more devoted to research.
Conclusion
Scars evolution through the maturation stage needs permanent adaptations with dif-
ferent treatments applied. The assessment of various parameters is linked to
inflammation; clinical signs associated to scars are subjective but reliable and vali-
dated with a risk of error depending on the operator. VSS, VAS, POSAS and
Manchester scales assess different scar characteristics; they can, be used in different
types of scars, easy to use but subjected to errors. Raters must be trained to use the
different clinical scales. We think that POSAS is more useful to assess linear or burn
scars than VSS and Manchester Scar Proforma for hypertrophic scars and keloids.
Technical tools exist to precisely evaluate the different parameters. Most of them
rate only one parameter, and they are more accurate; some of them are easy to use,
with a low cost and they can be used for routine clinical assess. Others more com-
plex for using, more expensive can be use in research or treatments evaluation.
310 L. Téot et al.
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Chapter 16
The Use of Biophysical Technologies
in Chronic Wound Management
Uwe Wollina, Birgit Heinig, and Luther Kloth
Keywords Wounds • Leg ulcer • Pressure ulcers • Diabetic foot syndrome

Therapeutic ultrasound • Topical negative pressure • Laser • Light-emitting devices
• Infrared • Shock waves • Electrotherapy • Current
Introduction
Chronic wounds are a common clinical challenge. Their aetiology is heterogenous.

The most common types are leg/ foot ulcers and pressure ulcers. In a meta-analysis
of population-based prevalence studies, prevalence rates of open ulcers ranged from
0.12% to 1.1% of the population. The prevalence rate of open or healed ulcers was
reported to be 1.8% [1]. Venous leg ulcers are the most common cause of chronic
wounds in the western world. An estimate of the prevalence of venous ulceration is
approximately 0.3% of the adult population [2] with individuals aged 60 years and
over at higher risk [3].
U. Wollina, M.D. (*)

Department of Dermatology and Allergology, Academic Teaching Hospital
Dresden-Friedrichstadt,
Friedrichstrasse 41, 01067 Dresden, Germany
e-mail: wollina-uw@khdf.de
B. Heinig, Dipl-Med
Department of Wound Healing, Centre of Physical and Rehabilitative Medicine, Academic
Teaching Hospital
Dresden-Friedrichstadt, Friedrichstrasse 41, 01067 Dresden, Germany
L. Kloth, PT, MS, FAPTA, CWS, FACCWS
Department of Physical Therapy, College of Health Sciences, Marquette University,
Milwaukee, WI, USA

314 U. Wollina et al.
According to the World Health Organization (WHO) the total number of people
with diabetes was 171 million in 2000 and is projected to rise to 366 million in
2030. Among people with diabetes, the annual incidence of developing a foot ulcer
ranges from 1% to 4.1% and the prevalence ranges from 4% to 10%, which suggests
that the lifetime incidence may be as high as 25% [4]. It is estimated that approxi-
mately 15% of the more than 150 million people with diabetes world-wide will at
some stage develop diabetic foot ulceration with the highest prevalence (>11%) in
some African countries [5, 6].
Pressure ulcers occur with an estimated prevalence of 5–9% and more than 70%
occurring in patients over 70 years of age. They can be regarded as a potentially
preventable complication of an acute immobility illness [7]. The overall estimate of
the prevalence of pressure ulcers in all healthcare institutions across Canada was
26.0% [8]. Two cross-sectional surveys among 21,574 German hospital patients and
nursing home residents (147 institutions total) in 2002 and 2003 found that among
all persons at risk, pressure ulcer prevalence was 21.1%. The most common pres-
sure ulcer locations were the sacrum and the heels [9].
The burden of chronic wounds for patients and society is enormous. From the
patient’s perspective, a major negative impact on quality of life (QOL) is related to
ulcer pain [10]. Other important factors contributing to impaired QOL are mal-
odour, exudates discharge, loss of mobility and impaired social and professional
activities [11]. It behoves care providers and clinicians to examine and exhaust all
avenues of therapy to this population of patients with chronic wounds. Physical
techniques based on sound biophysical principles have been used in medical man-
agement for some considerable time for example the use of infra red radiation (heat)
to treat muscular sprains. There are some of more recent origin such as the use of
focused energy to reduce stones in the bladder. This chapter is focused on the use of
biophysical principles to treat chronic wounds.
The cost of wound care is high and rising based on cost estimates from different
countries. The most important components are the costs of hospitalisation for
treatment of complications arising from wounds (in particular in diabetic foot
ulcers), and the cost of nursing time. Care pathways to avoid hospitalisation offer an
important means of reducing costs [12].
The treatment of chronic wounds must take into account the underlying
pathology (i.e. vascular, infectious. etc.) as well as any concurrent co-morbid
conditions. The process of chronic wound healing process differs in many impor-
tant respects from that in acute wounds. Wound bed preparation is the manage-
ment of a wound that includes interventions that will accelerate endogenous
healing or facilitate the effectiveness of other therapeutic measures. The major
principles of wound bed preparation are encapsulated by the acronym TIME
(European Wound Management Association 2004 [13]). TIME stands for tissue
(non viable or deficient), infection/inflammation, moisture (imbalance) and edge
(non advancing or undermined). Debridement can and does play a vital role in
wound bed preparation and the removal of barriers that impair wound healing. In
accordance with the TIME principle, debridement can remove nonviable tissue,
16 The Use of Biophysical Technologies in Chronic Wound Management 315
Table 16.1 Levels of evidence-based medicine in clinical studies

Level 1: evidence is available for meta-analysis about several randomized controlled studies
Level 2: evidence is available from at least on randomized controlled trial
Level 3: evidence is available from good methodological studies without randomization
Level 4a: evidence is available from clinical reports
Level 4b: this represents a consensus of respected experts or expert committees
Adapted from Sackett et al. [14]
decrease bioburden thereby controlling inflammation or infection, decrease

excess moisture, and stimulate a nonadvancing wound edge. Continued debride-
ment, as part of a multifaceted treatment strategy, will also keep any present
biofilms in a weakened state [15, 13, 16].
In this chapter we will review the role of biophysical technologies in wound
management. The evidence available will be analysed and related to components of
the TIME concept to describe the potential inherent in different techniques. It is
interesting that with the exception of the use of shock waves all the biophysical
technologies addressed in this chapter fall under the clinical practice domain of the
profession of Physical Therapy and are used by Physical Therapist professionals
involved in wound management in the United States and Canada. This is different
in other countries. In Germany, Physical Therapists educated at Technical Colleges
and specialized wound nurses are involved in wound care in hospitals and outpatient
settings. Physicians of various specialties are able to earn a sub-specialization in
Physical Therapy. In France Physical Therapy may be studied in Universities. A
close collaboration of medical doctors and therapists is essential for the benefits of
patients with chronic wounds.
The following paragraphs provide an overview on biophysical techniques and
evaluate respective scientific background as suggested by the principles of evidence-
based medicine (Table 16.1).
Acoustic Energy
In this chapter, both extracorporeal shock wave therapy (ESWT) and low-frequency
ultrasound (LFUS) are discussed. Whereas ESWT has been introduced only recently
to treat chronic wounds, LFUS has a longer tradition, is in greater clinical use and
supported by more research evidence.
Low Frequency Ultrasound
Low-frequency (kHz) ultrasound (LFUS) (20–120 kHz, 0.05–1.0 W/cm2) is deliv-

ered by typically coupling the probe using normal saline and with light contact with
Fig. 16.1 Principle of

low-frequency ultrasound Ultrasonic transducer
application for chronic
wounds
Ultrasonic gel
Hydrocolloidal/-gel wound treatment
Ulcer surface
Long wave of
low-frequency ultrasound
Contacting appliction if low-frequency ultrasound
Table 16.2 Cellular effects of low-frequency ultrasound

Cell type Effect(s)
Fibroblasts Increased synthesis of collage and non-collagenous matrix proteins
Increased expression of keratinocytes growth factor (KGF) and vascular
endothelial growth factor (VGEF)
Activation of activation of extracellular regulated kinase (ERK) and c-Jun
N-terminal kinase (JNK) pathways
Increased proliferative activity
Osteoblasts Increased proliferative activity
Increased synthesis of fibroblast growth factor (FGF), VGEF, and interleu-
kin-8 (IL-8)
Monocytes Increased synthesis of VGEF
Adapted from Uhlemann et al. [17]
necrotic fibrinous wound tissues. It may be used to debride or in noncontact mode

in a water bath ([17–19]; Fig. 16.1). At adequate dosage, therapeutic high-frequency
(MHz) ultrasound (HFUS) and LFUS have been reported to induce in vitro cell
proliferation, protein synthesis, and production of cytokines by fibroblasts, osteo-
blasts, and monocytes ([20]; Table 16.2).
The clinical effects of LFUS occur when acoustic mechanical energy resulting
from cavitation is created on the wound surface. It is argued that the energy is
released from implosion of microbubbles (cavities) that causes fibrinolysis (debri-
dement) leading to decreasing bioburden through fragmentation of bacteria and
biofilms [21]. Both thermal and nonthermal effects occur in deeper tissue layers,
however these effects are mild compared to therapeutic HFUS (1 and 3 MHz) that
is capable of increasing tissue temperatures about 3 degree C [18]. Thus, on wound
surfaces the physical effects of cavitation and micro-streaming are important modes
of action. Stable (non-destructive) cavitation that is more pronounced when using
HFUS, may produce visible gas bubbles that can be seen in fluids as long as the
ultrasound energy (US) is being applied. Few if any of these bubbles implode, so
Table 16.3 Technical details of some low-frequency ultrasound devices

Device Frequency (kHz) Remarks Clinical trials
PainShield 100–300 Supracutaneous application Uncontrolled trials
with disposable patches
Bandelin Ultrapuls 43 Pulse ultrasound for supracuta- Single RCT
H neous and subaqual application (venous leg ulcers)
UAW Sonoca-180 20–80 Noncontacting device with a Uncontrolled trials
special type of handpieces
(Sonotrode) uncontrolled trials
Schülke Sonics 20–80 Noncontacting device with Uncontrolled trials
Sonotrode and disinfectant
irrigation
MIST therapy 40 Noncontacting device with RCT (diabetic foot
system special handpieces ulcers)
Qoustic wound 35 Noncontacting device with Uncontrolled trials
therapy system special handpieces
RCTs randomized controlled trials in wound healing
cavitation effect is weak. However, research has shown that HFUS causes degranu-
lation of mast cells with release of histamine [22] and other cellular effects
(Table 16.2) which may occur when HFUS is applied to intact periwound skin.
Transient cavitation, that is more pronounced with LFUS (e.g. 22.5, 25 and 35 kHz)
[20] occurs only in a liquid medium such as normal saline. In water, transient cavi-
tations develop at 0.5 W/cm² for LFUS. Transient cavitation is selectively destruc-
tive of fibrinous necrotic tissue via fibrinolysis. It is thought that the higher viscosity
of healthy tissues protects them from cavitation produced by LFUS. This method of
debridement has been reported to be very effective on venous leg ulcers [23] and
other wounds [24].
In the use of therapeutic HFUS dosage parameters intensity in W/cm² and appli-
cation time in minutes are important. In addition, application frequency (number
of times per day or per week) and series (total number of treatments) impact on
therapeutic efficacy. The effects of therapeutic ultrasound are dependent to the
frequency of the probe used for treatment. LFUS generates long waves that allow
deeper penetration [17] while higher frequencies are more active at superficial
levels (Table 16.3).
An in vitro cellular morphology study evaluated the effects of 40 kHz ultrasound
(US) on mitogenic activities, expression of keratinocyte growth factor (KGF) and
transforming growth factor beta-1 (TGF-beta1), and activation of extracellular regu-
lated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways of der-
mal fibroblasts. Ultrasound treated fibroblasts exhibited a much earlier increase in
KGF expression, ERK activation, and JNK activation. The ERK/JNK ratio was
increased markedly in ultrasound-treated fibroblasts. The data suggest that ultra-
sound induces cellular responses that may be beneficial to wound healing [25]. Low
power LFUS removes biofilms on implants by cavitating and non-cavitating power
densities. This has been proved with animal experiments on inserted Foley urinary
catheters. LFUS maintained the urine sterile for up to 9 days compared to 2 days in
Fig. 16.2 Low-frequency

a
ultrasound device UltraPulse
H (Bandelin electronic,
Berlin, Germany) with
43 kHz. (a) The device,
(b) subaqual application for
chronic venous leg ulcer
controls [26]. LFUS is effective in controlling wound bacterial colony in vitro: 0%

of sham treated and 33% of Pseudomonas aeruginosa, 40% of Escherichia coli and
27% of Enterococcus faecalis were dead after one ultrasound application. Minimal
effects on Staphylococcus aureus and methicillin-resistant S. aureus were observed.
Eleven patients with pressure ulcers with bacterial counts >105 CFU/g of tissue
received 2 weeks of 40 kHz noncontact LFUS. The quantities of seven bacterial
organisms were reduced substantially from baseline to 2 weeks post treatment.
Taken together, these studies indicate that 40 kHz noncontact ultrasound can be
used to reduce bacterial quantity [27].
In the clinical setting, three methods of application of LFUS are available. In
Germany, for cutaneous and under water application the ultraPuls H (BANDELIN
Electronic, Berlin; Germany) is widely used (see Fig. 16.2a) . This 43 kHz device
uses pulsed LFUS (pulse frequency 15 Hz). Intensities between 0.02 and 0.05 W/
cm2 (cutaneous and subaqueous application) and 0.2–0.3 W/cm2 (only for subaque-
ous application) are possible (Fig. 16.2b). The maximum application time should
not exceed 15 min per site [28].
Fig. 16.3 With the MIST

therapy system™
(Celleration, Eden Prairie,
Minnesota; USA) the
acoustic energy is delivered
to the wound as a vapour or
mist
The PainShield device (from NanoVibronix, Nesher; Israel) is another LFUS

device that delivers acoustic energy to tissues through the skin. This system utilizes
a patch that contains a battery that powers the ultrasound product. The device con-
sists of a disposable patch through which localized acoustic energy intended is
delivered to induce soft tissue healing. The acoustic energy sets up vibrations at
frequencies of 100–300 kHz with an acoustic intensity of 200 mW/ cm2. The tech-
nique is reported to have been used on diabetic foot ulcers in an open pilot trial
[29, 30] where the patch was applied on adjacent intact periwound skin. Noncontact
LFUS devices include the 40 kHz MIST Therapy System (Celleration, Eden Prairie,
Minnesota; USA), 35 kHz Qoustic Wound Therapy System (Arobella Medical,
Minnetonka, Minnesota; USA; Fig. 16.3), 25 kHz UAW Sonoca 180 (Söring UAW,
Quickborn; Germany) (Fig. 16.4a, b) or 25 kHz Schülke Sonicx (Schülke & Mayr,
Norderstedt; Germany).
For the subaqueous treatment of leg ulcers application of LFUS is widely used
since water is an excellent medium for transmitting acoustic waves. The tempera-
ture of the water bath should be between 32°C and 37°C. The applicator held at
approximately 1 cm from the tissue is moved over the whole wound surface in a
distance of several centimetres for about 10–20 min a day with daily application for
2–4 weeks. Adding an aqueous antiseptic agent to the coupling medium permits the
antimicrobial activity to be enhanced [31].
In uncontrolled trials LFUS has been used for debridement of chronic wounds
such as leg ulcers and pressure ulcers [32]. In a study of Uhlemann et al. [32] under
water application of LFUS (Intensity 0.6 W/cm2; frequency 43 kHz; 8 min per
25 cm2) resulted in an improvement of the Wollina Wound Score [33] . There was
less fibrin coverage, less exudate, and improved granulation [32].
a Söring
b Twinball Hoof Spatula
Fig. 16.4 The 25 kHz UAW Sonoca 180 (Söring UAW, Quickborn; Germany). (a) The device,
(b) the application on a chronic leg ulcer for debridement
A single-blind, randomised, prospective trial was performed on venous leg ulcer

patients (n = 24) with LFUS added to conventional wound care of outpatients. The
LFUS treatment consisted of 10 min under water application of 30 kHz continuous
ultrasound at 100 mW/cm2 intensity three times a week. Ulcer area was measured
by planimetry. After 12 weeks of treatment the control group showed a mean
decrease of 16.5% in the ulcer area. In contrast the mean ulcer area decreased by
55.4% in the LFUS group (P < 0.007). Patients reported only minor side-effects such
as a tingling and occasionally pinhead-sized bleeding in the ulcer area [34].
Sub aqueous LFUS, noncontact 40 kHz LFUS may be used in clinical settings.
A retrospective review of charts for 76 patients treated with noncontact LFUS as an
adjunct to conventional wound care was conducted. For this device (MIST Therapy
System™, Celleration, Eden Prairie, Minnesota; USA) the acoustic energy is deliv-
ered to the wound as a vapour or mist (Fig. 16.3). The treatment was administered
for a mean of 5.1 min per session for a mean of 2.3 times per week. The median
duration of therapy was 4.3 weeks. The median wound area was reduced by 79%
from the start to the end of LFUS treatment. The proportion of participants with
greater than 75% granulation tissue increased from 32% before LFUS to 46% after
LFUS [35].
Ultrasonic MIST™ debridement uses acoustic energy to slowly remove devital-
ized tissue from the wound bed and to promote wound healing. In a retrospective
analysis 163 patients who received MIST Therapy plus standard of care (treatment
group) and 47 patients who received the standard of care alone (control group) have
been evaluated. The follow-up of patients was 6 months. In the treatment group,
MIST Therapy was administered to wounds three times per week for 90 days or
until healed. A significantly greater percentage of wounds treated with MIST
Therapy plus standard care healed as compared with those treated with the standard
of care alone (53% vs. 32%; [31] P = 0.009). The data also indicated a faster rate of
healing in the MIST-treated wounds [36].
The same research group performed a feasibility study on MIST Therapy in leg
ulcer patients in a clinical case series. Patients were initially treated with standard of
care before the addition of or the switch to noncontact LFUS (n = 51, age median ±
1 SD 72 ± 15 years). Patients with various wound pathologies were included in the
study. Of all the wounds, 63% had a multifactorial etiology, and 65% had associated
transcutaneous oximetry levels below 30 mmHg. The mean ± 1 SD treatment time
of wounds during the baseline standard of care control period versus the noncontact
LFUS therapy period was 9.8 ± 5.5 weeks versus 5.5 ± 2.8 weeks (P < .0001). The
mean ± 1 SD percent volume reduction in the baseline standard of care control
period versus the noncontact LFUS therapy period was 37.3% ± 18.6% versus
94.9% ± 9.8% (P < .0001) [37].
A prospective parallel group randomized, controlled trial in patients with non-
healing leg and foot ulcers associated with chronic critical limb ischemia compared
MIST Therapy plus standard wound care for 12 weeks or until fully healed (treat-
ment group; n = 50) with standard wound care alone (control group; n = 35). MIST
Therapy was administered three times per week for 5 min per treatment. The authors
used the baseline transcutaneous oxygen pressure when assessing the potential to
heal ischemic wounds. A significantly higher percentage of patients treated with
standard wound care plus MIST Therapy achieved greater than 50% wound healing
at 12 weeks than those treated with standard care alone (63% vs. 29%; P < .001).
Thus, failure to achieve the minimum wound healing requirement occurred in 37%
of patients in the treatment group and 71% of patients in the control group [38].
A noncomparative clinical outcome trial of MIST therapy enrolled 23 patients.
Control data were obtained from a prospectively collected database from the same
clinic. During an 8 month period, a total of 29 lower extremity wounds in 23 patients
were treated with noncontact LFUS therapy. When MIST Therapy was used as a
stand-alone device, median time to healing was 7 weeks. Historic controls were
healed during a median time of 10 weeks; however, a statistically significant num-
ber of these patients required wound related hospitalization and surgical procedures
to achieve closure compared with wounds treated with MIST Therapy. Treatment
with MIST Therapy achieved healing in chronic wounds when used as a standalone
device or in combination with moist wound care in 69% of cases. Response to
treatment was evident within 4 weeks of therapy. Earlier transition to secondary
procedures and decreased utilization of inpatient care might result in more cost-
effective wound healing than the current standard of care [39].
A retrospective chart review and analysis of reported pain scores of 15 consecu-
tive patients (age range 28–88 years) with painful, nonhealing, lower extremity
wounds treated for 2–4 weeks with MIST Therapy has been reported. The mean pain
scores decreased from 8.07 (±1.91) pre-treatment to 1.67 (±1.76) post-treatment
(P = 0.0003). No patients reported worsening pain after treatment commenced [40].
A meta analysis suggested that noncontact, LFUS ultrasonic MIST Therapy pro-
motes wound healing when used in conjunction with standard wound therapy. Only
few mild adverse effects have been noted [41].
Tan et al. [42] used LFUS at 25 kHz delivered by a portable Sonaca-180™ via a
handheld probe, using normal saline as the irrigation/coupling medium. The ultrasound
was applied for 10–20 s per probe head area onto the ulcer. Each leg underwent treat-
ment at an interval of 2–3 weeks with compression bandages reapplied at the end of the
treatment. Nineteen patients with leg ulceration of at least 6 months were recruited.
Each patient received on average 5.7 treatments, each ranged from 5 to 20 min depend-
ing on the ulcer size. Symptomatic relief (pain and odour reduction) was achieved in six
patients. Seven patients achieved complete ulcer healing (mean ulcer size = 4.72 ± SD
1.872 cm2) but no treatment effect was observed in eight patients. There were no major
complications of the treatment but emergency facilities for treatment of acute wounds
should be available. Although LFUS was relatively painless dysaesthesia during appli-
cation may be reduced with the help of analgetic ointments. The authors concluded that
LFUS (25 kHz) debridement may heal some recalcitrant ulcers when standard com-
pression regimens have failed. It is cheap and may be done at outpatient level. The role
of simple wound cleansing warrants further investigation [42]. In contrast to leg/foot
ulcers, the evidence for LFUS in pressure ulcers is insufficient [43] a conclusion also
supported by the EPUAP/NPUAP Pressure Ulcer Treatment Guide 2009.
In conclusion, there is increasing evidence for the usefulness of LFUS as an
adjunct to good wound care in order to improve healing. Most data available relate
to improved tissue viability and granulation. However, the treatment parameters
(best frequency, intensity, and series) are yet not standardized. The level of evidence
for LFUS is 3.
Shock Waves
Extracorporeal shock wave therapy (ESWT), also known as acoustic pressure

wound therapy (APWT), is a new approach to soft tissue wound healing. Studies in
streptozotocin-induced diabetic Wistar rats showed that wound size was significantly
reduced while perfusion increased in the ESWT treated rats compared with the
control group (P < 0.01). Immunohistochemical stains showed increased neo-angio-
genesis and tissue regeneration after treatment [44].
Two hundred and eight patients with complicated, nonhealing, wounds both
chronic and acute were prospectively enrolled into a feasibility study. Treatment
consisted of debridement, ESWT [100–1,000 shocks/cm2 at 0.1 mJ/mm2, according
to wound size, every 1–2 weeks over a mean of three treatments] at outpatient level
after which the wounds were covered with moist wound dressings. Thirty-two
(15.4%) patients dropped out of the study following the first ESWT but were included
in the Intention to Treat (ITT) data analysis that Intention-To-Treat . Of the 208
patients who were enrolled, 156 (75%) experienced 100% wound coverage as a
result of the treatment.. During a mean follow up period of 44 days, there was no
treatment related toxicity, infection, or deterioration of any ESWT treated wound.
Intent-to-treat multivariate analysis identified age (p = 0.01), wound size < or =10 cm2
(p = 0.01; OR = 0.36; 95% CI, 0.16–0.80), and duration £1 months (p < 0.001;
OR = 0.25; 95% CI, 0.11–0.55) as independent predictors of complete healing [45].
A retrospective analysis of 41 patients (with 52 chronic wounds) who received
ESWT at least two times per week revealed that 38% of wounds (N = 20) healed
completely in a mean 6.8 weeks. Median wound area and volume decreased
significantly (88% [P < .0001] and 100% [P < .0001], respectively) from start to end
of ESWT. The proportion of wounds with greater than 75% granulation tissue
increased from 26% (n = 12) to 80% (n = 41) (P < .0001), and normal periwound skin
increased from 25% (n = 13) to 54% (n = 28) (P = .0001). The presence of greater
than 50% fibrin slough decreased from 50% (n = 24) to 9% (n = 4) of wounds
(P = .006) [46].
These results are in concord with those from an uncontrolled prospective study
in leg ulcer patients (n = 30). Sixteen of 32 wounds healed completely within six
sessions of ESWT. In all of the unhealed wounds, a decrease of the amount of exu-
dates, increased percentage of granulation and decrease of wound size were statisti-
cally significant after four to six sessions of ESWT (p < 0.01). A significant decrease
in pain associated with the treatment was also reported (p < 0.001) [47].
A randomized, prospective, controlled study evaluated whether ESWT is effec-
tive in the management of neuropathic diabetic foot ulcers (n = 30). One group
received standard care and shock wave therapy, the other only standard care. After
20 weeks of treatment, 53.3% of the ESWT treated patients had complete wound
closure compared with 33.3% of the control patients. In this study, time taken to
heal were 60.8 and 82.2 days, respectively (p < 0.001) [48]. In conclusion, the first
published clinical trials demonstrated the efficacy of ESWT in healing of chronic
leg ulcers of various aetiologies. The clinical effects include faster healing, improved
granulation, reduced wound size and a higher rate of complete healing compared to
controls. On the basis of the current evidence it may be suggested that ESTW has a
beneficial role in wound management.
It must be emphasised that the dose and duration of application (shocks/ area and
frequency of application) remain to be clarified. Some guidance of what type of
patients and chronic wounds will benefit most from is awaited. On the basis of these
report level 2 evidence is assigned to ESWT.
Negative Pressure Wound Therapy or Topical Negative

Pressure Therapy
Negative pressure wound therapy (NPWT) or Topical negative pressure (TNP)

which is the generic term has been evaluated both in vitro an in vivo. This modality
of treatment has gained widespread use from the Vacuum Assisted Closure tech-
nique (VAC™; Kinetic Concepts Inc. San Antonio, TX) that applies localized nega-
tive pressure to the wound bed through a polyurethane reticulated open-cell foam
dressing (V.A.C. GranuFoam™ Dressing – see Fig. 16.5a,b) or a polyvinyl alcohol
foam (V.A.C. WhiteFoam) dressing [49]. In recent years, several other makes and
types are available to treat chronic wounds using topical negative pressure through
different materials e.g. foam, gauze and so on. The mechanisms of action are not
completely understood, though the biophysical and biochemical effects are impor-
tant. In essence the technique is based on delivering topical negative pressure
through a material that is applied to a wound.
Maier et al. [50] measured exudate secretion at different pressures using both poly-
vinyl (PVA) and black polyurethane (PU) foam pads. This group studied 8 wounds
with surface areas greater than 20 cm2. With pressures of 40 kPa (or 300 mmHg),
wound excretion decreased by 50% when the black PU foam was used.
Recent research in advanced wound treatment technologies suggests that that
microdeformations induced by the application of sub-atmospheric (negative) pressure
through VAC GranuFoam Dressing is instrumental in regulating the mechanobiology
of granulation tissue formation [51]. While the clinical response is unequivocal, mea-
surement of deformations at the wound-dressing interface has not been possible due
to the inaccessibility of the wound tissue beneath the sealed dressing. In a bench test
wound model microcomputed tomography (microCT) imaging of deformation
induced by NPWT was performed and an algorithm set for quantifying the 3D strain
field at sub-millimeter resolution developed. Microdeformations induced in the tissue
phantom revealed average tensile strains of 18–23% at sub-atmospheric pressures of
−50 to −200 mmHg (−6.7 to −26.7 kPa). The authors propose that strain signals
quantified using this system can then be used in future research aimed at correlating
the effects of mechanical loading on the phenotypic expression of dermal fibroblasts
in acute and chronic ulcer models [52].
It has been postulated that reduction of local and interstitial tissue edema,
increased perfusion of the peri wound area, changed bacterial composition, and
mechanical stimulation of the wound bed contribute to the clinical success of TNP
or NPWT. Wound fluid samples of 33 wounds treated with either NPWT (n = 15) or
conventional therapy (n = 18) were analysed for albumin, pro- and activated matrix
metalloproteinase MMP-9, and its tissue inactivator TIMP-1, and the ratio of total
MMP-9/TIMP-1. Albumin levels were found to increase significantly in acute
wounds compared with chronic wounds; however, no difference could be found on
comparing NPWT with conventional therapy. There were significantly lower levels
of pro-MMP-9 and lower total MMP-9/TIMP-1 ratio in TNP treated wounds during
the follow-up of 10 days. These data strongly suggest that TNP or NPWT therapy
influences the microenvironment of the wound [53].
Fig. 16.5 VAC™ (Kinetic

Concepts Inc. San Antonio, a
TX) applies localized
negative pressure to the
wound bed through a
polyurethane reticulated
open-cell foam dressing.
(a) The device, (b) the foam
on a chronic leg ulcer
In addition to these findings, lymph vessel proliferation was noted in all types of
wounds treated by TNP or NPWT even at the first dressing change. This therapy
seems to be inducing morphological and quantitative alterations on the lymph ves-
sel network in a wound. An increase in the density of lymph vessels manifested
histologically correlates with a better clinical outcome, in terms of healing rates and
hospitalisation time [54].
Diabetic foot wounds, particularly those secondary to amputation, are very com-
plex and difficult to treat. Armstrong et al. [55] investigated whether NPWT improves
the proportion and rate of wound healing after partial foot amputation in patients
with diabetes. One hundred sixty-two patients were enrolled into a 16-week, multi-
centre randomised clinical trial. Inclusion criteria consisted of partial foot amputa-
tion wounds up to the transmetatarsal level and evidence of adequate perfusion.
Patients who were randomly assigned to NPWT (n = 77) received treatment with
dressing changes every 48 h. Control patients (n = 85) received standard moist
wound care according to consensus guidelines. NPWT was delivered through the
VAC Therapy System. Wounds were treated until healing or completion of the 112-
day period of active treatment. Analysis was by intention to treat method. It was
reported that more patients healed in the NPWT group than in the control group (43
[56%] vs. 33 [39%], p = 0.040). The rate of wound healing, based on the time to
complete closure, was faster in the NPWT group than in controls (p = 0.005). The
rate of granulation tissue formation, based on the time to 76–100% formation in the
wound bed, was faster in the NPWT group than in controls (p = 0.002). The fre-
quency and severity of adverse events (of which the most common was wound
infection) were similar in both treatment groups [55]. There was no difference
between groups for in-patient hospital stay (number of admissions or length of stay).
More surgical procedures (including debridement) were required in the control
group (120 vs. 43 NPWT, P < .001). The average number of dressing changes per-
formed per patient was 118.0 (range 12–226) for controls versus 41 (6–140) for
NPWT (P = .0001). The control group had 11 (range 0–106) outpatient treatment
visits during the study versus 4 (range 0–47) in the NPWT group (P < .05). The aver-
age direct cost per patient treated for 8 weeks or longer (independent of clinical
outcome) was $27,270 and $36,096 in the NPWT and control groups, respectively.
The average total cost to achieve healing was $25,954 for patients treated with
NPWT (n = 43) compared with $38,806 for the control group (n = 33). Thus, treat-
ment of diabetic patients with post amputation wounds using NPWT resulted in
lower resource utilization and a greater proportion of patients obtaining wound heal-
ing at a lower overall cost of care when compared to moist wound therapy [56].
This would suggest that NPWT delivered by the VAC Therapy System seems to
be a safe and effective treatment for complex diabetic foot wounds, and could lead
to a higher proportion of healed wounds, faster healing rates, and potentially fewer
re-amputations than standard care [57].
Continuous NPWT delivered at −125 mmHg has been recommended, despite
consistent research findings suggesting potential advantages to the use of lower
pressures and intermittent therapy. To enhance understanding and document the
disparity between the body of NPWT science and current practice with respect to
negative pressure levels and modes of therapy, a meta-analysis was conducted.
Thirty-six publications found to contain directly relevant information of in vivo and
in vitro studies were examined. While lower negative pressures and intermittent
therapy were associated in earlier studies with improved microvascular blood flow
in porcine wound models and with reduced pain in patients, early system shortcom-
ings discourage adoption of intermittent therapy. Subsequent preclinical studies
confirmed the beneficial effects of intermittent therapy compared to continuous
therapy on blood flow and granulation tissue formation and lower pressures
(−75 mmHg or −100 mmHg) compared to higher pressure (−125 mmHg) on soft
tissue blood flow. Considering the available preclinical evidence, reported patient
pain, and common use of high-pressure continuous NPWT in clinical practice,

high-quality randomized controlled clinical trials must be conducted to help clini-
cians optimize care [58].
In an another uncontrolled study, the data from 25 patients with cutaneous
wounds undergoing vacuum assisted closure (V.A.C.) therapy were analysed for
pain, patient autonomy, and consumption of analgesic drugs. The average pain score
without V.A.C. was (4.2 ± 2.4) during the first day of therapy the average pain score
was (6.2 ± 2.8). Before and after V.A.C. therapy, a similar number of patients (17/25
vs. 19/25) used pain-killers, but the dosage had to be increased. There were five
drop outs due to uncontrollable pain. The therapeutic response to the pain killers
was poor. In addition, there was a higher degree of dependence during the V.A.C.
therapy. Increased levels of pain associated with the loss of autonomy during V.A.C.
therapy increase nurses’ workload and worsen patients’ quality of life [59].
For home treatment with NPWT, a systematic education of patients and relatives
is necessary to ensure the same level of efficacy and safety as in the hospital setting
[60]. Although most trials on NPWT have been conducted in adult patients, there is
also some evidence for its usefulness in pediatric patients with chronic wounds [61].
In a single centre analysis, 24 pediatric patients (mean age 8.5 years [range 14 days–
18 years old]) with 24 wounds were treated with NPWT. Every second wound was
infected at baseline. Sixteen patients had hypoalbuminemia and six had exposed
hardware and bone in their wounds. Twenty-two wounds reached full closure in a
median time of 10 days (range 2–45) following negative pressure wound therapy and
flap closure (11), split-thickness skin graft (3), secondary (4), and primary (4) clo-
sure. Pressures used in this population ranged from 50 to 125 mmHg and most
wounds were covered with reticulated polyurethane foam. One patient developed a
lymphatic fistula during the course of negative pressure wound therapy [62].
A systematic literature review was undertaken to assess the effectiveness and
safety of NPWT. Randomized controlled trials (RCTs) assessing NPWT were
included. A total of 14 RCTs were included. Trials covered patients with pressure
wounds (2), post-traumatic wounds (3), diabetic foot ulcers (4) and miscellaneous
chronic ulcers (5). In all trials NPWT was at least as effective and in some cases
more effective than the control treatment.
Most evidence supports the effectiveness of NPWT on chronic leg ulcers and
posttraumatic ulcers. NPWT appears to be a safe treatment, and serious adverse
events have been rarely reported. Only two trials were classified as high quality stud-
ies whereas the remaining trials were classified as having poor internal validity.
Tentative evidence indicates that the effectiveness of NPWT is at least as good as
or better than current local treatment for wounds. The need for large high-quality
randomised studies is apparent [63].
In a Cochrane analysis seven trials involving 205 participants were identified.
The seven trials compared NPWT with five different comparator treatments. Four
trials compared NPWT with gauze soaked in either 0.9% saline or Ringer’s solu-
tion. The other three trials compared NPWT with hydrocolloid gel plus gauze, a
treatment package comprising papain-urea topical treatment, and cadexomer iodine
or hydrocolloid, hydrogels, alginate and foam.
These data do not show that NPWT significantly increases the healing rate of
chronic wounds compared with comparators. Data on secondary outcomes such as
infection rate, quality of life, oedema, hospitalisation and bacterial load were not
reported [64].
In another literature search of the same group, 15 publications on 13 RCTs were
identified. These reported on patients with chronic wounds, diabetic wounds, pres-
sure ulcers, skin grafts and acute wounds. In chronic and diabetic wounds, NPWT
did not allow earlier complete wound healing. It was, however, associated with a
1–10 day reduction in the time needed to prepare the wound for secondary closure
surgery [65].
Augustin and Herzberger [66] established the benefits and limitations of NPWT
on the basis on “best evidence” using an internet-based literature search based on the
criteria of the Cochrane Collaboration, supplemented by an additional manual search.
The therapeutic benefit was determined using the current benefit definitions of
German social law SGB V and criteria of quality control bodies (GBA and IQWiG),
with emphasis on the patient’s perspective and on daily routine. The online search
revealed 674 publications. After additional manual searching, a total of 269 original
papers filled the criteria for evaluation. A majority of analyzed studies indicate an
effectiveness and clinical benefit for NPWT. These authors conclude that in Germany,
NPWT is a ‘standard’ approach for treating problematic acute and chronic wounds.
It is often an essential treatment option given its effectiveness in many indications.
NPWT also has marked clinical benefits from the patient’s perspective [66].
Campbell et al. [67] analysed retrospectively clinical data of 30 patients treated
with the Chariker-Jeter gauze-based negative pressure systems (V1STA, Versatile-1
and EZ-Care; Smith & Nephew, Inc.). A pressure setting of −80 mmHg was used.
The mean age of the patients was 72 years. The wounds consisted of chronic (n = 11),
surgical dehiscence (n = 11) and surgical incision (n = 8). Discontinuation of therapy
was instigated upon closure through secondary intention or when size and exudate
were sufficiently reduced that the wounds could be managed by conventional wound
dressing (median 41 days). An overall median reduction in wound volume of 88%
(p < 0.001) and a 68% reduction in area (p < 0.001) compared with baseline were
observed over the course of NPWT [67].
Mesh graft transplantation is a surgical procedure in the treatment of recalci-
trant leg ulcer of venous, mixed venous-arterial, and neuropathic origin. In an
uncontrolled retrospective trial, the usefulness of NPWT to increase the take rate
of transplants was investigated. A consecutive case series of 54 patients with
chronic leg ulcers who received a total of 74 mesh grafts was carried out.
Postoperative VAC therapy was performed in 28 mesh grafts treatment while 46
mesh grafts were treated with standard gauze therapy. In the VAC group, 92.9% of
grafts showed complete healing, compared to 67.4% in the control group without
postoperative VAC therapy. Differential analysis revealed a negative correlation of
the take rate in patients over 70 years of age or in patients suffering from diabetes
mellitus or lipodermatosclerosis. Patients with diabetes mellitus and of greater age
exhibited improved take rates (100%) in the VAC group compared to 50 and 62%,
respectively [68]. The results of this retrospective study demonstrate for the first
time the significant benefit of VAC therapy after skin grafting in chronic leg ulcer
patients as evaluated in a clinical trial.
The usefulness of NPWT in other types of chronic wounds such as vasculitic
ulcers or malignant wounds has yet not been studied systematically. There is some
causal evidence for the use of NPWT in pyoderma gangraenosum as an adjunct to
immunosuppressive treatment [69, 70]. We have successfully treated two patients
with lymphoedema and leg ulcers using NPWT or TNP; this suggests that this ther-
apeutic approach is effective in reducing oedematous [71].
A prospective clinical randomised trial compared the costs of NPWT with
conventional therapy (moist gauze) in the management of full-thickness wounds
that required surgical closure. The direct medical costs of the total resources
needed to achieve a healthy, granulating wound bed that was ‘ready for surgical
therapy’ were calculated. Fifty-four patients admitted to a department of plastic
and reconstructive surgery were recruited into the trial. NPWT had higher mate-
rial costs. However, these were compensated by the lower number of time-con-
suming dressing changes and the shorter duration until they were ‘ready for
surgical therapy’, resulting in the therapy being equally as expensive as conven-
tional moist gauze [72].
NPWT adverse effects include discomfort, pain, and excessive tissue growth
into the dressing. Complications are limited if the device is used properly [73]. In
light of the current treatment options, NPWT may be considered useful as adjuvant
therapy for chronic wounds; however, there is inadequate definitive evidence that
wound healing is substantially better with these devices than with traditional
therapy.
All chronic wounds are contaminated by bacteria and other microorganisms,
some are at risk of infection or infected. Before initiating NPWT on critically colo-
nized or infected wounds the following rules should be applied: (1) the patient has
to be free of most systemic signs of gross infection; (2) all necrotic tissue needs to
be debrided and abscesses drained in the wound; and (3) the wound needs an ade-
quate perfusion (necrosis can occur when negative pressure is applied on an isch-
emic wound). If there is any doubt, a re-examination of the wound should be done
in 12–24 h before starting NPWT. In deep wound infection, NPWT should always
be used in combination with antibiotics after having obtained a bacterial swab pre-
or intraoperatively to avoid the consequences of bacteremia. The GranuFoam
Dressing (R) (KCI International, San Antonio, Tx) is recommended in contaminated
or colonized shallow wounds with no exposed bone or foreign body. It can also be
used either in wounds with low risk of infection or in infected wounds [74].
In a prospective open trial the effect of NPWT, delivered using VAC, on the
microbiology of chronic, non-infected venous leg ulcers has been investigated.
Seven patients receiving compression therapy were recruited. The ulcer was swabbed
and VAC was applied at −125 mmHg continuous pressure on day 1 for 6 days.
Standard methods for bacteriological sampling and measuring the wound surface
area were applied at baseline and at the VAC dressing changes on days 3 and 6. Log
median colony forming units (CFUs) per cm2 data were used for statistical analyses.
The bacterial species were identified. The median log10 CFU/cm2 on days 1, 3 and
6 were 3.5, 4.7 and 5.1 respectively. There was a significant increase in bacterial
colonisation between days 1 and 6 (p < 0.02). No change was observed in the
identified microbiological species during therapy with VAC. This pilot study sug-
gests that VAC therapy increases absolute numbers of bacteria colonising non-
infected leg ulcers [75]. Surveillance of the wound bacteriology is therefore
recommended.
The rate of complications of NPWT is low and the treatment is usually well tol-
erated. The most common adverse effect is some temporary pain that can be man-
aged by pain killers. Other possible rare complications in chronic wounds include
wound infections, bleeding, and toxic shock syndrome. Rare cases of wound sepsis
have been seen in case of pump failures or retained foam material [76].
TNP has been evaluated in numerous trials. Although different devices are avail-
able, most of the evidence has gained using the VAC system. Treatment costs is an
important issue for the VAC system, but total costs may be lower compared to stan-
dard therapies depending on various factors like health system, reimbursement, hos-
pitalization versus outpatient therapy, and patient’s characteristics. Comparative
trials for such systems are not available. Best evidence has gained for diabetic foot
ulcers. Evidence is much weaker for the other main types of chronic wounds.
Therefore, randomized controlled trials comparing healing, costs of care, patient
pain, and quality-of-life outcomes of this treatment to non-gauze type dressings and
other treatment technologies are needed. The use of NPWT to treat diabetic foot
ulcers has gained evidence level 2 and for indications such as venous leg ulcers or
pressure ulcers the evidence is level 3.
Electromagnetic Thermal Energy Technologies
The use of heat in wound healing has mainly been focused on surgical wounds and
the prevention of secondary infection. In this section the use of non-contact normo-
thermic wound therapy and infrared irradiation for the treatment of chronic wounds
is presented. Wound infection is a contraindication for use these techniques.
Infrared Radiation (Non-contact Normothermic Wound Therapy)
Both systemic and local warming have been shown to reduce the risk of surgical site
infection in patients undergoing clean, acute wound operations [77]. Chronic
wounds typically are at risk for deeper wound infections because of heavy bacterial
colonization. Noncontact normothermic wound therapy (NNWT) (Warm-Up®
Active Wound Therapy; Augustine Medical, Eden Prairie, Minnesota; USA;
Fig. 16.6 Noncontact

normothermic wound
therapy: (Warm-Up® Active
Wound Therapy; Augustine
Medical, Eden Prairie, MN,
USA)
Fig. 16.6), also known as non-contact radiant-heat bandage consists of a noncon-

tact, domed wound cover into which a flexible infrared heating card is inserted. A
battery pack powers the device and warms the wound to a pre-determined tempera-
ture. The inside of the wound cover contains a foam ring which acts as a wick to
drain away exudate.
McCulloch and Knight [78] examined the effect of NNWT in the treatment of
neuropathic foot wounds in diabetics (n = 36). Patients received NNWT plus
offloading compared with offloading alone for a period of 8 weeks or until healing.
Wounds of subjects in the treatment group healed at a rate of 0.019 ± 0.019 cm2/day
compared with that of 0.008 ± 0.009 cm2/day in the control group (p = 0.049).
Kloth et al. [79] studied the effect of NNWT versus standard wound care on
patients (n = 40) with 43 Stage III and IV pressure ulcers. A sterile noncontact
wound NNWT dressing was applied to 21 wounds for 24 h per day, 7 days per
week. Each day after the wound was irrigated and the noncontact dressing was
changed, a heating element in the dressing was activated for 31-h periods for
12 weeks or until wound closure. Twenty-two control wounds were treated with
standard, moisture-retentive dressings. The healing rate for the treatment group
was significantly greater than that for the control group (0.52 and 0.23 cm2 per
week, respectively; p < 0.02). However, the difference in the incidence of closure
among wounds that completed the entire 12-week protocol between treatment and
control groups was not significant.
In a prospective, randomized, controlled study, Alvarez et al. [80] compared dia-
betic foot ulcer healing in patients being treated with either NNWT applied for 1 h
three times daily until healing or 12 weeks, or standard care. Surgical debridement
and adequate foot off-loading was provided to both groups. Twenty patients com-
pleted the study. After 12 weeks, 70% of the wounds treated with NNWT were
healed compared with 40% for the control group. However, the differences were not
significant (p < 0.069, ns). The same group performed a single centre randomized
study with 49 patients with diabetic neuropathic foot ulcers with improved healing
in the NNWT group [81].
In a randomized controlled study, Thomas et al. [82] examined the effectiveness
of radiant heat bandage (NNWT) on the healing of stage 3 or stage 4 pressure ulcers.
A total of 41 subjects with a stage three or stage four truncal pressure ulcer greater
than 1.0 cm2 were recruited from outpatient clinics, long-term care nursing homes,
and a rehabilitation centre. The experimental group was randomized to a radiant-
heat dressing device and the control group was randomized to a hydrocolloid dress-
ing, with or without calcium alginate filler. Subjects were followed until healed or
for 12 weeks. Eight subjects (57%) in the experimental group had complete healing
of their pressure ulcer compared with 7 subjects (44%) with complete healing in the
control group (p = 0.46). The authors noted that although a 13% difference in heal-
ing rate between the two arms of the study was found, this difference was not statis-
tically significant [82].
In conclusion, the evidence in favour of NNWT in chronic wounds is insufficient
although minor beneficial effects in wound healing have been observed in small
studies in diabetic neuropathic ulcers and pressure sores. The evidence level
is 4a.
Other Infrared Thermal Radiation Devices
Infrared radiation has been investigated in wound healing with water-filtered infra-
red light (WIRA).
Water-filtered infrared-A (WIRA) and visible light are emitted by the Hydrosun®
radiator device (Hydrosun® Medizintechnik, Mülheim; Germany). The unique prin-
ciple of operation involves the use of a hermetically sealed water-filter in the radia-
tion path that absorbs or decreases those infrared wavelengths emitted by
conventional infrared lamps that would otherwise lay a thermal burden on the skin
(especially infrared-B and -C and water absorption bands within infrared-A). With
this principle high irradiation intensities are perceived as pleasant and therapeutic
heating of deeper tissue layers over longer periods of time can be achieved.
Water-filtered infrared-A (WIRA) is a special form of heat radiation with a high
tissue-penetration and with a low thermal burden to the surface of the skin. Mercer
et al. [83] performed a prospective study (randomised, controlled, blinded, de facto
with one exception only one cohort possible) using WIRA in the treatment of
patients with recalcitrant chronic venous leg ulcers. Ten patients (median age
62 years) with 11 recalcitrant ulcers of the lower legs were treated with water-
filtered infrared-A and visible light radiation [WIRA (+VIS), Hydrosun® radiator
type 501, 10 mm water cuvette, water-filtered spectrum 550–1,400 nm] or visible
light radiation (VIS; only in a single patient). The uncovered wounds of the patients
were irradiated two to five times per week for 30 min at a standard distance of 25 cm
(approximately 140 mW/cm2 WIRA and approximately 45 mW/cm2 VIS). Treatment
continued for a period of up to 2 months. The study showed a complete or nearly
complete healing of lower leg ulcers in seven patients and a reduction of ulcer size
in another two of ten patients, a reduction of pain and pain medication consumption
(e.g. from 15 to 0 pain tablets per day), and a normalization of the thermographic
image (up to 4.5°C temperature difference). In one patient the therapy of an ulcer of
one leg was performed with the fully active radiator [WIRA(+VIS)], while the ther-
apy of an ulcer of the other leg was made with a control group radiator (only VIS
without WIRA), showing a clear difference in favour of the WIRA treatment.
Failures to achieve complete or nearly complete wound healing were seen only in
patients with arterial insufficiency, in smokers or in patients who did not have
venous compression garment therapy. WIRA can alleviate pain considerably (with
an impressive decrease of the consumption of analgesics) and accelerate wound
healing or improve a stagnating wound healing process and diminish an elevated
wound exudation and inflammation both in acute and in chronic venous leg ulcers
including infected wounds. WIRA is a non contact easy-to-use device that is pleas-
ant to the patient. Wound healing and infection defence (e.g. granulocyte function
including antibacterial oxygen radical formation of the granulocytes) are critically
dependent on a sufficient energy supply (and on sufficient oxygen). The beneficial
clinical effect of WIRA on wounds and wound infection may be explained by the
improvement of both the energy supply and the oxygen supply (e.g. for the granu-
locyte function). WIRA causes as a thermal effect in the tissue an improvement in
three decisive factors: tissue oxygen partial pressure, tissue temperature, and tissue
blood flow. Besides this non-thermal effects of infrared-A by direct stimulation of
cells and cellular structures with reactions of the cells have also been described. It
is concluded that WIRA can be used to improve wound healing, to reduce pain,
exudation, and inflammation and to increase quality of life [83].
Another device is used with WIRA for whole body hyperthermia is
IRATHERM-2000 (l 760–1,400 nm) (Von Ardenne Institute for Applied Medical
Research, Dresden; Germany). It has mainly been used for chemotherapy [84, 85].
There has been no data reported yet on wound healing with this device.
In conclusion, the principle of hyperthermia by WIRA has been used for various
indications including venous leg ulcers. The procedure seems to be safe. The
reported data, however, are insufficient.
The level of evidence is 4a.
Electromagnetic Non-thermal (Photobiomodulation)

Energy Technologies
Laser and Light
Laser (monochromatic light) and phototherapy (polychromatic light) are two

sides of the coin of light-based therapies. Laser has been used in the low level
range for wound treatment in many ways. Phototherapy with visible light shows
anti-microbial activity.
Low Level Laser Therapy
Recent studies using advanced experimental techniques have attempted to define the
precise biostimulative effects of low level laser therapy (LLLT). Zhang et al. [86] dem-
onstrated with cDNA microarray technology an upregulation of genes that led to an
increase in the proliferation of fibroblasts. Irradiation of human fibroblasts at 628 nm
caused an increase in genes already known to play roles in enhancement of cell prolif-
eration and suppression of apoptosis [86]. Alexandratou et al. [87] used single cell
confocal microscopy to show in real time that LLLT irradiation can cause intracytoso-
lic increases in pH and calcium as well as changes in reactive oxygen species [87].
Genetically diabetic mice (C57BL/Ksj/db/db) were used as an animal model for
a wound healing study for LLLT. An argon dye laser (630 nm; 20 mW/cm2) irradia-
tion enhanced the percentage of wound closure over time as compared to the nega-
tive control group (58.4% vs 40.8% at day 10 and 95.7% vs. 82.3% at day 20,
p < .01). Histological evaluation showed that laser irradiation improved wound epi-
thelialization, cellular content, granulation tissue formation, and collagen deposi-
tion in laser-treated wounds as compared to the negative control group [88].
Sixteen residents of a nursing home were treated (27 sites, 23 open wounds and 4 at
risk areas). Twenty one open wounds (pressure ulcers, venous leg ulcers, and lower
extremity diabetic wounds) were treated with LLLT. At the end of the 9-week trial, the
majority (61.9%) of the 21 wounds achieved significant improvement (> or = 50% wound
closure), 9 (42.8%) had 100% closure. Chronic and acute wounds had similar improve-
ment [89]. This study included a small number of patients with various ulcer pathologies
and duration with controls permitting no conclusion to drawn from the results.
Kopera et al. [90] compared the effectiveness of LLLT (continuous wave 685 nm
low level diode laser light, 200 mW, 4 J/cm2) or visible light from an incoherent
polychromatic nonlaser commercial light-emitting diode (placebo light source).

Forty-four patients with venous leg ulcers were assigned to two treatment groups
(laser and placebo) or a third group (standard wound care only) to quantify the
effect of the laser therapy. Patients in all three groups received standard wound care
(disinfection, a hydrofibre dressing and compression bandaging). The difference in
the reduction in wound size was not statistically significant in any of the three
groups. A positive effect of using the non-laser device (placebo effect) was demon-
strated in some patients. These study results suggest that LLLT does not stimulate
wound healing in venous leg ulcers [90].
Lagan et al. [91] performed a placebo controlled study on low intensity laser and
combined phototherapy in venous leg ulcer patients (n = 15; mean age = 69.9 years).
The treatment was performed in an outpatient “leg ulcer clinic” once per week for
irradiation/sham treatments for 1 month. A multisource diode array (660–950 nm)
was used for irradiation; parameters were kept constant for the duration of the study
(532 mW; 5 kHz; 12 J/cm2). There was no statistically significant difference between
treatment and placebo groups in wound healing and pain [91].
Recently, Sobanko and Alster [92] reviewed the data on controlled trials with
LLLT. Randomized controlled trials have been performed in humans with venous
leg ulcers, pressure ulcers, and delayed postoperative wounds. Most trials included
only a small number of patients. Treatments varied in terms of laser source (HeNe
– 632 nm, GaAs – 904 nm, diode – 980 nm etc., frequency of application from once
a week to six times a week; the duration of treatment was four to 10 weeks). The
power was 4 J/cm2. Twelve trials were identified with a total of 198 LLLT-patients
but most trials could not demonstrate significantly improved wound healing. The
authors concluded that their data suggest that the addition of LLLT in humans does
not improve wound healing. Oddly in vitro and animal studies suggest LLLT should
facilitate wound healing [92].
Phototherapy
Light-Emitting Diodes
Light has photobiomodulatory (PMB) effects. Light-emitting diodes (LED), origi-

nally developed for plant growth experiments conducted by NASA, were investi-
gated on cells grown in culture, ischaemic and diabetic wounds in rat models, and
human acute and chronic wounds. In the in vitro and in vivo (animal and human)
studies a range of LED wavelengths, power intensity, and energy density parameters
were tried to identify conditions for each biological tissue that are optimal for bio-
stimulation. LED energy applied in vitro increases cell growth 140–200% in mouse-
derived fibroblasts, rat-derived osteoblasts, and rat-derived skeletal muscle cells,
and increases in growth of normal human epithelial cells by 155–171%. Wound size
decreased up to 36% when this technique was used in conjunction with hyperbaric
oxygen therapy (HBO) in ischemic rat models. LED decreased wound healing time
in crew members aboard a U.S. Naval submarine. NASA LED for light therapy
alone, and in conjunction with hyperbaric oxygen, seems to have a potential in

wound healing process [93].
Vinck et al. [94] investigated the efficacy of green LED irradiation on fibroblast
proliferation and viability under hyperglycemic circumstances. LED irradiation was
performed on three consecutive days with a probe emitting green light (570 nm) and
a power output of 10 mW. Each treatment lasted 3 min, resulting in a radiation
exposure of 0.1 J/cm2. The proliferation rate was significantly higher in all irradi-
ated cultures in comparison with the controls (p = 0.001). Future in vivo investiga-
tions should be done in diabetic patients [94].
The effect of PBM on cutaneous wound healing was studied in an animal model
of type II diabetes, Sand rats. Irradiation was performed with a 632-nm light on full-
thickness skin wounds while a diabetic control group received no irradiation.
Significant improvement in wound healing histology and wound closure were found
following treatment with 4 J/cm2 (16 mW, 250 s treatments for four consecutive
days; p < 0.05). The 4 J/cm2 dosage significantly improved histology and closure of
wounds in the diabetic group in comparison to the non-irradiated diabetic group.
Quantitative analysis of basic fibroblast growth factor (bFGF) expression at 36 h
post-injury revealed a threefold increase in the diabetic and non-diabetic Sand rats
after PBM. The results demonstrate that PBM is effective in improving the healing
of cutaneous wounds in an animal model of type II diabetes, suggesting that PBM
(632 nm, 4 J/cm2) would be effective in treating chronic cutaneous wounds in dia-
betic patients [95].
It has been proposed that production of low level reactive oxygen species (ROS)
following illumination is the first step of PBM. It was also shown that white light
(400–800 nm) has similar stimulatory effects as lasers and LEDs. ROS at higher
levels are toxic to cells and bacteria. In an in vitro study, the phototoxicity of broad-
band (400–800 nm, 120 J/cm2) visible light on the survival of several pathogenic
bacteria often isolated from chronic wounds has been investigated: Staphylococcus
aureus 195, Pseudomonas aeruginosa 1316, Escherichia coli 1313, and Serratia
marcescens. Illumination with white light, 120 J/cm2, caused a reduction of 62%,
83%, and 56% in the colony count of E. coli 1313, S. aureus 195 and S. marce-
scens, but not of P. aeruginosa 1316. The phototoxic effect was found to involve
induction of ROS production by the bacteria. It was also found that illumination of
S. aureus 195 and E. coli 1313 in the presence of yocyanin, known to be secreted
by P. aeruginosa, had a stronger bactericidal effect compared to illumination alone.
The study demonstrates that visible light at high intensity can kill bacteria in
infected wounds [96].
A double-blind randomized placebo controlled design was used to study 23
diabetic leg ulcers in two groups of 14 patients. Group one ulcers were cleaned,
dressed with 1% silver sulfadiazine cream and treated with “placebo” photo-
therapy (<1.0 J/cm2) twice per week, using a Dynatron Solaris 705® (Dynatronics,
Salt Lake City, Utah, USA) device. Group two ulcers were treated similarly but
received 3 J/cm2 dose. At evaluation points from day 15 to day 90 the mean ulcer
granulation and healing rates were significantly higher for group two than the
“placebo” group (p < 0.02). While “placebo” treated ulcers worsened during the
initial 30 days, group two ulcers healed rapidly; achieving 56% more granulation
and 79.2% faster healing by day 30, and maintaining similar higher rates of gran-
ulation and healing compared with the “placebo” group. By day 90, 58.3% of
group two ulcers had closed and 75% had achieved 90–100% healing. In con-
trast, only one “placebo” treated ulcer closed by day 90; no other ulcer attained
³90% healing [97]. Although interesting effects have been documented by in-
vitro and animal experiments, in particular with relevance to diabetic wound
models, at present the clinical evidence is unimpressive. The evidence-based
medicine level is 4a.
Electric Field Stimulation (Conductively Coupled Method)
The use of electric fields for wound healing has gained impetus by the discovery of
the biological wound current that is inherent in the tissues. The capacitively coupled
method is much more evidence based and therefore has achieved broader accep-
tance than the inductively coupled (no electrode contact) method that will be dis-
cussed later.
Conductively Coupled Method
The Section on Clinical Electrophysiology (SCE) of the American Physical Therapy

Association differentiates three types of current: direct current (DC), alternating
current (AC), and pulsed current (PC) [98]. The reason the SCE adopted PC is to
provide clearer descriptions of pulsed waveforms that are delivered by the majority
of electrotherapeutic devices used by clinicians. The adoption of PC is not meant to
imply that there is an additional type of basic current. Direct Current: DC is a con-
tinuous, unidirectional flow of charged particles for 1 s or longer. In the tissues, the
negatively charged ions move toward the anode and positively charged ions moving
toward the cathode (Fig. 16.7). The electrode polarity remains constant until it is
changed manually on the electrical device (ES) device. Continuous DC is steady
without pulses. When DC is used to treat wounds, care must be taken to avoid unde-
sirable responses by delivering more than 1.0 mA to the electrodes. Alternating
Current: AC is continuous bidirectional flow of charged particles in which a change
in direction of flow occurs at least once every second. The most common AC wave-
form is the sine wave. Pulsed Current (PC) is the brief unidirectional or bidirec-
tional flow of charged particles (electrons or ions) in which each pulse is separated
by a longer off period of no current flow. PC is described by its waveform, ampli-
tude, duration, and frequency. PC can have two waveforms: monophasic or bipha-
sic. High voltage monophasic PC (HVPC) typically has very short-duration
(2–20 ms) twin triangular pulses that have single-phase charges on the order of 1.6
mq. Although HVPC is unidirectional this current, because of the low phase charge
Fig. 16.7 Schematic Direction of ions

presentation of the flow of
ions and the technical anion
direction of electricity in − cation
electrotherapy +
−
+
−
+
anode cathode
Technical Direction of electricity
+ −
quantity, unlike DC (also known as galvanic current) it does not cause electro-
chemical skin irritation ([99]; Fig. 16.8).
Historically, conductively coupled electrical stimulation has been used for
decades by physical therapists as a treatment for wound care. Several studies have
assessed the effectiveness of electrical stimulation on chronic wounds. The evi-
dence suggests it is a useful, accessible and cheap therapy, which can play a valu-
able role in everyday practice [100].
There are two methods used to apply the capacitively coupled (electrodes make
contact with the body) electric field currents namely, the direct method in which one
electrode is placed in the wound and the second (return) electrode of opposite polar-
ity is placed on intact periwound skin (see Fig. 16.8). In the second method, three
electrodes are used with two having the same polarity placed on adjacent intact
periwound skin on opposite sides of the wound, while the third (return) electrode of
opposite polarity is placed away from the wound on intact skin (Fig. 16.9).
Hinsenkamp et al. [101] examined the effects of low frequency pulsed electrical
current on epidermal repair in vitro. Charge-balance current stimuli proposed for
chronic wound treatment were tested on skin keratinocytes cultured at an air-liquid
interface on dead human dermis. The results suggested that the balance between
proliferation and differentiation in electrically treated samples is significantly
modified in favour of differentiation. More advanced differentiation, shown through
epidermal histology, was obtained in cultures exposed to electrical current, in which
the culture growth, the result of keratinocyte migration and proliferation, was greater
in control samples [101].
Bullock et al. [102] investigated effects of biphasic pulsed currents on re-epitheli-
alization using a keratinocyte colony-forming model and a reconstituted skin wound
healing model. They found that growth of keratinocyte colonies and keratinocyte
migration in an in vitro wound bed were not significantly affected by induced short
duration biphasic pulsed currents at a frequency of 0.5 Hz of 100 and 200 mV/mm
Fig. 16.8 High voltage monophasic PC (HVPC). Electrical stimulation being used to enhance
healing of a left gluteal pressure ulcer
[102]. Other investigators have found that physiologic electric fields with polarity
caused migration of cultured corneal epithelial cells and stromal fibroblasts [103].
On the basis of increased blood flow, protein denaturation, and stimulation of
cellular defense, an antibacterial effect of ES is to be expected. Although the
antibacterial effect of ES already has been demonstrated in vitro, little attention has
been paid to the direct antibacterial effect of changing polarity of the applied current.
Daeschlein et al. [104] aimed to investigate the antibacterial effect of positive and
negative monophasic low-voltage pulsed current on typical Gram-positive and Gram-
negative germs of chronic wounds. Using the Dermapulse-System (Gerromed,
Hamburg; Germany; Fig. 16.10), three Gram-negative (Escherichia coli, Pseudomonas
aeruginosa, Klebsiella pneumoniae) and three Gram-positive (Staphylococcus
aureus, Staphylococcus epidermidis, Escherichia faecium) organisms were tested
against positive and negative polarity low voltage pulsed current. All tested organ-
isms were significantly reduced by ES. The reduction differed significantly between
positive polarity and control and negative polarity and control, with the highest log10
reduction factor (RF) was achieved with positive polarity. Using positive polarity, the
maximum RF was measured for E. coli (median log10 RF 0.83; 25th percentile 0.59,
75th percentile 0.98) and the lowest for S. epidermidis (median log10 RF 0.20; 25th
percentile 0.17, 75th percentile 0.24). Yet, there was no significant difference with
positive ES against Gram-positive or Gram-negative organisms [104].
Fig. 16.9 Three electrodes are used with two having the same polarity placed on adjacent intact
periwound skin on opposite sides of the wound, while the third (return) electrode of opposite polar-
ity is placed away from the wound on intact skin
ES seems to work on different levels important for normal wound healing

(Table 16.4).
In a meta-analysis Gardner et al. [105] attempted to quantify the effect of electrical
stimulation on chronic wound healing. Fifteen studies, which included 24 electrical
stimulation samples and 15 control samples, were analyzed. Rate of healing per week
was 22% for electrical stimulation samples and 9% for control samples. The net effect
of electrical stimulation was 13% per week, an increase of 144% over the control rate.
The 95% confidence intervals of the electrical stimulation (18–26%) and control
samples (3.8–14%) did not overlap. Electrical stimulation was most effective on pres-
sure ulcers (rate of healing net effect = 13%). The findings regarding the relative effec-
tiveness of different types of electrical stimulation device were inconclusive.
Strong support for the use of ES in promoting healing of pressure ulcers comes
from the evidence based Treatment of Pressure Ulcers: Quick Reference Guide,
developed and released electronically in December 2009 (http://www.epuap.org/
guidelines/Final_Quick_Treatment.pdf). This guide concluded a 4-year collabora-
tive effort between the European Pressure Ulcer Advisory Panel (EPUAP) and
American National Pressure UlcerAdvisory Panel (NPUAP): quick reference guide,
developed and released electronically in December [106]. A rigorous and explicit
Fig. 16.10 Low-voltage monophasic pulsed current (Dermapulse; Gerromed, Hamburg; Germany)
Table 16.4 Cellular and antimicrobial effects of electrostimulation

Cell type Effect(s)
Fibroblasts Increase in protein synthesis (including collagen)
Stimulated migration
Keratinocytes Stimulated migration
Macrophages Stimulated migration
Neutrophils Stimulated migration
Vascular endothelial cells Neoangiogenesis
Bacteria Bacteriostatic, bacteriocidic
Kloth [99]
methodology was used in the development of these guidelines (See the Clinical
Practice Guideline for a more detailed description). All evidence was reviewed for
quality, individual studies were classified by design and quality.
The EPUAP/NPUAP recommendation and strength of evidence rating for the
use of ES in the treatment of pressure ulcers is:
• Conductive Electrical Stimulation : Consider the use of direct contact (capacita-
tive) electrical stimulation (ES) in the management of recalcitrant Category/
Stage II, as well as Category/Stage III and IV pressure ulcers to facilitate wound
healing. (Strength of Evidence = A)
• Inductive Electromagnetic Electrical Stimulation: Consider the use of pulsed

electromagnetic field (PEMF) treatment for recalcitrant Category/Stage II, III,
and IV pressure ulcers. (Strength of evidence = C)
Although electrical stimulation produces a substantial improvement in the heal-

ing of chronic wounds, further research is needed to identify which electrical stimu-
lation devices are most effective and which wounds respond best to this treatment.
Improved rates of wound healing and incidence of closure may be improved if
thermal energy applied to wound and periwound tissues were to precede or accom-
pany electrical stimulation treatment that employed more than two electrodes. It is
possible that the use of heat to help dilate blood vessels thereby improving electrical
conductivity would more evenly distribute the higher density electric field around
the wound and surrounding tissues.
The use of a three electrode system was reported. Current was delivered via a
biphasic sine wave at a frequency of 30 Hz and pulse duration of 100 ms. Stimulation
electrodes 5 × 5 cm and 5 × 10 cm were placed at 10 cm and 15 cm separation dis-
tances above the quadriceps muscle. Skin currents were dispersed more evenly and
more deeply than with two-electrode ES (p < 0.05). In wounds, there was almost
complete healing in 1 month, and current was uniform in the wound [107].
A three electrode ES device was tested in combination with local warming of the
wound in non-healing chronic ulcers. Eighteen subjects (mean ± SD age, 35.7 ±
21.3 years) with chronic ulcers (no healing for 26.1 ± 24.6 months) received ES
treatment three times a week for 4 weeks. A heat lamp was used before and during
ES to keep the wound and periwound area warm (37°C). ES was applied for 30 min
with biphasic sine wave (charge balanced, no polarity) stimulation at a frequency of
30 Hz, pulse duration of 250 ms, and current of about 20 mA. Over the 1-month
period, the mean wound area significantly decreased by 43.4 ± 44.5% (p < 0.05),
and wound volume decreased by 57.0 ± 27.9% (p < 0.05). Skin blood flow
significantly increased after application of ES and local heat (p < 0.05). The skin
blood flow response decreased as time progressed and the wound healed. Thus, in
this pilot study, application of a three electrode ES system in combination with local
heat was effective in the healing of non-healing chronic wounds [108].
Jünger et al. [109] investigated 39 patients in a prospective, placebo-controlled,
double blind study on the effect of low-voltage monophasic pulsed current
(Dermapulse; Gerromed, Hamburg; Germany) on healing in chronic venous ulcers
during a 4-month course of treatment. The device is a battery operated device gen-
erating pulses of 140 ms with an impulse frequency of either 64 or 128 Hz (Fig. 16.10).
In the experimental group three ulcers healed while two healed in the placebo group.
Ulcer size was reduced significantly in each group, however, the difference of ulcer
area reduction between the experimental and the placebo group was not statistically
significant. In the experimental group, ES led to rapid and lasting reduction of pain
(p = 0.049). The authors concluded that for the subgroup of outpatients this treat-
ment method was economically effective. They further concluded that ES seems to
be a viable treatment option for therapy-resistant venous leg ulcers [109].
Neuropathic diabetic ulcers often heal slowly. This could be a consequence of

macrovascular and or microvascular disease. Lawson and Petrofsky [110] studied
the impact of room temperature and ES on skin blood flow in diabetic patients.
Recent evidence shows that skin blood flow may be increased if therapy is done
in a warm room. The purpose of this investigation was to compare healing rates
and skin blood flow of full-thickness wounds in people with diabetes and those
without diabetes using a warm room and ES. Twenty subjects with chronic full
thickness wounds were treated at an outpatient wound center, ten were diabetics.
Treatment consisted of biphasic electrical stimulation up to 20 mA for 30 min,
three times a week for 4 weeks at 32°C room temperature. Skin blood flow was
measured by laser Doppler imaging. Skin blood flow increased not only during
the ES – the increase in blood flow was greater for diabetics (87%) than non-dia-
betics (6%) at the periwound skin adjacent to the wound edge – but even at rest
before stimulation started after the initial treatment creating a carryover effect.
There was no increase in skin blood flow in the centre of the wound. Healing rates
over 4 weeks of up to 70% occurred in subjects with diabetes using biphasic cur-
rent [110]. This prompted the authors to compare the effect of general body heat-
ing versus using a local heat source to warm the wound. Twenty nine male and
female subjects participated in a series of experiments to determine effects on
wound healing associated with ES and the application of local heat with a heat
lamp compared to general heating of the subject in a warm room. Treatment con-
sisted of biphasic ES at currents at 20 mA for 30 min three times weekly for
4 weeks in either a 32°C room or, with the application of local heat, to raise skin
temperature to 37°C. Skin blood flow was measured by laser Doppler imaging.
Blood flow increased with both, local or general heating. During ES, blood flow
almost doubled at the periwound skin edge of the wound with a smaller increase
in the centre of the wound. However, the largest increase in blood flow occurred
in the subjects exposed to global heating. Further, healing rates, while insignificant
for subjects who did not receive ES were 74.5% with general body heating and
55.3% with local heat in 1 month; controls wounds got worse [111]. In conclu-
sion, the best healing effect occurred when ES was used with general body heat-
ing. Some benefits were obtained when ES was used along with local heat. It is
speculated that these effects may have occurred due to improved oxygenation
resulting from healing.
A prospective, uncontrolled study was conducted in elderly patients with stage
III or stage IV pressure ulcers. Topical hyperbaric oxygen (THBO) was applied
daily to the wounds of eight subjects of whom three also received ES. After 4 weeks
of treatment with THBO, wound size decreased an average of 34.4%. No significant
differences in healing were observed between patients receiving THBO alone and
those receiving THBO /ES [112].
Janković and Binić [113] studied the effect of a high voltage, frequency rhythmic
electrical modulation system with a biphasic, charge balanced waveform (FREMS,
Aptiva Ballet, Missauga, Ontario, Canada – see Fig. 16.11) on healing of chronic
painful leg ulcers (n = 35 patients with 43 leg ulcers). The subjects were separated
Fig. 16.11 High voltage,

frequency rhythmic electrical
modulation system with a
biphasic, charge balanced
waveform (FREMS, Aptiva
Ballet, Missauga, ON,
Canada)
into two random groups, one treated with FREMS and a control group. Outcome
measures were decreased in leg ulcer surface area, pain, leg ulcer score. It was
reported that ES using the FREMS technique accelerated ulcer healing, reduced pain
and demonstrated better effects in the treatment compared to the control group.
The effect of high voltage pulsed current (HVPC) on healing of chronic leg ulcers
of various aetiologies (diabetes, arterial insufficiency, venous insufficiency) was
evaluated in a randomized trial by Houghthon et al. [114]. Twenty-seven patients
with 42 chronic leg ulcers participated in the study and were randomly assigned to
receive either HVPC (100 ms, 150 V, 100 Hz) or sham HVPC treatment for 45 min,
three times weekly, for 4 weeks. Wound surface area and wound appearance were
assessed during an initial examination, following a 1–2-week period during which
subjects received only standard wound care, after 4 weeks of sham or HVPC treat-
ment, and at 1 month following treatments. The results indicated that HVPC applied
Fig. 16.12 POSiFECT®

(BIOFiSICA, Atlanta, GA,
USA) is a one-piece adhesive
dressing with integrated
bio-current that is intended to
mimic the physiologic wound
injury current
Cathode Paddle (−)

Anode Ring (+)
POSiFECT®RD
to chronic leg ulcers reduced the wound surface area over the 4-week treatment
period to approximately one half the initial wound size (mean decrease = 44.3%),
which was over two times greater than that observed in wounds treated with sham
units (mean decrease = 16.0%). The results of the study suggested that HVPC admin-
istered three times a week accelerated wound closure of chronic leg ulcers [114].
An open trial was undertaken to investigate the efficacy of ultra-low microcur-
rent delivered by the Electro Pressure Regeneration Therapy (EPRT; EPRT
Technologies, Simi Valley, California; USA) device for the management of chronic
wounds. In this study, 23 patients with chronic skin ulcers and two with abdominal
dehiscence that were present for an average of 16.5 months, and were not respon-
sive to standard wound care in a hospital setting, were treated with the EPRT device.
Wounds were treated with direct current (maximum of 3 mA) of one polarity for
11.5 min and then with a current of the opposite polarity for another 11.5 min.
Current in the mA to nA range was applied through special “wraps” applied above
and below the wound. The results revealed that 34.8% of cases achieved complete
wound healing after an average of 45.6 h of treatment, and 39.1% achieved ³50%
healing after an average of 39.7 h of treatment. Several patients achieved significant
results after one to two treatments. The EPRT device accelerated healing indepen-
dent of the patient’s age [115].
POSiFECT® (BIOFiSICA, Atlanta, Georgia; USA) is a one-piece adhesive dress-
ing with integrated bio-current that is intended to mimic the physiologic wound
injury current. The device delivers constant exogenous microampere current that is
1,000 times lower than traditional ES therapy. The therapy is powered by the
POSiFECT® Monitor. The anode is a flexible metal ring embedded in the outer cir-
cular adhesive part of the dressing. This is one of two contacts which are used to
maintain and deliver the micro-current to the wound (Fig. 16.12). The second
contact (the cathode) is on the end of a soft, insulated medical grade wire that is
attached to the power unit under the dressing flap. This contact is placed on the
wound bed during use. Both the anode and cathode are covered in a sodium-rich
hydrogel to ensure good electrical contact. The POSiFECT® Monitor embedded in
the dressing contains a low voltage battery supply and a tightly controlled microcur-
rent delivery system. This system continuously and automatically adjusts the micro-
current to levels to mimic the endogenous wound current. Each disposable dressing
has enough battery life to continue working for at least 48 h. An adhesive-backed
‘lid’ is fitted to cover the wound and maintain a moist wound environment.
A cost-utility assessment of POSiFECT® compared to standard wound care in
elderly patients with chronic, non-healing wounds of >6 months duration, has been
performed in the UK. Based on the study 33% of all wounds are expected to heal
within 16 weeks after the start of ES with this device. The study also suggested that
by using this device there could be a 51% decrease in the number of home clinician
visits, from 4.7 to 2.3 per week. The model also indicated that using the POSiFECT®
instead of standard wound care a 16% reduction in the National Health System
(NHS) cost of managing patient wounds from 2,287 pounds (95% CI: 1,838 pounds;
2,735 pounds) to 1,921 pounds (95% CI: 1,609 pounds; 2,233 pounds) which would
result in a health gain of 0.023 QALYs over 16 weeks [116].
Based on the present evidence from clinical trials, ES used adjunctively with
standard wound care is reported to enhance the healing of lower extremity wounds
of venous, arterial, and neuropathic etiologies as well as pressure ulcers. Available
data from in vitro, animal and human studies suggest that ES improves tissue oxy-
genation and functionality of various cells involved in normal wound healing. ES
would also appear to have some beneficial effects on wound colonization. ES works
on the principles enshrined in the TIME concept though high level evidence from
large controlled studies. The evidence level for ES in chronic wounds is 3.
Inductively Coupled Method
The inductively coupled method employs a coil of various configurations to trans-

mit a pulsed magnetic field into the wound tissues. The magnetic field then induces
an electric field current in the wound and periwound tissues.
The initial development of pulsed electromagnetic field (PEMF) therapy and its
evolution over the last century for use in clinical surgery has been slow, primarily
because of lack of scientifically-derived, evidence-based knowledge of the mecha-
nism of action.
PEMF was used in diabetic and normal wound healing in Db/db and C57BL6
mice. Cultured medium from human umbilical vein endothelial cells exposed to
pulsed electromagnetic fields was analyzed for fibroblast growth factor FGF-2 and
applied topically to wounds. Skin flaps were created on streptozocin-induced dia-
betic mice and exposed to pulsed electromagnetic fields. PEMF accelerated wound
closure in diabetic and normal mice. Cell proliferation and CD31 density were
significantly increased in PEMF groups. Cultured medium from human umbilical
vein endothelial cells in PEMF exhibited a three-fold increase in FGF-2, which
facilitated healing when applied to wounds. Skin of diabetic mice exposed to PEMF
did not exhibit tissue necrosis and demonstrated oxygen tensions and vascularity
comparable to those in normal animals. Results from research on normal and dia-
betic animals and in vitro findings demonstrate that PEMF’s are able to accelerate
wound healing under diabetic and normal conditions by up-regulation of FGF-2-
mediated angiogenesis and by preventing tissue necrosis in response to a standard-
ized ischemic insult [117].
Mitogen-activated autologous peripheral blood mononuclear cells (PBMC)
applied locally on the ulcer surface promote healing of chronic arterial and venous
leg ulcers. In vitro, extremely low frequency electromagnetic fields (ELFEF) interact
with PBMC via Ca++ channels, activating signal transduction cascades, promoting
cytokine synthesis, and changing cell proliferation patterns. ELFEF frequencies were
configured to interact in vitro with the proliferation patterns of PBMC obtained from
normal human volunteers. These ELFEF were then applied peripherally as the sole
treatment to 26 patients with 42 chronic leg ulcers of predominantly arterial or venous
aetiology unresponsive to previous medical and/or surgical treatments in a phase I
before-after design. Either wound healing or deleterious effect was observed in all
patients during the first 2 weeks after ELFEF exposure, permitting their previously
unresponsive ulcers to function as internal controls. After ELFEF exposure, 69% of
all lesions were cured or healed >50% in a period <4 months. Defective wound heal-
ing was observed in lesions associated with important arterial occlusion, uncontrolled
arterial hypertension, severe lipodermatosclerosis, non-pitting oedema, and obesity
(body mass index >30) or autoimmune diseases. Systemic effects are hypothetically
explained by ELFEF activation of PBMC and their subsequent transportation to the
ulcer site via humoral route [118]. PEMF therapy has been used successfully in the
management of postsurgical pain and oedema, the treatment of chronic wounds, and
in facilitating vasodilatation and angiogenesis ([119]; Fig. 16.13).
Fig. 16.13 Pulsed

electromagnetic field (PEMF)
therapy for a pre-sternal
chronic ulcer
Electromagnetic stimulation (EMS) is sometimes used as a treatment to assist

the healing of chronic wounds such as venous leg ulcers. A Cochrane review of the
effects of EMS on the healing of venous leg ulcers was done, a total of three eligible
RCTs were identified. Two trials compared the use of EMS with sham EMS and one
trial compared it with standard topical treatments. One trial found a difference in
healing rates at borderline statistical significance between EMS and sham EMS.
Although the direction of the treatment effect was in favour of EMS, the difference
was not statistically significant [120].
There is currently no reliable evidence of benefit of using EMS to treat venous
leg ulcers.
Conclusions
The management of human chronic wounds is a complex and clinically challenging.

This chapter has focused on the use biophysical forms of energy to treat wounds;
this approach is gaining interest. These different forms of physical energy have the
potential to offer adjunctive care . From the perspective of evidence based medicine,
there is a lack of RCTs; there is however some evidence. Future research should
address the need for high level evidence since physical techniques have good evi-
dence in in vitro and animal model studies. Their use in clinical wound management
needs to be addressed vigorously.
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Chapter 17
Research Studies in Wound Healing:
The Role of Outcomes/Endpoints
for the Evidence in RCTs
Finn Gottrup, Jan Apelqvist, and Patricia Price
Keywords RCTs • Wound management • Evidence based practice • Wound study

Non-healing wounds
Different types of measurements are used in the research studies in wound healing
primarily RCTs. Some of the most important measures in wound healing research:
outcomes/endpoints.
“The Patient Outcome Group (POG)” of the European Wound Management
Association (EWMA) established a working group published in 2010 a document
identifies criteria for producing rigorous outcomes/endpoints in both RCTs and
clinical studies was produced [1]. This chapter is based on this document.
Introduction: Aim of This Chapter
The aim of the document is to provide recommendations on how to achieve rigorous

outcomes/endpoints in studies on wound management. Furthermore to describe an
approach that will enable the design of RCTs and clinical studies to be both consistent
and reproducible in order to reach a higher quality of evidence in wound management.
F. Gottrup, M.D., DMSci (*)

Department of Dermatology, Copenhagen Wound Healing Center, D42,
Bispebjerg University Hospital,
DK-2400, Copenhagen NV, Denmark
e-mail: fgottrup@post4.tele.dk
J. Apelqvist, M.D., Ph.D.
Department of Endocrinology, Malmö University Hospital, Malmö, Sweden
P. Price, Ph.D., CHPsychol
School of Healthcare Studies, Cardiff University,
Cardiff, UK

356 F. Gottrup et al.
Is Evidence Based Practice Important in Wound Healing?
Non healing wounds are a significant problem for health care systems all over the world.
In the industrialized world, almost 1–1½% of the population has a problem wound at
any one time. As an example, the average cost per episode in Europe is 6,650 € for leg
ulcers and 10,000 € for foot ulcers. This accounts for 2–4% of the health care budget – a
figure which is likely to rise with an increasingly elderly and diabetic population.
There is a need for a review of strategies and treatments for this patient group to
reduce the burden of care in an efficient and cost-effective way. If patients at risk
were identified and aggressive interventions occurred before the development of
complications or progression of the wounds, patient morbidity and health care costs
could be significantly decreased. The question for wound care practitioners is which
type of intervention, which type of technology and which type of dressing materials
are the best from the point of view of a single patient or group of patients, with a
primary focus on healing and the absence of complications. Clinicians and clinical
scientists have concentrated on increasing the quality of the available evidence for a
given intervention from the patient perspective (e.g., through the CONSORT initia-
tive on reporting RCTs, www.consort-statement.org/).
Systematic reviews have indicated that there are substantial deficiencies in the
quality of clinical research (www.cochrane.org, www.nice.org.uk) such that all
stakeholders are concerned to increase the quality of work undertaken. Trials in
wound management should, whenever possible, adhere to the guidelines for con-
ducting and reporting clinical studies. However, wound management has a paucity
of high quality evidence, as the studies are often based on inadequate sample size,
short follow up, non-random allocation to treatment arms, non-blinded assessment
of outcomes, poor description of control and concurrent intervention.
This debate illustrates that there is a fundamental controversy about the best way
to evaluate the effectiveness of interventions in this complex patient population.
This confusion is illustrated by recent reviews regarding the value of various treat-
ment strategies for non healing wounds, which have focused on methodological
inconsistencies in primary research. This situation is confounded by the way
Regulatory and Reimbursement Bodies in various countries advice differently on
study design and the way they interpret the consequent results.
What Is Evidence Based Practice?
During the second half of the twentieth century there has been increased emphasis
on the application of evidence based practices to health care. Whilst we may use the
term ‘evidence’ quite informally in everyday use, Evidence Based Practice (EBP)
aims to apply the best available evidence to support clinical decision making with
practitioners reviewing information from rigorous data, instead of relying on single
observations or customs. Key components of this approach include the development
of important clinical questions and critically assessing the level and types of evidence
17 Research Studies in Wound Healing: The Role of Outcomes/Endpoints 357
Table 17.1 Levels of evidence

1a: Systematic reviews (with homogeneity) of randomized controlled trials
1a-: Systematic review of randomized trials displaying worrisome heterogeneity
1b: Individual randomized controlled trials (with narrow confidence interval)
1b-: Individual randomized controlled trials (with a wide confidence interval)
1c: All or none randomized controlled trials
2a: Systematic reviews (with homogeneity) of cohort studies
2a-: Systematic reviews of cohort studies displaying worrisome heterogeneity
2b: Individual cohort study or low quality randomized controlled trials (<80%
follow-up)
2b-: Individual cohort study or low quality randomized controlled trials (<80%
follow-up/wide confidence interval)
2c: ‘Outcomes’ Research; ecological studies
3a: Systematic review (with homogeneity) of case–control studies
3a-: Systematic review of case–control studies with worrisome heterogeneity
3b: Individual case–control study
4: Case-series (and poor quality cohort and case–control studies)
5: Expert opinion without explicit critical appraisal, or based on physiology, bench
research or ‘first principles’
(www.essentialevidenceplus.com/product/ebm_loe.cfm?show=oxford)
available. One potential difficulty in any field is the use of poor quality, contradic-
tory or incomplete evidence.
Different types of evidence are available and their relative importance for chang-
ing clinical practice has been organised into a hierarchy such that a well constructed,
meta-analysis of several well conducted randomised clinical trials is considered by
many to be the most robust type of evidence on which to base changes in clinical
practice (Table 17.1).
Which interventions, technologies and dressing materials are the best from those
available? Ongoing controversy surrounds the value of various approaches to wound
management and care. There is a need to consider alternative ways of achieving the
highest level of evidence required for this patient group.
Quality of evidence in wound management is interesting from different
perspectives:
1. From the clinical perspective the question is which interventions, technologies and
dressing materials are the best from the point of view of a single patient or group
of patients, where the primary focus is healing and the absence of complications.
Wound management has a paucity of high-quality evidence, as studies are often
based on inadequate sample sizes, have short follow-up periods, non-random
allocation to treatment groups, non-blinded assessment of outcomes, and poorly
described control groups and concurrent interventions;
2. From the policy maker and health-care system perspectives especially two issues
arise: a. Whether or not a particular product or intervention is safe and effective
when used as indicated — this is a question of regulatory approval and b. Whether
or not the product or intervention represents a cost-effective use of funds.
Too few good quality clinical or economic studies in wound care have resulted in
challenges to the reimbursement of modern dressings in favour of supposedly
better value traditional products.
3. From the industry perspective (medical device industry) the challenge is that the
standard of care and evidence requirements for reimbursement is different in
each country and that the large investments related to RCTs are rarely justified
by the pace of innovation and size of markets of most wound care products.
The Place of Outcomes/Endpoints in Evidence Based Practice
Study endpoints are the key stone measures in the outcomes discussion.
An endpoint is defined as the objective of an evaluation or study. The objectives
should include: (1) A precise statement of the degree of benefit expected from the
intervention, and its duration; (2) Clear statements on the time frame of the study
(especially in relation to how quickly the benefits might start); (3) A definition of
the patients for whom the benefit is sought.
In the past, the most commonly used clinical outcome (endpoint that directly
relates to outcome) was visible reduction in wound size, particularly intact skin (full
healing).
The development of tests and techniques to improve tissue sampling and analy-
sis, imaging technology and scientific progress in cellular and molecular biology
has enabled the development of more ‘objective’ wound outcome parameters (sur-
rogate outcome parameters) that relate to both the wound condition and the treat-
ment intervention being assessed (for example, rate of exudation, pain, granulation
rate, resolution of necrosis or infection).
A surrogate endpoint is defined as a physical sign or a laboratory measurement
that can be used as a substitute for a clinically meaningful endpoint, effectively
directly measuring how a patient feels, functions or survives.
The challenge, in non-healing wounds, is that these types of endpoints are difficult to
achieve and maintain. If the only gold standard was total wound closure, no therapy prob-
ably would ever be considered efficacious. Alternative endpoints are therefore needed.
Clinical Versus Surrogate Outcomes/Endpoints
The most commonly used clinical outcome (endpoints which are directly related to
outcome) has been visible reduction in wound size, including, in particular, intact
skin (full healing).
Over the past two decades there has been a marked growth in the interest in the
phases of wound healing with targeted treatment interventions being developed.
The development of tests and techniques to improve tissue sampling and analy-
sis, imaging technology and scientific progress in cellular and molecular biology
have enabled the development of more “objective” wound outcome parameters (sur-
rogate outcome parameters) linked to the targeted intervention and wound condition
(for example exudation rate, pain, granulation rate, resolution of necrosis or infec-
tion). As the literature fills rapidly with data validating tests of physiology and
molecular biology of wound healing, the translation of these technologies into the
clinical setting has been slow.
A surrogate endpoint is defined as a physical sign or a laboratory measurement
that can be used as a substitute for a clinically meaningful endpoint that measures
directly how a patient feels, functions or survives. Changes induced by a therapy to
achieve a surrogate endpoint are expected to reflect changes in a clinically meaning-
ful fashion. A valid surrogate endpoint is related to the outcome of interest, and is
affected by the treatment of interest to the same degree and in a manner that accu-
rately reflects the effect of the treatment on the true outcome.
The challenge, especially with regard to non healing wounds, is that this end-
point is difficult to achieve as well as maintain. If the only gold standard was the
achievement of total wound closure, no therapy would ever be considered efficacious.
Conversely, if a non-specific endpoint is chosen, positive findings in the clinical trial
may not translate into a clear clinical benefit at the bedside.
The ideal endpoint in, for example, debridement trials (e.g. topical enzymatic prod-
ucts) should be the achievement of a healthy and viable wound bed, consisting of good
quality granulation tissue. This wound bed would then be suitable for using novel treat-
ments such as tissue-engineered skin substitutes and growth factors requiring cell inter-
action or receptor binding sites. In addition, this would also provide a suitable bed for
skin grafting. An endpoint such as, ‘viable bed’ or ‘graft ready’ might therefore be more
appropriate for trials involving debriding agents rather than complete wound closure.
Alternative endpoints are needed especially when wound intervention is per-
formed for reasons other than healing (for example control of exudation, wound
debridement, reduction of pain, rate of granulation, dressing performance etc.) and
the primary outcome measure selected for any wound study should therefore be
appropriate to the intended purpose of the intervention. For this reason, it is impor-
tant that the study protocol clearly define the primary intent for the wound treatment
and provide a rationale for the outcome measures selected to reflect the primary
intent of the treatment or intervention.
Sometimes the term ‘intermediate’ endpoint has been used, for example to
describe a relative change in wound area. However, the present document uses the
terms surrogate and clinical endpoints.
What Types of Outcomes and Endpoint Measures Have Been

Used in Wound Healing Studies?
In 2002, an article search on wound studies from the period 1982 to 2002 was per-
formed. 28,301 published articles within the area of wound healing were found and
a number of these were analysed. All articles containing “wound healing” as the
subject heading in Medline were selected. The total number of studies included in
this analysis was 930. A number of outcome measures used in both clinical and
experimental settings were reported. For information about studies published before
2003, we refer to the article by Matousek et al. [2].
To achieve an updated status (2003–2009) on how outcome parameters are used,

defined and evaluated, we performed a literature search on chronic/problem wounds/
ulcers, with the objective to examine and register their use of endpoints, the quality
of endpoint definitions and level of robustness in method.
Following an evaluation of abstracts from the recent RCTs and comparative stud-
ies by three reviewers, 176 articles found in the literature search were selected for
analysis. Many studies measured multiple endpoints – in total, this analysis gener-
ated a list of 313 different endpoints.
The endpoints were divided into the following categories (the percentages repre-
sent each category’s proportion of the 313 registered endpoints):
• Reduction rate (24.1%)
• Wound closure (16.9%)
• Healing time (9%)
• Change in wound condition (9%)
• Biomarkers and bacteriology (4.5%)
• Circulation (1.9%)
• Infection signs (4.5%)
• Symptoms and signs (13.2%)
• Dressing performance (7.0%)
• Quality of life (5.8%)
• Costs and resources used (4.5%).
The findings of the analysis were used as a basis for discussing and suggesting
procedures for the successful measurement of each of the types of endpoint catego-
ries defined.
In general, it was found that a substantial number of endpoints (45%) were either
not predefined or insufficiently defined. As part of the analysis, the degree of robust-
ness of the measurement techniques used in studies and the degree of reproducibility
were evaluated. In 70% of cases, a standardised or clearly defined measurement tech-
nique was used to examine the endpoint (e.g. computerised planimetry or a standardised
evaluation method). These were defined as meeting the criteria for an acceptable degree
of robustness. However, 76% of these did not meet the criteria for reproducibility.
What Types of Biases Could Influence Outcome/Endpoint

Evaluation of Studies
When evaluating interventions in wound management it is a challenge to avoid bias.

Designing studies with the aim of obtaining sufficient information regarding out-
comes is particularly hazardous. The document therefore discusses and provides
recommendations regarding some of the considerations that should be made when
designing studies on interventions for the treatment of wounds.
Table 17.2 outlines some of the potential biases that can be introduced into study
design, unless great care is taken at the planning stage. In many cases the issues are
very similar regardless of intervention and are not specific to wound management stud-
ies, however, the key points related to the wound community are discussed below.
There is always the potential for bias to be introduced in studies, but the RCT
design puts the emphasis on reducing bias as much as possible. The above table
highlights the particular difficulties that may apply to wound management studies.
There is always a debate about the nature of the design of the study, dependent on the
Table 17.2 Potential sources of bias

Sources of bias
Accepted practice Application to wound management
Selection bias (all eligible Random allocation There are no particular issues for
patients should have the Concealed allocation wound management studies, and all
same chance of receiving efforts should be made to randomly
the intervention. Both allocate patients to groups
groups are similar at
baseline)
Performance bias (all Participants are blinded to The details of standard treatments
patients should receive treatment allocation should be made explicit. It may be
exactly the same Clinicians (who administer difficult to achieve, but the highest
treatment with the an intervention) are blinded level of blinding should be used
exception of the study to treatment allocation within an RCT. Double blind studies
intervention) are often difficult but blinded (or
independent) assessment of outcome/
endpoint should be mandatory
(especially if the study is not
blinded). Blinded analysis of data
should be feasible
Attrition bias (There All groups are followed-up Studies often have high drop out rates
should be no differences for the same time due to the nature of the participants,
between groups in the and these details must be reported
number of patients lost or Rates of drop-out are not Using a run-in period to address the
in the characteristics of high in either group change in wound status can help to
patients who are lost and Both groups should be increase homogeneity of groups, and
those who remain) similar in terms of patients reduce drop-out rates. Studies should
remaining in the analysis plan their recruitment to allow for
(and similar to baseline) potential drop outs due to the long
intervention periods
Detection bias Outcomes are precisely All outcomes/endpoints should be
(Outcomes should be defined precisely defined, with adequate
objectively measurable follow up periods allowing for
(and repeatable) or recurrence rates to be noted
investigators are blinded Valid and reliable methods Outcome assessors should be blinded
to treatment allocation) exist to measure outcomes to intervention, whenever possible
Length of follow-up is The risk for healthy selection bias has
adequate to identify to be considered in terms of
outcomes generalisability of the findings
Investigators (who assess
outcomes) are blinded to
initial treatment allocation
Investigators are blinded to
other important confound-
ing or prognostic factors
(continued)

Sources of bias
Accepted practice Application to wound management
Publication bias (All All research results should There is a concern that a substantial
data should be available undergo an independent number of studies regarding wound
through publication) peer review evaluation and intervention are not published or not
be made available to the available in indexed journals
public
Study conduct and data Ideally: independent study There is no reason why this should
analysis conduct, independent data not apply to wound management
analysis and reporting
audience for whom the data are prepared: for example, regulatory authorities require
the purist form of an RCT based on a restricted population in order to reduce the
heterogeneity of the population, to ensure that the study has sufficient internal valid-
ity to demonstrate efficacy. However, this restrictive approach to study design will
not allow for the generalisation of the findings to those patients who routinely present
at our clinics – where an effectiveness study, with the emphasis on whether or not the
treatment works pragmatically in routine practice may be more appropriate. There
are certain situations where the outcomes of an RCT may be very predictable, for
example, using healing as an outcome for certain dressing trials: this contradicts a
basic premise for conducting an RCT which states that the researchers should be in
a state of equipoise (i.e., uncertain about which intervention works best). In such
circumstances, a comparative cohort study may be more appropriate, as the resources
used to achieve similar outcomes is a more important question to investigate.
A further level of bias may be introduced if interventions are not used appropri-
ately, in line with the manufacturer’s instructions or as required by the wound condi-
tion. This is particularly difficult when a purist approach to RCT design requires
that the same intervention is used throughout the study period, which would directly
contradict the clinical need to adapt the treatment to the condition of the wound.
There is a real tension between the requirement to stick to a purist approach and
being pragmatic about the ways in which treatments are used in routine practice.
Recommendations for Use of Outcomes/Endpoints
The analysis presented in a previous section resulted in a number of rigorous recom-

mendation for outcomes/endpoints in studies on wound management RCTs and
comparative studies on non-healing wounds (Table 17.3).
Due to the increasing need for valid cost and resource utilisation studies with
regards to wound management, and the general lack of these studies, this outcome
parameter was described with further detail. Tables 17.4 and 17.5 provide some
further guidance on cost and resource utilisation study design.
Table 17.3 Statements on outcomes/endpoints

Wound closure, defined as total epithelialisation without discharge, is the most important
endpoint relating to ulcer healing. It must be confirmed by an independent source (photogra-
phy) and there must be sufficient follow-up to confirm healing
Wound area reduction is a valid endpoint with regard to wound healing but it must be
confirmed by tracing and include a predefined relevant cut-off to ensure that ‘reduction rate
error’ (described in section: ‘reduction rate’) does not occur
There is enough evidence to support the use of a 50% reduction in wound surface area over
time as a useful outcome, provided that the initial wound size and the measurement technique
are taken into consideration. The time interval used in such assessment will vary depending on
the wound type. Any reduction of less than 50% cannot be supported by the current literature;
in these instances, more objective measures of size reduction must be used
Time to heal is an important outcome. However, the study protocol must consider the substan-
tial methodological difficulties entailed, particularly confirmation of the exact date of healing
for each patient during the specified observation period. To date, the accepted time interval for
resource studies is 1 year
There is an urgent need for a validated scoring system with regard to wound condition
When using changes in the wound condition as an outcome parameter, they must be predefined
and measured in such a way that they can be validated independently, wherever possible (for
instance, by photograph)
When using biological markers as a primary outcome, they should be clearly predefined, and a
clinically relevant unit of change should be specified; reliable and valid quantitative assessment
methods should be used
When using wound infection as a primary outcome marker, it should be clearly predefined. At
present, this could be either a binary measure of presence/absence or a composite score
focusing on clinical signs and symptoms
Regardless of the assessment tool used, when using pain as an outcome measure it is important
to pre-define the amount of wound pain reduction that is clinically important
When surrogate parameters such as symptoms and signs, or composite endpoints such as
scales, are used as primary endpoints, it is essential that both their basic definition and what is
considered to be a clinically relevant difference are predefined. When used as an primary
endpoint, it is favourable for it to be verified by an independent evaluator
When assessing dressing performance in an objective manner, with a focus on a specific aspect
of symptom management, a comparative study may not be needed; the relevant data could be
better assessed using a cohort study with a standardised, reproducible and validated protocol
that includes resource utilisation (when appropriate)
HRQoL assessments must be based on tools with established psychometrics
The type of assessment must fit with the purpose of the data collection: if HRQoL data are to
be used for health technology assessment reviews, then generic and/or utility methods must be
included
When cost is used as an outcome parameter in wound management, it is essential to
measure all the quantities of resources used and then add the value of those resources,
according to a predefined protocol. It is recommended that resource use and cost are
shown separately
Table 17.4 Important items of resource utilisation in wound studies

Initial patient and wound assessment
Clinician time
Facility cost (e.g. outpatient clinic visit)
Diagnostic tests (e.g. X-ray)
Laboratory tests (e.g. microbiology)
Dressings, drugs and other disposables
Patient and carer travel time*
Patient out-of-pocket payments*
Patient/carer lost work time*
Wound treatments
Clinician time for dressing changes
Facility cost (clinic or outpatient setting)
Clinician travel time (to patient’s home)
Antibiotics
Diagnostic and laboratory tests
Special equipment (e.g. orthotic insoles)
Patient and carer travel time*
Inpatient costs
Inpatient bed-days
Antibiotics
Diagnostic and laboratory tests
Surgical procedures (theatre time, clinician time, disposables)
Rehabilitation costs
Outpatient follow-up visits
Special equipment (e.g. orthotic insoles)
* Depending on the perspective of the analysis (patient/care cost; social cost)
Table 17.5 A checklist for economic study design

Does the study involve explicit comparison of the costs and outcomes of at least two alternative
interventions?
Are the comparators relevant to the decision-problem that is to be addressed (e.g. are they
representative of normal clinical practice)?
Is the perspective of the analysis clear, and is the perspective relevant to the decision-problem?
Are all of the details of study design consistent with the chosen perspective, such as which
costs and outcomes to include?
Are costs and outcomes measured over a sufficiently long period of time such that all important
programme effects are captured?
Is resource use measured separately from the cost of resources? Are all relevant resources
included in the analysis?
Are resources valued appropriately, e.g. do prices adequately reflect the opportunity costs of
resources?
Are programme outcomes defined and measured appropriately, e.g. are outcomes clinically
meaningful and
Conclusion
This chapter has focused on the role of evidence in wound healing, particularly the
problems associated with achieving the highest level of evidence, the RCT. One of
the most important measures in the process of producing evidence is the use of out-
comes/endpoints. These measures have been described together with the importance
of choosing rigorous and robust outcomes/endpoints in order to be able to design
both consistent and reproducible RCTs and clinical studies in order to reach a higher
quality of evidence in wound management.
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Chapter 18
Models in Wound Healing
Ming Yuan Miao, Ting Xie, Shuliang Lu, and Raj Mani
Keywords Models • Wound healing • Systems modelling • In vitro models
Introduction
The field of wound healing traverses across most specialties. Models are developed
to improve our understanding of the disease process and to plan effective therapeu-
tic strategies. Models may be experimental or theoretical both approached based on
a reductionist philosophy. Systems models are also beginning to interest the wound
healing fraternity. This chapter, in a book that espouses the cause of measurements
in wound healing, presents a current look at experimental models before fleetingly
looking at theoretical models.
M.Y. Miao, M.D., Ph.D.

Burns Centre and Wound Healing Department, Ruijin and 9th Peoples’ Hospitals,
Shanghai, China
Wound Healing Group, Southampton University Hospital NHS Trust,
Southampton SO16 6YD, UK
T. Xie, M.D., Ph.D. • S. Lu, M.D., Ph.D.
Burns Centre and Wound Healing Department,
R. Mani, D.Sc., FACA (*)
Department of Clinical Measurements,
Southampton University Hospital NHS Trust,
Southampton, SO16 6YD, UK
e-mail: rm1@soton.ac.uk

370 M.Y. Miao et al.
Modeling is a difficult branch of science. For the better understanding of the heal-
ing process, wound healing models both in vivo and in vitro, have been developed
and redeveloped so that they could functionally mimic the natural process. In vivo
animal models of epidermal wound healing have been developed in several different
species including rats, mice, rabbits, and pigs. We have experience of different ani-
mal models, each with its specific advantages and disadvantages. Investigator need to
assess which model provides the best balance of pros and cons with regard to the
experimental objectives. Acute wound healing models, including excisional, inci-
sional and burn models, are most widely used for experiments. In the authors’ experi-
ence it is relatively easy to learn these methods. It cannot be emphasized too strongly
that that successful modeling depends on precise procedures: from anesthesia to skin
preparation and surgery are all essential. For a better understanding of these, the
reader is advised to read specific text books and reviews on modeling. The chapter
deals with developments in wound healing models. We are aware that some of the
models discussed need further design; however they are absolutely valuable in order
to understand the nature of healing.
In Vivo Animal Models of Impaired Wound Healing
Animal Models with Diabetes Mellitus
Diabetes is one of the most common underlying causes of impaired wound repair
and lower-extremity amputation in developed countries [1] and other societies are
following suit. Several models of wound repair have been developed in animals to
research potential mechanisms of diabetic wound healing. However, since diabetes
mellitus consists so many pathological variations, no single animal model can be
representative of the whole process. Each model mimics merely one aspect of this
complex disease. An investigator must select the most appropriate animal model to
achieve the experimental aim(s).
Methods and Validation
Generally there are two classifications of animal models of diabetes mellitus; one
induced by surgical or chemical modulation, the other is genetic diabetes mellitus.
Wounds have to be made on the skin of animal such as back, abdomen and neck by
burning, cutting or radiation in order to study healing as such wounds do not spon-
taneously appear on experimental animals.
Theoretically, pancreatic damage mediated by surgery and toxins offers a valu-
able tool with which to study the consequences of hyperglycemia though the current
guiding principles of animal research do not permit this approach. The availability
of much simpler animal models render it unnecessary [2].
18 Models in Wound Healing 371
Several toxins, including streptozotocin and alloxan, induce hyperglycaemia in

animals [3, 4]. Chemical agents produce a rapid development of hyperglycemia by
selective destruction of insulin-producing beta cells of the pancreas in mammals.
In general principles, the smaller the animal, the more manageable and cheaper is
the experiment. Hence, rats and mice are the most commonly induced by toxins.
In our experience, rodents are fasted with 12–16 h then induced through intra-
peritoneal or caudal vein by single or multiple injection of streptozotocin or
alloxan. The occurrence of polyuria, polydipsia, polyphagia, and elevated blood
glucose levels (higher than defined values) confirm the diabetic status of the ani-
mal. Rodents are allowed to manifest hyperglycemia for several weeks before
wounding [3].
Selective inbreeding permits the development of several strains of animal that
have such characteristics such as obesity and insulin resistance. Genetic animal
models of diabetes mellitus can also be divided into two types – spontaneous animal
models of type 1 diabetes include NOD (non-obese diabetic) mouse, BB (bio breed-
ing) rat and Chinese hamster. Spontaneous animal models of type 2 diabetes include
Ob/Ob mouse, db/db mouse, Zucker (fa/fa) rat [2].
Models of Pressure Ulcer
Use of the Model
Pressure ulcers, also known as decubitus ulcers or pressure sores, continue to be a

major health care problem affecting a large proportion of the population which
includes individuals with spinal cord injury, immobility and the elderly [5]. The most
important pathophysiological cause of pressure ulcers are ischemia-reperfusion
injuries and prolonged unrelieved pressure over susceptible areas such as bony
prominences. The latter varies with the tissue at risk. Pressure ulcers range in clini-
cal severity the lowest starting from stage I and progressing to stage IV. Inspite of
the widespread nature and magnitude of this clinical problem, research into pressure
ulcer is still developing [6].
It is impossible to develop an experimental pressure ulcer on healthy tissue. No

single model can adequately reproduce all the pathological aspects of pressure
ulcers on account of the great variability. The most suitable animal model best to
mimic the pressure damage experimentally has been became a subject of debate for
many years [6]. The fuzzy rat model with computerized-controlled pressure deliv-
ery system has attracted wide attention. [7].
Fuzzy rats are chosen to characterize pressure-induced damage to cutaneous and
subcutaneous tissues. The reasons for choosing hypotrichotic rats are to minimize
the effect of hair on skin and subcutaneous tissue pressure. After being anaesthe-
tised, the rats are placed in a custom-made saddle and held in situ using restraining
equipment. Selected level of pressure is applied by metal columns on both sides of
the back. The contact surfaces of the columns should be parallel to the skin surfaces
over the greater trochanter. When the columns are properly positioned over skin, the
positioning swivel joint is tightened. After the first pressure session is completed,
the indentations on the skin surface caused by applied pressure are marked to ensure
proper placement of the mental columns in subsequent pressure inducing sessions.
During each daily procedure, pressure of 145 mmHg is applied for 6 h. Pressure is
measured at the skin surface and applied over the trochanter region. Unless indi-
cated otherwise, pressure is applied for five consecutive daily sessions of 6 h dura-
tion each. The pressure is maintained by a computer-controlled miniature stepper
motor with the aid of interactive software. The histological exam should include
biopsies of skin, subcutaneous tissue and muscles.
Ischemic Wound Model
Use of the Model
Ischemia is one of the most important factors in the development of chronic wounds
[8]. Chronic ulcers in patients with clinical conditions, such as trauma, atherosclero-
sis, diabetes, vascular diseases and peripheral neuropathy, are aggravated and per-
petuated by tissue hypoperfusion and hypoxia [9]. Additional, research shows that
the response of wound healing to topical agents is deranged in the elderly demon-
strating the impact of physiological change of blood supply on wound healing [10].
Cutaneous ischemia can be produced by skin banding and vessel ligation. Skin
banding is often used in the guinea pig model [11, 12]. Vessel ligation can reduce
the blood supply substantial to a region of skin or other tissue. This type of opera-
tion requires well developed surgical skills and a keen knowledge of the vascular
anatomy. Flap surgery may be used to perform vessel ligation on the rabbit ear or
lower limbs [13].
The ischaemic rabbit ear wound model was first establish by Mustoe and col-
leagues. This model has been extensively used especially in the last decade for
growth factor studies [14–16]. To develop an ischaemic ulcer, using an electric clip-
per, shave both sides of each ear and the base of one ear circumferentially. Then use
a depilatory agent to clean the tissue of all hair. This process will render one ear
ischaemic while the other serves as a paired control. Then locate the three major
vessels (?) the rostral, central, and caudal. After incision, dissect under a micro-
scope, each vessel from the surrounding tissue. The rostral and central arteries are
transected and ligated. Ligation of two of the three supplying arteries slows the rate
of healing considerably [14]. However, the loss of blood supply is not irreversible,
and collateral circulation develops in about 2 weeks. Care should be taken to pre-
serve the caudal artery and all associated veins to avoid venous congestion.
After suturing the incision, four or more wounds are created on the inside of each
ear by punch biopsy. Granulation and epithelization occur on avascular cartilage.
Take care not to perforate the cartilage. Similar models may also be set up in the rat
and mouse ear with some experimental modification of the technique [17].
Modeling an Infected Wound
Use of the Model
Although practices of wound management and care continue to improve, wound

infections remain an important cause of morbidity and mortality in surgical and
trauma patients, especially when caused by multidrug-resistant germs [18, 19]. It
has been thought that chronicity in wounds begins with a persistent contamination
of bacteria and endotoxin prolonged proinflammatory cytokines production. This
condition causes an elevated level of matrix metalloproteases with decreased growth
factors secretion [20–22]. Interactions between microbes and host tissue determine
whether colonizing bacteria progress to invade the wound and cause infection.
An infected wound model is usually combined with other wound such as burn and
cutting. After creating wounds on porcine, guinea pig or rodents, bacterial suspen-
sions such as Staphylococcus aureus or Pseudomonas aeruginosa are applied to the
surface of the wound. The concentration of bacteria used depends on the virulence
and pathogenicity of the organism and the immune response ability of the host.
Occlusive dressings are often used to prevent cross contamination and to ensure
optimal growth conditions for the applied bacteria.
A successful infected wound model can be demonstrated by maintaining a bacte-
rial level below 105/g of tissue on tissue biopsies and clinical features [23].
Radiation Wound Model
Use of the Model
In certain clinical situations such as tumor on or near body surface, radiation admin-
istered prior to surgery has several advantages compared to post operation irradia-
tion. However, the main shortage of preoperative radiation is that it increases the
risk of postoperative wound complications [24]. Irradiation causes impairment
tissues healing after surgical operation. The major injuries in irradiated tissue is
characterized a dose-dependent decreased collagen formation, and impaired neo-
vascularization [25]. In order to decrease the incidence of impaired wound healing
following radiation, scientists have used some animal models to study mechanistic
investigation.
After anaesthetizing, animals such as rats and mice are placed ventral surface down-
wards into a container. Animals are placed in a certain angle to irradiate the side of
the back. Irradiation is done with Gamma rays or X-rays. Animal containers are
covered by a lead shield so the irradiation was just applied on a local area for each
animal. The dose and duration of radiation used depend on the animal species, radi-
ation type and wound depth. There is a time window needed for early acute skin
reaction following radical exposure. For this reason, animals are irradiated several
days before the wounding procedure. The wound are usually performed in the cen-
ter of the irradiated skin area through incision or excision.
Sometimes in contrast to local irradiation, total body radiation technique will be
used. Systemic radiation will affect marrow precursors which are critical to wound
repair. Systemic radiation model can be used to compare local with systemic effects
of wound therapy 12 [13].
In Vitro Models
Scratch Wound Model in Cultured Cells
The Scratch wound model was used in astrocyte injury research [26] where the
repair cells especially the fibroblast proliferate around injury area and move to the
center of wound. The scratch wound model well mimics this lateral movement. This
system allows the observation of the wound cellular response to an injury in a cul-
ture environments thereby excluding interactions with other cells.
Target cells are incubated in serum free medium or even adding proliferation
inhibiting drugs. In these culture conditions, the cells can keep viable in a nonpro-
liferating state [27]. The confluent monolayers are wounded by either scratching
with a plastic pipette tip or microinjection needle. The remained monolayers are
washed by PBS (expand PBS before using the acronyme –ED) to remove the most
detached cells and debris. This washing is done to preventing changing the medium
by cell debris and factors released from detached cells. Observation after scratch
includes morphology studies, response of the cytoskeleton elements, proliferation
and mobility.
The scratch wound model is suitable for epithelial and fibroblast-like cells [26].
These cells have polarity and capacity to move fairly quickly. They also possess the
property to endure serum free conditions.
Matrigel Angiogenesis Model
Angiogenesis, the formation of new blood vessels, is a key process for wound repair.
During the phase of tissue repair, new capillaries extend from pre-existing vessels.
The formation of new vessels is important for granulation tissue metabolism.
Angiogenesis in vitro, the spontaneous organization of endothelial cells into capillary-
like structures, was first observed by Folkman [28]. Matrigel is the trade name for a
mixture of basement membrane proteins and growth factors secreted by Engelbreth-
Holm-Swarm (EHS) mouse sarcoma cells. This mixture protein resembles the com-
plicated extracellular environment. The Matrigel angiogenesis model is a two
dimensional model that permits endothelial cells organization to be studied [29].
Matrigel is thawed at 4°C overnight and then pipetted into a tissue culture wells until
polymerized. Endothelial cells are harvested with trypsin, re-suspended in medium with
Fetal bovine serum (FBS) and plated onto a layer of Matrigel at a certain density. Matrigel
cultures were incubated at 37°C for a certain length of time. Capillary like structure for-
mations have been filmed using microscopy. It is important to choose the lowest
magnification of the microscope that will permit the whole area to be analyzed. Microscope
images are acquired and sequential frames captured for subsequent analysis.
Three Dimensional Culture Model
Granulation tissue formation and re-epithelialization during the wound repair

includes the migration, proliferation, with degradation and synthesis of the extracel-
lular matrix [30].
Collagen and/or fibrin appear more complex when cultured with extracellular
matrix (ECM) than as cells on two dimensional surface. The results obtained from
two dimensional assays do not necessarily reflect the situations in vivo due to the
lack of cell-ECM interaction [31]. Engineered biological systems that exhibits
in vivo-like structural and functional characteristics may provide a model for kinetic
and mechanistic research of wound repair.
The main differences in terms of modeling between three-dimensional and two-
dimensional cultures are more complex in scaffold design and longer observation
time [31]. The consideration in scaffold design should be focused on the ability to
create scaffolds with predefined and precisely controlled room to enable optimiza-
tion of structure that can accommodate restricting parameters such as mechanical
properties and nutrient transport [32]. The material of scaffold can divided into
natural polymer and inorganic materials. The former includes collagen, gelatin, fibrin,
chitosan and glycosaminoglycan. The inorganic materials are usually used in bone
tissue engineering covering hydroxyapatite, calcium phosphate and calcium phos-
phate cement. The most popular trade scaffolds are Matrigel and collagen complex.
A Human Wound Healing Model: Human

Skin Tissue Culture System
Reepithelialization is a main goals of healing which implies recovering of the

stratified squamous epithelium. During the epidermal wound healing process, kera-
tinocytes undergo a succession of physiological changes including cell migration,
proliferation and differentiation at the wound margins. To study the process of re-
epithelialization of an epidermal wound, a human supravital skin organ culture
model has been developed. It is centrally de-epidermized and kept in culture [33].
This skin model consists of keratinocytes grown on a de-epidermized dermis and
maintained at the air-liquid junction.
Samples of human skin may be obtained from prepuce, thoracoabdominal or
amputated extremities with punch biopsies. Weight and size standard skin are trimmed
of excess fat tissue. Next step is to fixing each skin sample with needles before remov-
ing a punch biopsy of tissue including the whole epidermis and the upper dermis from
the center of the specimen Each skin sample is placed dermis-side down on gauze in
a culture dish in a culture dish containing Dulbecco’s Modified Eagle Medium
(DMEM) (expand this please –ED) supplemented with FBS (expand please). Ensure
that the medium is only in contact with the underside of the sample and the epidermis
remained constantly exposed to the air. These cultures are incubated at 37°C in air
containing for several days, depending on the experimental procedure [17].
This model has been used in studies investigating complex aspects of cutaneous
dendritic cells [34] and it offers the potential to investigate on epidermal damage by
ultraviolet and keratinocyte transplantation [33].
Models of Cultaneous Scars
Keloid Model in Athymic Nude Mice
Keloid scars are benign fibrous proliferations in the dermis that are proud of the
skin surface [35]. Grafting human keloid tissue to an immunodeficient animal
host has proved to be more successful in creating an accurate animal model which
is prone to induce keloid scarring [36]. A model of human scar tissue implantation
in athymic mice was described some 30 years ago [37, 38]. The histological
examination demonstrated that the implantation retained a healthy graft with

preservation of histological characteristics on both keloid and normal skin
tissue.
Fresh keloid specimens were obtained as excess material from plastic surgery
procedures. These tissue obtained under sterile conditions and placed in aseptic
containers, kept refrigerated until samples were transported to the laboratory for
implantation into athymic mice [39].
The implantation procedure was carried out under sterile conditions. Implants
are made over the scapular area which is swabbed with 70% ethanol in advance,
followed by 1 cm incision made on both sides of back. A fine-tipped scissors was
inserted into the incision for about 2 cm until a subcutaneous pouch is created. The
tissue pieces of standard size usually 10 × 5 mm and no more than 5 mm thick were
inserted into the pouch [40, 41]. The incisions were closed using wound clips or
suturation. After implants are made animals were housed one to each cage. The
mice were observed everyday and the implants are at various times. Keloid history
may be observed for up to 246 days post implantation [41].
Anastomosis of vessels within the implant to the murine vasculature often
occurred at approximately within the first 16 days [42]. Remodeling of the periph-
ery of the implant occurred much more early. So it is suggested that day 16 is the
ideal time to set up experiments in this model [43]. Systemic therapies for keloid
scar also can be evaluated.
Excessive Dermal Scarring Model in Rabbit Ear
This hypertrophic scar model is induced in a anatomic specific site, which is based
on the observation that scars would endure for months on rabbit ears after wounding
[44]. This model can be used to evaluate age related changes in scar formation.
The rabbit model of hypertrophic scar is created on young adult female New
Zealand white rabbits. All of the surgical procedures are performed on the ventral
surface of each ear. This ear area is minimally hairy, this minimizes the possibility
of skin appendage influences on wound healing and scar formation [44]. Animals
are anesthetized by ketamine and xylazine with an intramuscular injection. Four or
more wounds are created down to the bare cartilage using a dermal biopsy punch.
The diameter of punch affects the formation and duration of scar. 5 mm punch
biopsy may fail to generate hypertrophic scar, 6 mm wounds are less hypertrophic
due to faster re-epithelialization [45]. The cartilage must be scored without full
thickness dissection, as the whole deficiency of cartilage would cause undirected
granulation tissue and epithelial ingrowth resulting in expected histology. Complete
removal of the perichondrial layer is needed as it will inhibit epithelialization.
Electro-cauterization should be used discreetly except in unappeasable bleedings.
Scar tissue is usually harvested within 1 month although the persistent elevation of
scars for over 280 days has been documented.
Female Duroc Pig Model for Hypertrophic Scar
Hypertrophic scarring often occurs after deep dermal wound. In 1972 Silverstein
et al. reported that deep donor sites in female Durocs healed with thick scar forma-
tion, but no additional studies followed from this research. Porcine models may
offer the best promise for recreating human wounds using animal models. Female
Duroc pig scars are thick, hairless, firm, and hypo- or hyper-pigmented. Besides
these scars contain disorganized collagen bundles and lack elastin. The disoriented
collagen fibers form in this model is close to the characteristic patterns for human
hypertrophic scar [46].
The hair on the back was clipped and skin cleansed with povidone-iodine solu-
tion and rinsed with 70% alcohol. Rectangular wounds were created with a standard
electric dermatome. For the deeper wounds it was necessary to utilize two or more
passes of the dermatome, which introduces some error in wound depth. The wound
margins are with a minimum of 2.5 cm between each wound [47]. The wound can
be created in different dermis level: shallow, intermediate and deep [48]. Scar tis-
sues are collected on weeks or months post-wounding. Samples are taken from the
center of each wound and adjacent skin at each time point.
The settings on the dermatomes should be very precise. However, this is difficult
to translate into precise wound depths for multiple, uncontrollable reasons. Therefore,
total dermatome settings rather than wound depths are preferred. Additionally, it is
not possible to create wounds with uniform depths. Usually the wounds are deeper
in the middle than at the surrounding [49].
Keloid Heteograft in the Hamster Cheek Pouch
This model has positioned keloid implants into a immune privileged site. The
histological assessment displayed the presence of blood vessels, inflammatory
infiltrate and collagen deposition, similar with human keloid scarring. This model
is easy to handle and more economic than athymic mice model [50]. In terms of
keloids, since Hamster (Mesocricetus auratus) is the only known species in which
this scar model is developed, this is a major limitation for obtaining more experi-
mental model in vivo.
Hamster cheek pouches are highly distensible diverticles of the jugal mucosas,
whose main functions are the storage and transportation of food. The mocosas are
extended under the skin from the posterior border of the oral cavity. The cheek wall
is constituted by two epithelium layers. Each pouch, after being everted, presents two
walls or epithelium layers. Each layer is formed by epithelial cells strata, which are
supported by naturally immunodeficient areolar conjunctive tissue, without lym-
phatic vessels [51]. Therefore, the space between both epithelial walls is considered
an “Immunological Privileged Site”. The ultra-structure of pouch wall epitheliumis
resembles the human skin, which has been named “skin without follicles and
glands” [52].
Keloid samples have to be transported from the surgical suite to the experimental
laboratory immerse into a flask with 0.9% cold physiologic solution. Tissue frag-
ments are obtained from a 2 mm circular punch and should be keep cold. Animals
are preferably anesthetized via the intraperitoneal system. Cheek pouches have to
be washed from the oral cavity through water with a plastic syringe without needle,
removing any residual food or sawdust. Then, each pouch is everted with two
Adson-Brown forceps, followed fixing with 13 × 4.5 needles on a coated styropor
surface of the operating field.
A 5 mm incision is performed on a proximal avascularized area related to the
animal mouth, from the first epithelial layer of the pouch wall, in order to not
touching the pouch retractor muscle fibers, which are inserted into its proximal
third, with the aim of avoiding a possible contact with lymphatics [53]. Through
this incision with the aid of dissection scissors, a rhomboid diversion (???) is
made in the sub-epithelial conjunctive tissue (to serve as a tunnel), up to the
most distal point of the pouch. With thin and long pincers, the cutaneous frag-
ment is inserted between two layers of the pouch epithelium in the pouch, up to
its most distal position. Once positioned, the fragment is slightly compressed
between the two epithelial layers with one of the index fingers. This maneuver
aims to allow the fragment naturally fixed by the adjacent areolar conjunctive
tissue without the need of sutures. After being invaginated, the pouch wall
accommodates the grafts tissue and protects them from external traumas. Thus,
the use of protective dressing is not needed and several animals are allowed in
one cage.
Wound Healing Models in Transgenic and Knockout Mice
The development of transgenic and knockout technology and advances in the genetic
alteration of the murine species have permitted several complex pathologic pro-
cesses in wound repair to be delineated. These animals allow gain-of-function
experiments (overexpression of ligands and receptors) and loss-of-function exper-
iments (gene knockouts by homologous recombination or over-expression of
dominant-negative-acting molecules) [54]. They represent a useful tool for explor-
ing the molecular pathways behind the wound healing.
Transgenic and knockout mice hold the promise of increasingly sophisticated
linkage to promoters that control the gene expression of specific cell types and loca-
tions. Such genetic adjustment allows for studies more clearly on the impact of
single genes in wound healing. For example, this genetic manipulation has already
been used to link genes to a keratin promoter, restricting their expression to only
epithelial cells [55]. Moreover, green fluorescent protein (GFP) and luciferase
transgenic mice are also allow for the detailed dissection of the stem cell biology in
wound healing [56].
However, some of the targeted gene functions might not be revealed due to
redundancy or compensation. This hypothesis is supported by the lack of obvious
wound-healing abnormalities in some knockout mice, such as mice deficient in
KGF or TGF-a [57, 58]. It may also can be reasoned that these proteins are indeed
but not basilic for wound repair [54].
Surgical models in transgenic and knockout mice include standard and impaired
models. The former contain head punch model, ear epithelial wound, back punch
and back incisional model. Tube flap and transverse rectus abdominal myocutane-
ous flap model come under the category of Impaired modela [59].
Expanded Polytetrafluoroethylen (ePTFE) Implants Models
Use of Model
For the limitation of ethically acceptable sampling procedures, Occasionally clini-

cal observation of wound tissue has been limited to qualitative,subjective measure-
ments [60]. However, several researchers have used the ePTFE tube model for
wound healing studies because it is simple, safe and minimally invasive. CellstickR
(Viscose Cellulose Sponge Model – (Interchim, France.) – please check ED) may
also serve as an implant model. However, ePTFE is a non-dynamic model. ePTFE
model can be used for determination of amino acids, hydrolysis and subcutaneous
collagen deposition. Moreover, Cellstick can be used for analysis of a constant leak-
age of wound fluid [61].
The ePTFE model is a tube with three times larger pore size than used in commer-
cial vascular prosthesis implants; it is commercially available as Impra (Insert trade-
mark details) [62]. The deposition levels of collagen in drawn-off wound fluids
detected from assays of hydroxyproline, protein, and DNA can be determined using
the ePTFE implants model [63].
After the disinfecting and anesthestising the skin, perforate the skin with the
large cannula distal to the disinfected site. Insert the suture through the distal end of
the cannula and pull it through the proximal end. It is usual to leave 1 cm of the tube
perforating through the skin at the proximal area [61]. The duration of implantation
can be 1 week or more, depending on the aims of the study [63]. In comparison,
Cellstick use is often over after the first day because the fluids usually exude during
inflammatory stage only.
Discussion
Models Using Systems Biology
The role of experimental models was presented in the preceding sections. Clearly
each model has some uniqueness, there are highlights and limitations. A great deal
of information has been derived from such modeling though the use of animals is
under constant review. Experimental modeling is based on the assumption that an
understanding of a process exists (be it limited). This reductionist philosophy is less
appealing to systems biology who consder known aspects of a process to explain
healing (or nonhealing) in mathematical terms. There are several good examples of
the use of systems approach. [64] and future efforts should seek to combine both
experimental and mathematical modeling.
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Appendix
Technique Manufacturer Website

Ankle brachial pressure Huntleigh, Getinge Group http://www.huntleigh-diagnostics.
index (ABPI) com/diagnostics/
LifeDop, SummitDoppler http://www.summitdoppler.com/
Duplex ultrasound Sonosite http://www.sonosite.com/
PHILIPS http://www.healthcare.philips.
com/main/products/ultrasound/
GE http://www.gehealthcare.com/
euen/ultrasound/index.html
Monofilament assessing Bailey Instruments Ltd http://www.baileyinstruments.co.
uk/
Smith-Nephew Corporate http://global.smith-nephew.com/
master/6600.htm
Vibration perception Neurothesiometer, Horwell http://www.scientificlabs.co.uk
Scientific Laboratory Supplies
Biothesiometer, Bio-Medical http://www.biothesiometer.com/
Instrument Company
Transcutaneous oxygen Moor Instruments http://www.moor.co.uk/
tension PHILIPS http://www.healthcare.philips.
com/
main/
products/patient_monitoring/
Perimed A B http://www.perimed-instruments.
com/
Radiometer Medical http://www.radiometer.com/
Wound tracing Visitrak, Smith-Nephew http://global.smith-nephew.com/
Corporate master/6600.htm
Wound debridement The Versajet Hydrosurgery http://global.smith-nephew.com/
System, Smith-Nephew master/6600.htm
Corporate
High resolution ultrasound ATYS Medical http://www.atysmedical.com/en/
VisualSonics http://www.visualsonics.com/

DOI 10.1007/978-1-4471-2987-5, © Springer-Verlag London 2012
386 Appendix
Technique Manufacturer Website

Optical coherence Carl Zeiss International http://www.zeiss.com/
tomography (OCT)
Video microscopy Flexbar Machine Corporation http://www.flexbar.com/
IScope Corp http://www.amscope.com/
Laser Doppler flowmetry Transonic Systems Inc http://www.transonic.com/
Moor Instruments http://www.moor.co.uk/
Perimed A B http://www.perimed-instruments.
com/
Tristimulus systems Konica Minolta Sensing, Inc. http://www.konicaminolta.com
Hunter Associates Laboratory http://www.hunterlab.com/
DermaSpectrometer Cortex Technology http://www.cortex.dk/
Pressure measurements Medtronic Xomed http://www.medtronic.com/
Rex Gauge Company http://www.rexgauge.com/
Skin torsion Dia-stron http://www.diastron.com/
Footprint impression PressureStat, Visual Footcare http://visualfootcare.com/home.
Technologies, LLC asp
Foot pressure Electrodynogram System, http://www.langerbiomechanics.
measurements Langer Biomechanics Group com
EMED system, Novelusa http://www.novelusa.com/index.
php?fuseaction=home.main
F-scan System, Tekscan, Inc http://www.tekscan.com/
Digital Rheo Dopplex II PPG, http://www.huntleigh-diagnostics.
photoplethysmography Huntleigh Diagnostics Ltd com/diagnostics/
Vasoquant, ELCAT GmbH http://www.elcat.de/index_US.
php
Index
A epidermolysis bullosa, 55–58

Abnormal reactive hyperaemia, 100 external causes
ABPI. See Ankle brachial pressure index chemical burns, 54
(ABPI) factitial dermatitis, 54
Acute wounds radiation dermatitis, 54
antibiotics, 120 spider bites, 53–54
causes, 116 hematoxylin and eosin, 40
corrupt matrix, 117–118 immunofluorescence, 41
diabetic foot ulceration, 147 infectious causes
microbial analysis, 127 atypical mycobacterial infection,
neutrophils, 116 45–46
trauma/excisional surgery, 73 buruli ulcer, 46–48
wound measurement, 74 chromoblastomycosis, 48
Adipofascial flap, 83, 85–86 deep fungal infections, 48
Alport syndrome, 140 mycetoma, 49
Alternating current (AC), 337 necrotizing fasciitis, 50–51
Ambulatory venous pressure measurement paracoccidioidomycosis, 49
(AVP), 3 sporotrichosis, 48
American National Pressure Ulcer vibrio vulnificus infection, 49–50
Advisory Panel (NPUAP), malignancies, 52–53
340–342 metabolic disorders, 52, 53
Ankle brachial pressure index (ABPI), microbiology culture, 41
3, 22–23, 211–213 polymerase chain reaction, 41–42
Antibiotics, 120 pyoderma gangrenosum
Anti-cancer drugs, 119 inflammatory cytokines, 44–45
Anticoagulant drugs, 120 systemic diseases, 46
Anticonvulsants, 27–28 treatment options, 47
Antiphospholipid antibody syndrome, 52 special stains, 40–41
Antiplatelet drugs, 120 swab, 40
Arteriosclerosis, 160 tissue biopsy, 40
Arterio-venous (AV) response, 210 vasculitis
Aspirin, 120 clinical manifestations, 43
Atypical mycobacterial infection, 45–46 diagnostic tests, 44
Atypical wounds potential etiologies, 43
drug-induced causes, 55 tissue biopsy and tissue culture, 44
electron microscopy, 42 treatment options, 45

DOI 10.1007/978-1-4471-2987-5, © Springer-Verlag London 2012
388 Index
Atypical wounds (cont.) Charged couple device (CCD) camera, 233

vasculopathies Chemical burns, 54
antiphospholipid antibody syndrome, 52 Chromoblastomycosis, 48
cryofibrinogenemia, 51 Chronic intermittent hypoxia (CIH), 228
cryoglobulinemia, 51 Chronic ulcers
malignancy, 167–169
vasculitis and vasculopathy, 166–167
B Chronic wound management
Bethlem myopathy, 136 conductively coupled method
Biofilms adjacent intact periwound skin, 340
antimicrobial tolerance, 129–130 alternating current, 337
chronic wound, 130–131 cellular and antimicrobial effects, 341
diagnosis, 131–132 conductive electrical stimulation, 341
therapeutic strategies, 132–133 direct current, 337, 338
Biomarkers, impaired healing electrical device, 339
acute traumatic wounds, 247–249 electrotherapy, 338
bacterial bioburden, 254, 255 FREMS, 343, 344
biofilms and bacterial and fungal species, HVPC, 337–339, 344, 345
254–256 inductive electromagnetic electrical
chronic wounds, 249–252 stimulation, 342
rapid point-of-care detectors, 252–253 POSiFECT®, 345, 346
Brain injury, 94 pulsed current, 337, 338
Burns THBO, 343
adequacy of resuscitation,272–273 electromagnetic non-thermal energy
depth, 270 technologies
blood flow, 267–268 low level laser therapy, 334–345
burn wound pathology, 264–265 phototherapy, 335–337
sources of error, 268–270 electromagnetic thermal energy
structural integrity, 266–267 technologies
surface appearance, 265–266 NNWT, 330–332
extent, 260, 263–264 WIRA, 332–334
Lund & Browder chart, 261, 262 evidence-based medicine, 315
serial halving, 262 inductively coupled method
sources of error, 263 ELFEF, 347
Wallace Rule of Nines, 262 EMS, 348
healing, 277–279 PBMC, 347
intra-arterial monitoring, 273 PEMF, 346–347
microbiological assessment, 273–275 low frequency ultrasound
nutritional status, 276–277 cellular effects of, 316
outcomes, 279–281 chronic venous leg ulcer, 318
severity scores, 270–271 clinical effects, 316
Buruli ulcer, 46–48 ERK/JNK ratio,317
25 kHz UAW Sonoca 180, 320
MIST therapy system™, 319, 321
C PainShield device, 319
Calciphylaxis principle of, 316
histopathology, 164–165 transient cavitation, 317
metabolic disorders, 52 UltraPulse H, 318
Carbohydrates, 67 NPWT/TNP
Carbon monoxide, 120 adverse effects, 329
Carboxyhaemoglobin, 272 clinical randomised trial, 329
Center for Disease Control and Prevention complications of, 330
(CDC), 274, 275 deep wound infection, 329
Cerebral palsy (CP), 94 diabetic foot wounds, 325
Index 389
granulation tissue formation, 326 D

home treatment, 327 Deep fungal infections, 48
lymph vessel proliferation, 325 Deep venous thrombosis (DVT), 10
mesh graft transplantation, 328 Delayed healing/non-healing
MMP-9/TIMP-1, 324 antibiotics, 120
rate of wound healing, 326 anti-cancer drugs, 119
RCTs, 327 anticoagulant drugs, 120
VAC™, 324, 325 antiplatelet drugs, 120
shock waves, 322–323 biofilms
Chronic wounds antimicrobial tolerance, 129–130
antibiotics, 120 chronic wound, 130–131
biofilms, 129–130 diagnosis, 131–132
causes, 73 therapeutic strategies, 132–133
characteristics, 118 carbon monoxide, 120
collagen degradation, 117 colchicine, 120
comorbid ailments, 115 corrupt matrix, 117–118
comorbid factors, 116 corticosteroids, 119
definition, 113 diabetic ulcers, 115
diabetes, 147 epidemiology, 114
diabetic ulcers, 115 erythromycin, 120
ECM component, 121 growth factor problems, 118–119
elastin degradation, 117 hydroxyurea, 119
epidemiology, 114 infection development, 126–127
epidermis and dermis, 116 local factors, 113–118
HBOT, 34 methotrexate, 119
ischemia, 116 nicotine, 120
lower extremities, 145 pathophysiology, 116–117
Marjolin’s ulcers, 53 predisposing factors, 115–116
microbial profile, 125 pressure ulcers, 115
microbiological analysis, 127 smoking, 120
microorganisms infection, 124 systemic factors, 114
MMPs, 118 therapeutic intervention, 120–122
morbidity, 124 vasoconstricting drugs, 120
natural acellular matrix, 122 venous ulcers, 114
neutrophils, 116 wound contamination, 126–127
old age, 115 wound infection
pathophysiology, 120 chronic ulcers, 124
physical trauma, 116 historical background, 123
pressure ulcers, 115 MRSA, 124–125
radiation ulcers, 169–170 potential wound pathogens, 124
resident cells, 118 surgical wound infection, 123–124
senescent (unresponsive) fibroblasts, 121 Dermal Torque Meter, 309
venous leg ulcer, 145–146 DFU. See Diabetic foot ulcer (DFU)
venous ulcers, 114 Diabetes mellitus, 150, 370–371
wound area, 113 Diabetic foot ulcer (DFU)
wound fluid, 117 diabetic peripheral neuropathic pain
CIH. See Chronic intermittent hypoxia (CIH) anticonvulsants, 27–28
CM. See Confocal microscopy (CM) glycaemic control, 25
Colchicine, 120 opioids, 28
Confocal microscopy (CM), 205–206 pathogenesis, 26
Corticosteroids, 119 pharmacological therapy, 25–26
Coumadin necrosis, 55 physical therapy, 28
Cryofibrinogenemia, 51 selective serotonin-reuptake
Cryoglobulinemia, 51 inhibitors, 27
390 Index
Diabetic foot ulcer (DFU) (cont.) pressure measurement

topical treatment, 28 out of shoe measurements, 180–181
tricyclic antidepressants, 26–27 peak pressures, 179–180
effective management, 17 shear forces, 180
epidemiology in shoe measurements, 181
annual incidence, 148 pressure reduction, 176
geographic location, 148–149 felted foam dressings, 187–188
health care costs, 150 half shoe, 186–187
hospital discharges, 149 removable cast walker, 186
LEA and medical complications, 147 total contact cast, 185–186
peripheral arterial disease, 147 prevention
etiology, 175 insoles, 183–184
histopathology shoes, 184–185
arteriosclerosis, 160 socks, 183
collateralization and angiogenesis, 160 prevention strategies, 17
factor VIII, 162 repetitive moderate stress, 175
features, 161 wound assessment
hyperglycaemia, 160 elliptical wound measurement
mal perforans, 162 method, 25
Masson Trichrome strain, 162 ruler technique, 24
neuropathy and ischemia, 160 wound tracing, 25
oxidative stress, 160 Diabetic shoes, 184–185
infection treatment Digital photoplethysmography (d-PPG),
antibiotics, 31, 32 6, 8
G-CSF, 33 Direct current (DC), 337, 338
honey,33–34 Doppler shift, 3, 4, 268
hyperbaric oxygen therapy, 34 DVT. See Deep venous thrombosis (DVT)
PDGF (Regranex), 32–33
microcirculation,209–210
neuroischaemic lesions E
debridement, 29–30 EBP. See Evidence based practice (EBP)
dressings, 30 Ehlers-Danlos syndrome, 135
footwear, 30–31 Electrodynogram system, 181
offloading, 30–31 Electromagnetic non-thermal energy
optimum glycaemic control, 28 technologies
topical negative pressure, 30 low level laser therapy, 334–335
neuropathy phototherapy, 335–337
deep tendon reflex, 21 Electromagnetic stimulation (EMS), 348
10-g monofilament (10-g MF), 18–20 Electromagnetic thermal energy technologies
heat/cold discrimination, 21 NNWT,330–332
modified neuropathy disability score, WIRA, 332–334
20–21 Electron microscopy, 42
pinprick and tuning fork, 21 Electron paramagnetic resonance (EPR)
vibration perception threshold, 21–22 laboratory to bedside, 238–239
offloading, 162 principles, 236–237
peripheral arterial disease vs. TCOM, 238
ankle-brachial pressure index Electron paramagnetic resonance imaging
measurement, 22–23 (EPRI), 235–237
Doppler ultrasound waveforms, 23–24 ELISA. See Enzyme-linked immunosorbent
toe blood pressure and toe-brachial assays (ELISA)
pressure index, 23 Emotional stress, 115
transcutaneous oxygen tension, 23 EMS. See Electromagnetic stimulation (EMS)
peripheral neuropathy, 176–178 Endovenous ablation, 11
pressure evaluation and modulation, 176 Endovenous laser ablation (EVLA), 11
Index 391
Enzyme-linked immunosorbent assays G

(ELISA), 245, 246 Gamma-amino butyric acid (GABA), 27
Epidermolysis bullosa, 55–58, 136 10-g monofilament (10-g MF), 18–20
EPR. See Electron paramagnetic resonance
(EPR)
EPRI. See Electron paramagnetic resonance H
imaging (EPRI) Harris-Beath foot printing technique, 180
Erythromycin, 120 HBOT. See Hyperbaric oxygen therapy
European Pressure Ulcer Advisory Panel (HBOT)
(EPUAP), 340–342 Healing potential index (HPI), 200
Evidence based practice (EBP) Heel, 98
clinical perspective, 357 HIF. See Hypoxia inducible factor (HIF)
industry perspective, 358 High-frequency ultrasound (HFUS), 198, 199,
levels of, 357 316, 317
non healing wounds, 356 High voltage monophasic PC (HVPC),
outcomes/endpoints 337–339
clinical vs. surrogate, 358–359 HPI. See Healing potential index (HPI)
economic study design, 364 HT. See Hyperspectral technology (HT)
non-healing wounds, 362, 363 Hydroxyurea, 119
resource utilisation, 364 Hyperbaric oxygen therapy (HBOT), 34
types of biases, 360–362 Hyperglycaemia, 160
wound healing studies, 359–360 Hyperspectral technology (HT), 232
policy maker and health-care system Hypertensive leg ulcers, 159–160
perspectives,357–358 Hypertrophied scars, 137
EVLA. See Endovenous laser ablation (EVLA) Hypoxia
Excessive healing, 136–137 biological significance, 225–227
Expanded polytetrafluoroethylen (ePTFE) infection, 227
implants models, 380–381 inflammation, 227
Extracellular matrix (ECM), 244, 245, 375 microRNA, 226
Extracellular polymer substances (EPS), 128 mitochondrial respiration, 227
Extracellular regulated kinase (ERK), 317 nitric oxide, 227–228
Extracorporeal shock wave therapy (ESWT), oxygen saturation, blood
322–323 indicator clearance methods,
Extravasation, 55 228–229
Extremely low frequency electromagnetic laser Doppler flowmetry, 229–231
fields (ELFEF), 347 molecular reporting, 233–234
NIRS and hyperspectral imaging,
230–232
F redox signaling, 227
Factitial dermatitis, 54 systemic limitations, 228
Fascial flap, 84, 90 Hypoxia inducible factor (HIF), 233, 234
Felted foam dressings, 187–188
Female Duroc pig model, 378
Fetal bovine serum (FBS), 375 I
Fluorescent resonance energy transfer (FRET), Immunofluorescence, 41
252 Inflammatory cytokines, 44–45
Foam sclerotherapy (FS), 10–11 Infrared thermography, 205
Forefoot, 97 Insoles, 183–184
Frequency rhythmic electrical modulation International Working Group on the Diabetic
system (FREMS), 343, 344 Foot (IWGDF), 19
FRET. See Fluorescent resonance energy Ischemic wound model
transfer (FRET) methods and validation, 372–373
FS. See Foam sclerotherapy (FS) use of, 372
F-scan system, 181–182 Ischium, 99
392 Index
J clinical effects, 316

c-Jun N-terminal kinase (JNK), 317 ERK/JNK ratio, 317
25 kHz UAW Sonoca 180, 320
MIST therapy system™, 319, 321
K PainShield device, 319
Keloid scars, 137 principle of, 316
Knee ulcer, 100 transient cavitation, 317
Knobloch syndrome, 136 UltraPulse H, 318
Low level laser therapy (LLLT), 334–335
LSV. See Long saphenous vein (LSV)
L
Laser-Doppler flowmetry (LDF)
blood flow changes, 201 M
burn depth,268 MAC. See Medial arterial calcification (MAC)
development of, 200 Manchester scar proforma, 296, 297
hypoxia, 229–231 Manchester scar scale, 296–298
LDPI, 200 Marfan syndrome, 135
use of, 200–201 Matrigel angiogenesis model, 375
Laser Doppler imaging (LDI), 229–231, Matrix metalloproteinases (MMPs)
268–270 acute wound fluids,250
Laser Doppler perfusion imaging (LDPI), 200 levels of MMP-9, 247
Lateral malleolar defects, 77 measurement of, 253
Lateral malleolus, 98 MMP-2, MMP-3, and MMP-7, 248
Lathyrism, 134 rapid point-of-care detector, 253
LDF. See Laser-Doppler flowmetry (LDF) serum proinflammatory, 248
LDPI. See Laser Doppler perfusion imaging TIMPs, 246, 251
(LDPI) Medial arterial calcification (MAC), 23
LED. See Light-emitting diodes (LED) Metabolic disorders, 52, 53
Leg ulcer management Methicillin resistant Staphylococcus aureus
assessment, 2–3 (MRSA), 124–125
chronic ulceration, 1 Methotrexate, 119
chronic venous insufficiency, 1, 2 Microvascular ischemia, 114
compression therapy, 12 MMPs. See Matrix metalloproteinases
digital photoplethysmography, 6, 9 (MMPs)
district nurses, 1 MND. See Motor neurone disease (MND)
Doppler ultrasound examination, 3–4 Moor laser Doppler flowmeter (MBF3), 212
Doppler validity and reliability, 4–5 Motor neurone disease (MND), 93
duplex ultrasound, 5 MRSA. See Methicillin resistant
endovenous ablation, 11 Staphylococcus aureus (MRSA)
foam sclerotherapy, 10–11 Multiple sclerosis (MS), 93
NHS, 1 Mycetoma, 49
pinch skin grafting, 12–13
surgery, role of, 9
vascular and non-vascular aetiologies, 2 N
venous reflux and indications, 9 National Institute for Clinical Excellence
LFUS. See Low frequency ultrasound (LFUS) (NICE), 210
Light-emitting diodes (LED), 335–337 NDS. See Neuropathy disability score (NDS)
LLLT. See Low level laser therapy (LLLT) Near infrared spectroscopy (NIRS)
Long saphenous vein (LSV), 9 absorption coefficients, 230, 231
Lower extremity amputation, 147–150 advantage of, 231
Low frequency ultrasound (LFUS) clinical applications, 232
cellular effects of, 316 ischemia, 231
chronic venous leg ulcer, 318 limitations of, 232
Index 393
Necrotizing fasciitis, 50–51 O

Negative pressure wound therapy (NPWT), Obesity, 64
248 Observer scar assessment scale (OSAS),
adverse effects, 329 299–300
clinical randomised trial, 329 Obstructive sleep apnea (OSA), 228
complications of, 330 Opioids, 28
deep wound infection, 329 Optical coherence tomography (OCT), 267
diabetic foot wounds, 325 Optical oximetry, 235
granulation tissue formation, 326 Optical pedobarograph, 181
home treatment, 327 Optimumnutrition, 67
lymph vessel proliferation, 325 Oxidative stress, 160
mesh graft transplantation, 334
MMP-9/TIMP-1, 324
rate of wound healing, 326 P
RCTs, 327 Paracoccidioidomycosis, 49
VAC™, 324, 325 Patient and observers scars assessment scale
Neuropathy disability score (NDS), 20–21 (POSAS), 298–301
Nicotine, 120 PBMC. See Peripheral blood mononuclear
NIRS. See Near infrared spectroscopy (NIRS) cells (PBMC)
N-methyl-D-aspartate (NMDA) receptors, 27 PDGF (Regranex), 32–33
Noncontact normothermic wound therapy Peak pressure gradient (PPG), 179
(NNWT), 330–332 PEMF. See Pulsed electromagnetic field
Non-steroidal anti-inflammatory drugs (PEMF)
(NSAIDs), 120 Peripheral blood mononuclear cells (PBMC),
Normal reactive hyperaemia, 100 347
NPUAP. See American National Pressure Peripheral neuropathy
Ulcer Advisory Panel (NPUAP) detection
NPWT. See Negative pressure wound therapy biothesiometry, 177–178
(NPWT) Semmes-Weinstein monofilament, 177
Nutrition tuning fork, 176–177
cost analysis, 69 pressure, 178
etiological factor, 63 Photobiomodulatory (PMB), 335, 336
intervention trials, 69 Photoplethysmography (PPG), 228–229
malnutrition Pinch skin graft, 12–13
awareness, 69 Planimetry, 304–305
collagen synthesis, 66 PMB. See Photobiomodulatory (PMB)
growth factor synthesis, 65 Podotrack system technology, 180
impaired, 65 Pole test, 213, 214
lactic acid production, 66 Polymerase chain reaction (PCR), 41–42, 254,
macro nutrients, 65, 66 256
medical condition,64 Poor healing/improper healing
microcellular environment, 65 collagen synthesis and polymerization,
micro nutrients, 65, 68 133–135
under nutrition, 64 genetic disorders, 135–136
obesity, 64 POSAS. See Patient and observers scars
oral supplements, 66, 69 assessment scale (POSAS)
overnutrition, 64, 65 PPG. See Peak pressure gradient (PPG)
poor nutritional intake and status, 65 Pressure ulcers
protein deficiency, 65 clinical assessment
screening and diagnosis, 66 abnormal reactive hyperaemia, 100
total body nutrition, 65 normal reactive hyperaemia, 100
nutritional procedure, 64 delayed healing/non-healing, 115
wound care managers, 68–69 histopathology, 162–163
394 Index
Pressure ulcers (cont.) dissected flap, 79

incidence/prevalence, 95–96 distally based fasciocutaneous/
methods and validation, 371–372 musculoneurofasciocutaneous sural
neurological conditions, 93–94 flaps, 89–90
physiopathology, 94–95 fascial flap, 84, 90
preventative measures, 101–103 fasciocutaneous flap, 79, 81–84
recurrence of, 99 flap transfer, 78
sites large gluteal defect, 88
forefoot, 97 lateral malleolar defects, 77
heel, 98 post operative care, 91
ischium, 99 precautions, 90
knee ulcer, 100 propeller flap/scalitonized perforator flap,
lateral malleolus, 98 86, 88–89
sacrum, 97 retrograde fasciocutaneous flap, 77
scrotum, 100 shallow defect, 78
trochanter, 99 skin graft, 76
treatment and post-healing mobilisation sutured and detached flap, 79
conservative management, 103 Reduction factor (RF), 339
fasciocutaneous flaps, 104 Removal cast walkers (RCW), 31
mobilisation, healing, 109 Retrograde fasciocutaneous flap, 77
musculocutaneous flap, 104 Rex Durometer Max Hand, 308
neurologically impaired patients, 103 RF. See Reduction factor (RF)
preoperative preparation, 104 ROS. See Reactive oxygen species (ROS)
reconstruction and surgical options, 104
use of, 371
Protein deficiency, 134 S
Protein intake, 67 Sacrum, 97
Pulsed current (PC), 337, 338 Scar management
Pulsed electromagnetic field (PEMF), 346–347 assessment by photographs, 302–303
Pyoderma gangrenosum, 169 biomechanical properties, 307
inflammatory cytokines, 44–45 clinical evaluation, 292–293
systemic diseases, 46 color–vascularization, 302
treatment options, 47 extension, 309
extensive burn scars, 292
hypertrophy, 305–307
Q laser Doppler, 304
Quality of life (QOL), 314 Manchester scar scale, 296–298
narrow band simple reflectance meters,
303–304
R planimetry, 304–305
Radiation dermatitis, 54 POSAS, 298–301
Radiation ulcers, 169, 170 reliability, 294
Radiation wound model scar height and volume, 305–307
methods and validation, 374 Seattle scar scale, 296
use of, 373–374 Stony Brook scar evaluation scale, 298,
Radio frequency ablation (RFA), 11 301
Randomized controlled trials (RCTs) subjective assessment, 294
NPWT, 327, 328 suction, 308–309
role of evidence, 358, 360–362 technical assessment, 302
RCW. See Removal cast walkers (RCW) three dimensional systems, 305, 306
Reactive oxygen species (ROS), 116 Tonometer, 308
Reconstructive surgery torsion, 309
adipofascial flap, 83, 85–86 transversal image reconstruction, 307
composite tissue (flap), 80 Tristimulus systems, 303
Index 395
TUPS, 305, 306 medical treatment, 196

validity, 293–294 morphometric analysis, 195
Vancouver scar scale, 294–296 objective characterization, 196
SCE. See Section on Clinical pharmacological scientific research,
Electrophysiology (SCE) 196
Scratch wound model, 374–375 portable laser scanner, 196
Scrotum, 100 ulcerative cutaneous lesions, 195
Seattle scar scale, 296 Skin graft, 76
Section on Clinical Electrophysiology (SCE), Smoking, 120
337 Socks, 183
Selective serotonin-reuptake inhibitors Spider bites, 53–54
(SSRIs), 27 Spinal cord injury, 94
Semmes-Weinstein monofilament (SWM), 177 Sporotrichosis, 48
Shallow defects, 78 Squamosus eccrine metaplasia, 169
Skin and vascular assessments SSIs. See Surgical wound infection (SSIs)
ABPI and TBI, 211–214 Stony Brook scar evaluation scale, 298, 301
angiography, 216 Stroke, 94
chronic ulcers, 194 Surgical wound infection (SSIs), 123–124
confocal microscopy, 205–206 Syringosquamous metaplasia, 169
Duplex testing, venous function, 215–216
Duplex ultrasound, 214–215
high frequency ultrasound, 198, 199 T
infrared thermal imaging systems, 204–205 TCAs. See Tricyclic antidepressants (TCAs)
laser Doppler systems TCC. See Total contact cast (TCC)
advantage of, 200 TCOM. See Transcutaneous oxygen
backscattered signal, 200 measurement (TCOM)
blood flow changes, 201 TcPO2. See Transcutaneous oxygen tension
capillaries and dermal vessels, 200 (TcPO2)
concomitant evaluation, 201 THBO. See Topical hyperbaric oxygen
development of, 200 (THBO)
HPI, 200 Thermography, 204–205
LDPI, 200 Tissue inhibitors of metalloproteinases
photons of monochromatic light, 199 (TIMPs), 246, 251
radiation, 199 Tissue ultrasound palpation system (TUPS),
vasodilating effects, 201 305, 306
magnetic resonance imaging, 216 TNP therapy. See Topical negative pressure
MBF3, 212 (TNP) therapy
microcirculation, 209–210, 216–219 Toe blood pressure index, 211–214
mixed arterio-venous ulcer, 207, 208 Toe-brachial pressure index, 23
Perimed PIM3 system, 213 Tonometer, 308
peripheral arterial disease, 208 Topical hyperbaric oxygen (THBO), 343
pH measurement, 202–204 Topical negative pressure (TNP) therapy,
pole test, 213–214 324–330
transcutaneous oximetry, 201–202 Total body surface area (TBSA),
typical venous leg ulcer, 207 260–263, 271
venous haemodynamics, 216 Total contact cast (TCC), 185–186
wound bed colour assessment Transcutaneous oxygen measurement (TCOM)
non invasive tristimulus colorimeter, clinical applications, 234
197 vs. EPR oximetry system, 238
range of colour, 196, 197 Transcutaneous oxygen tension (TcPO2)
wound size analysis epidermal barrier, 202
digital mappings, 194–195 measurement, 202
3D scanning devices, 195 post-heating reactive hyperaemia, 202
epithelial proliferation, 195 sensors, 216–219
396 Index
Trans-epidermal water loss (TEWL), 278, 279 WIRA. See Water-filtered infrared-A (WIRA)
Transesophageal echocardiography (TEE), 272 World Health Organization (WHO), 314
Transthoracic echocardiography (TTE), 272 Wound bed colour assessment, 196–197
Tricyclic antidepressants (TCAs), 26–27 Wound healing
Tristimulus systems, 303 acute
Trochanter, 99 hemostasis, 243
inflammation, 243, 244
molecular and cellular events, 244
U remodeling, 244
UK National Health Service (NHS), 1 repair, 244
Urokinase plasminogen activator (uPA), 117 biological significance, 225–227
complications
delayed healing/non-healing, 113–133
V excessive healing, 136–137
Vacuum-assisted closure (VAC), 30 normal wound repair, 111–113
Valve dysfunction, 114 poor healing/improper healing,
Vancouver scar scale (VSS), 294–296, 301 133–136
Vasculitis cultaneous scars
clinical manifestations, 43 excessive dermal scarring model, 377
diagnostic tests, 44 female Duroc pig model, hypertrophic
potential etiologies, 43 scar, 378
tissue biopsy and tissue culture, 44 keloid heteograft, Hamster cheek
treatment options, 45 pouch, 378–379
Vasculopathy keloid model, athymic nude mice,
antiphospholipid antibody syndrome, 52 376–377
cryofibrinogenemia, 51 cytokines, growth factors and proteases,
cryoglobulinemia, 51 245–247
Vasoconstricting drugs, 120 dynamic reciprocity and regulation,
Venous leg ulcer, 146–147 244–245
Venous ulcers, 114 ePTFE implants models, 380
biological treatment, 159 evidence based practice, 356
endothelial damage, 156 clinical perspective, 357
growth factors, 156 clinical vs. surrogate outcomes/
hematoxylin and eosin, 157 endpoints, 358–359
immunohistochemical marker D2-40, 159 economic study design, 364
phosphotungstic acid hematoxylin, 158 industry perspective, 358
pseudovasculitis/pseudovasculopathy, 156 levels of, 357
punch biopsies, 156 policy maker and health-care system
tumor necrosis factor alpha, 158 perspectives, 357–358
Vibration perception threshold (VPT), 21–22, resource utilisation, 364
177 types of biases, 360–362
Vibration threshold (VT), 176 wound healing studies, 359–360
Vibrio vulnificus infection, 49 nutrition(See Nutrition)
VPT. See Vibration perception threshold oxygen cost, 227–228
(VPT) systems biology, 381
VSS. See Vancouver scar scale (VSS) transgenic and knockout mice, 379–380
in vitro models
human skin tissue culture system, 376
W Matrigel angiogenesis model, 375
Water, 67–68 scratch wound model, 374–375
Water-filtered infrared-A (WIRA), 332–334 three dimensional culture model,
Widened scars, 137 375–376
Index 397
in vivo animal models nitric oxide, 227–228

diabetes mellitus, 370–371 redox signaling, 227
infected wound, 373 systemic limitations, 228
ischemic wound model, 372–373 tissue oxygen tension
pressure ulcer, 371–372 EPR, 238–239
radiation wound model, 373–374 EPRI, 235–237
Wound tissue oximetry optical oximetry, 235
hypoxia TCOM vs. EPR oximetry system, 238
biological significance, 225–227 transcutaneous gas measurements,
infection, 227 234–235
inflammation, 227
laser Doppler flowmetry, 229–231
mitochondrial respiration, 227 Z
molecular reporting, 233–234 Zinc, 68
NIRS and hyperspectral imaging,
230–232

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Colin Davies - Measurements in Wound Healing - Science and Practice-Springer-Verlag London (2013)

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Measurements in Wound Healing

Raj Mani • Marco Romanelli • Vijay Shukla

ISBN 978-1-4471-2986-8 ISBN 978-1-4471-2987-5 (eBook)

Library of Congress Control Number: 2012942730

© Springer-Verlag London 2012

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

When is an idea formed? At the moment it is actively discussed or somewhere in the

1 The Importance of Vascular Investigation and Intervention

12 Wound Tissue Oximetry: A Cornerstone in Wound Care ................ 225

Nilesh. K. Agrawal, M.S., M.Ch. Department of Plastic Surgery, Institute of

Daniel J. Gibson, M.S. Department of Biochemistry and Molecular Biology,

Sarah A. Pape, MB, ChB, FRCSEd(Plast), MClinEd Department of Plastic

Uwe Wollina, M.D. Department of Dermatology and Allergology,

Colin Davies and Keith Poskitt

Keywords Leg ulcer • Photoplethysmography • Vascular • Venous • Foam

Chronic ulceration of the leg is a common condition which is time consuming to

C. Davies, B.Sc.(Hons), M.Sc.

R. Mani et al. (eds.), Measurements in Wound Healing, 1

Fig. 1.1 Venous ulcer

Fig. 1.2 Differential Other causes*

Mixed arterial &

Doppler Ultrasound Examination

The use of handheld Doppler ultrasound as a diagnostic tool is now mandatory

Fig. 1.3 Hand-held Doppler ultrasound assessment of arteries

Table 1.1 Interpretation of ankle brachial pressure indices

Validity and Reliability of Doppler

Other potential limitations to the validity of Doppler ultrasound can be derived

Duplex ultrasonography has become the investigation of choice in patients present-

Digital photoplethysmography (d-PPG) is a non-invasive test, which measures the

Leg Ulcer Diagnosis and Further Intervention

Fig. 1.6 Venous reflux and Superficial and

Superficial and Isolated

The Role of Surgery

Fig. 1.7 Foam sclerotherapy

Compression Therapy after Surgery and Sclerotherapy

Graduated compression stockings or bandages are recommended after surgery to

Pinch Skin Grafting for Venous Leg Ulcers

Fig. 1.8 Pinch skin grafting

Agbor Ndip and Edward B. Jude

Keywords Diabetic foot ulcers • Neuropathy • Infection • Wound healing •

A. Ndip, M.D., Ph.D.

R. Mani et al. (eds.), Measurements in Wound Healing, 17

facilitates benchmarking therapeutic response which makes it essential that ulcer

Initial Assessment of Diabetic Foot Ulcers

As far as diabetes neuropathic complications go, loss of protective sensation (LoPS)

The 10-g Monofilament (10-g MF)

The standard 10-g Semmes-Weinstein monofilament is designed to buckle at a force

Table 2.2 The modified neuropathy disability score (NDS)

Fig. 2.1 Technique for

The Modified Neuropathy Disability Score (NDS)

Deep Tendon Reflex

Vibration Perception Threshold (VPT)

This is assessed using a neurothesiometer (powered by a rechargeable battery) or a

Fig. 2.2 Technique for assessing neuropathy using the biothesiometer

Peripheral Arterial Disease

Ankle-Brachial Pressure Index (ABPI) Measurement

Toe Blood Pressure and Toe-Brachial Pressure Index (TBI)

Transcutaneous Oxygen Tension (TcPO2)

The transcutaneous oxygen tension (TcPO2) is essentially a research tool that is

Doppler Ultrasound Waveforms

The detection of abnormal waveforms on Doppler ultrasound probing of peripheral

(abnormal) or triphasic (abnormal). Any abnormal waveform recorded warrants