Principles and Practice of Burn Care

Pathophysiology of Burn Shock
37
Pathophysiology of Burn Shock
4
Arvind Vartak
Chapter
INTRODUCTION
Burn injury is the commonest form of injury that afflicts
the human beings. Extensive burns are the severest form of
trauma. It is the most painful of all injuries, most
complicated to treat, most expensive to manage and leaves
behind permanent memories, both physical and
psychological. Over the last several decades, better
understanding of physiological changes which occur
following a burn injury has led to great improvements in
treatment. This has significantly improved the results by
reducing the morbidity and mortality in burn injured patients.
Following a severe burn, the patient passes into a state
of shock.1 The success in adequate management of shock
can be achieved with anticipation, recognition and suitable
treatment of shock. Hypovolemic shock is a disparity
between the circulating blood volume and the capacity of
vascular tree (the intravascular compartment). Neurogenic
shock appears as a state of profound mental and physical
depression consequent to severe physical injury and/or an
emotional disturbance (Stedman’s 1976).2 In case of burn
injury, this may be a result of intense pain associated with
emotional disturbance immediately after the burn injury
occurs. It is characterized by pallor, dizziness, restlessness
and rarely unconsciousness. This state passes off
spontaneously and usually is not noticed by the time the
patient reaches a burn care facility. It does not need any
specific treatment.
PATHOPHYSIOLOGICAL CHANGES
FOLLOWING BURNS
Hypovolemic shock appears as a result of loss of liquid
component of the blood (plasma) in to the tissues leading to
edema formation in surrounding tissues. After burn injury
there is an immediate transient decrease in the blood flow in
the involved tissues. This is followed by pronounced arterial
vasodilatation in patent microcirculation. Cotran (1965)3
demonstrated through the electron micrographic studies that
the burn induces an endothelial cell gap in capillary
microvasculature. This leads to increased capillary
permeability from intravascular to extravascular
compartment. Arturson (1966)4 demonstrated the
extravasations of fluorescein-labelled dextran with molecular
weight around 150,000 from post capillary venules within
10 minutes after a thermal injury. This leads to burn edema.
In extensive burns, due to large volume of fluid lost from
intravascular compartment, systemic hypovolemia develops.
This causes fall in the pressure at the arterial end of capillary
network. In an untreated or inadequately treated burn injury
edema formation is retarded. After adequate resuscitation is
carried out, the circulation is restored. With restoration of
the blood flow, edema formation begins as a result of gradual,
prolonged fluid flux into interstitial spaces.
The changes in a burn wound take place due to the
effect of the heat and superimposed by acute inflammatory
reaction (Arturson 1996).5 There is rapid edema formation
which reaches its peak in 1 to 3 hours post burn. Increased
permeability may prolong up to 16 hours post burns. Severe
interstitial edema formation may cause significant ischemia
due to the compression of subdermal vessels and can lead
to progression of necrosis. This leakage from intravascular
to extravascular compartment may continue for a long time,
even for 24 hours. Baxter (1967, 1974) 6,7 feels that the
increased capillary permeability following a burn injury
continues for about 24 to 36 hours and recommends that
the colloids should be avoided for first 24 hours.

38
Principles and Practice of Burn Care
Capillary permeability, following direct exposure to heat
and in response to certain substances released in the tissues
which include histamine and serotonin, increases severely,
leading to loss of fluid from intravascular compartment to
extravascular compartment. These changes can occur even
in unburnt tissues. This increased permeability in unburnt
tissues like skin and muscles may remain elevated for as
long as 12 hours after the burn injury for the molecules of
the size of albumin. The sustained increase in water content
and elevated lymphatics flow in these tissues is probably
caused by sustained hypoproteinemia (Arturson 1996).5
In superficial dermal burns, the fluid eventually exudes
out of the wound in the form of blisters. There is
preponderance of albumin in this exudate due to smaller
size of albumin molecules. This permits their escape into
extravascular compartment more easily. Sometimes clotting
occurs in blister fluid indicating that large fibrinogen
molecules have also leaked out of the vascular tree. This
loss of protein-rich fluid from plasma after burn leads to a
clinical condition of shock.
The protein content of the exudate may be as high as
80% of plasma levels (Muir, Barkley and Settle (1987).8
Plasma
Blister Fluid
Albumin
3.9 gm%
3.7 gm%
Globulin
2.6 gm%
1.3 gm%
Total Proteins
6.5 gm%
5.0 gm%
After 24 to 36 hours, the capillary endothelial integrity
returns spontaneously. The large molecules of colloids
which leaked into the interstitial spaces can not be brought
back into the circulation. This additional colloid load has to
be handled by the lymphatics.
Under normal circumstances, the lymph returns to the
circulations in volume comparable to intravascular pool.
About 50% of circulating plasma protein is carried daily by
lymphatics. Following a burn injury, quite obviously, the
lymphatic system is put to a very severe increase in its
work load and can not function efficiently. This occurs due
to increase in the volume as well as the process of coagulation
due to leakage of fibrinogen and its products in interstitial
spaces leading to lymphatic obstruction (Arturson 1996).5
Extensive extracellular edema after the burn injury may
occur as a result of increased capillary hydrostatic pressure.
Reduction in protein concentration of plasma causes
reduction of colloidal osmotic pressure and together with
lymphatic blockage and increased permeability of capillaries
leads to leakage of protein rich fluid into the extravascular
compartment. Dilatation of capillaries causes a dramatic
fall in peripheral resistance and increase in the blood flow.
The altered endothelial permeability leads to the leakage of
larger protein molecules into the interstitial compartment
(Arturson 1996).5
In the absence of treatment, the natural defense
mechanisms of the body counter the development of shock
in the following ways:
1. Proportionately less loss of protein than that of
electrolytes. This leads to relative increase of plasma
osmotic pressure, which reduces further leakage.
2. By withdrawing fluid from extracellular space of
undamaged tissues which may be result of increased
osmotic pressure.
3. By general visceral vasoconstriction reducing the volume
of vascular compartment.
4. Development of intense thirst leads to increased ingestion
of fluids and rapid absorption from the gut.
By these mechanisms, the body may be able to
compensate for the rate of fluid loss in minor and moderate
burns. The patient may pass safely through the shock phase.
However, in extensive burns, the rate of loss outweighs
these in-built defense mechanisms and the plasma volume
continues to fall. When untreated or inadequately treated,
the patient may progress into a state of irreversible shock.
Edema formation is retarded in larger injury as a result
of systemic hypovolemia until adequate fluid resuscitation
is carried out. On many occasions, loss of plasma volume
occurs in excess of replaced fluid in spite of massive infusion
over several hours.
In minor and moderate burns, it may be possible to
achieve the replacement of lost fluid by oral ingestion alone.
In extensive burn injury, however, the rate of loss of fluid
from the intravascular compartment is so high that it may
not be possible to replace the fluid at the rate at which it is
lost by oral route alone. Replacement by intravenous route
is then necessary. Adults with 25% or more Burn Surface
Area (BSA) require intravenous replacement of lost fluids.
Children and elderly patients are likely to require aggressive
resuscitation even with a burn injury as little as 10% BSA.
It will be useful to understand the fluid shift in normal
tissues. Under physiological conditions, the high pressure
in the arterial end of capillaries causes the filtration of plasma
into the tissue interstitial spaces. At the venous end of
capillaries this fluid is partially reabsorbed and returns to
the circulation. Remaining fluid is handled by the lymphatic
system which drains finally to the venous system.

Pathophysiology of Burn Shock
39
Burn injury is only to the skin; however secondary
involvement of all organs can occur as a result of reduced
tissue perfusion due to a state of shock. In untreated severely
burned patients the changes become more severe and
widespread and finally lead to death due to multiorgan failure.
EFFECTS OF OLIGEMIA ON VARIOUS ORGANS
(TABLE 4.1)
As a result of reduction of plasma volume, vasoconstriction
and decrease in venous return, there is a marked fall in
cardiac output in patients with severe burn. There is severe
tachycardia. With proper fluid replacement, the cardiac
output promptly returns to normal. Without adequate fluid
therapy the cardiac output continues to fall at a slower rate
until death occurs as a result of multi-organ failure.
As a result of vasoconstriction and increased heart rate,
blood pressure is maintained till late and is not a reliable
guide for the assessment of state of shock. Moreover, in
these patients, due to formation of severe edema, blood
pressure readings cannot be taken accurately.
Table 4.1: Pathophysiologic effects of major burn shock
System
Early
Late
Cardiovascular
↓
Cardiac Output (CO) due to decreased
↑
CO due to sepsis
circulating blood volume, impaired cardiac
↑
CO may be 2-3 times > baseline hypertension,
contractility
tachycardia
↑
Systemic vascular resistance (SVR)
↑
SVR
Respiratory
Upper airway obstruction due to edema
•
Bronchopneumonia
Lower airway obstruction due to edema,
•
Tracheal stenosis, laryngeal damage (as
bronchospasm, particulate matter
complication of intubation)
↓
FRC
↓
Chest wall compliance
↓
Pulmonary compliance
↓
Pulmonary edema (as a consequence of
overzealous resuscitation )
Renal
↓
Renal blood flow
↑
Renal blood flow
↓
GFR: due to
↑
GFR 2o to ↑CO
a. ↓ Circulating blood volume
Tubular dysfunction
b. Myoglobinuria
c. Hemoglobinuria
Hepatic
↓
Function due to ↓ circulating blood volume,
↑
Functions due to hyper metabolism, enzyme
hypoxia, hepatotoxins
induction, ↑ CO
↑
Functions due to sepsis-drug interaction.
Hepatitis
Coagulation and
Hemoconcentration
Anemia
Hematology
Hemolysis
↓
Platelets
↑
Fibrin split products, consumptive
coagulopathy, anemia
Infection and
Impaired immune function (burn wound sepsis,
Multiple organ failure, endotoxemia
immunity
pneumonia)
Neurologic
Encephalopathy
Encephalopathy
Seizures
Seizures
↑
ICP
ICU psychosis
Metabolic
↓
ionized calcium
↑
metabolic rate
Impaired thermoregulation
↑
CO2 product and O2 utilization.
Gastrointestinal
Stress ulceration (Curling’s ulcer)
Stress ulceration (Curling’s ulcer)
Impaired intestinal barrier function
Impaired intestinal barrier formula
Endotoxemia
Endotoxemia
Skin
↑
Heat, fluid, electrolyte loss
Contractures, scar formation
Pharmacokinetics
Altered volume of distribution
Tolerance to narcotics, sedatives,
Enzyme induction, altered receptors.
Drug interaction.
↓, decrease in; ↑, increase in; CO, Cardiac output; FRC, Functional residual capacity; GFR, glomerular filtration rate; ICP, intracranial pressure;
ICU, intensive care unit
Adopted and modified from: McLennan N, Heimbach DM, Cullen BF. Anesthesia for major thermal injury. Anaesthesiology 1998, 84: 749-770.

40
Principles and Practice of Burn Care
In the intestines, vasoconstriction may be so intense
that the blood flow may be inadequate to satisfy the needs
of cells of capillary walls. Absorption of fluid from intestine
cannot occur, peristalsis ceases and paralytic ileus
develops. At this stage, the patient who is intensely thirsty
starts vomiting after any attempt at drinking fluids and this
important defense mechanism fails. Presence of peristalsis
indicates that the visceral circulation is adequate. Similarly
in the stomach, the intense vasoconstriction and
hemoconcentration resulting in micro-thrombi may lead to
acute ischemic damage to gastric and duodenal mucosa
resulting in acute mucosal lesions called ‘Curling’s ulcer”.
Brain continues to receive significant contribution of
blood flow for a considerable time and consciousness is
not lost till late.
There is severe reduction in microcirculation in lungs
though rapid and continuous blood flow in larger vessels is
noticed. The ventilation rate increases and is usually
proportional to the severity of burn injury and declines
gradually. Oxygen consumption also increases but is not
affected by the ventilation rate if the respiratory exchange
is not impaired. However, in case of inhalational injury the
oxygen tension decreases and the blood gas changes are
noticed. This is discussed in detail in chapter on Respiratory
tract injury in burns. Interstitial edema is present in varying
degrees which later leads to a classical condition called
ARDS (Acute Respiratory Distress Syndrome). Both these
conditions lead to inadequate oxygen transfer resulting in
marked hypoxia in various organs.
Of all the viscera, the kidneys are most likely to be
damaged by persistent shock. Secretion of urine is a valuable
guide to renal function and indirectly to the blood flow of
kidneys and thus to the tissue perfusion is general. During
the shock phase, there is a marked rise in Antidiuretic
Hormone (ADH). This results in conservation of body fluids
when oligemia is present. Antidiuresis may be the combined
effect of ADH, hypovolemia and reduction of plasma flow
due to release of vasoactive substances (Wilkinson, 1966).9
After a prolonged, uncontrolled shock, renal failure ensues.
After about 48-72 hours, if hypovolemia is adequately
corrected, reabsorption of interstitial fluids starts, the level
of ADH falls and the phase of diuresis starts.
Liver, pancreas, endocrines and in fact all other
organs are directly or indirectly affected due to the effects
of oligemia, fall in cardiac output and vasoconstriction. The
liver enzymes (SGOT, SGPT and Alkaline phosphatase) rise
promptly following burns, peak at 2-3 days and persist at
high levels till several days post burns due to the liver damage
caused by oligemia.
In the endocrine system there are variable changes in
the different hormonal levels. There is decrease in insulin
production in early post burn period lasting for 2 to 3 weeks.
Glucagon is elevated but fasting blood sugar is not grossly
increased. Growth hormone increases in the first 2 weeks
and then decreases to a lower level which is still above
normal for another 6 to 8 weeks. This can lead to slight
increase in blood glucose levels. ACTH level increases
proportional to burn size, especially in children. Cortisol
levels increase two to four fold. The Gonadotrophic
hormones are only slightly affected.
The effects of thermal injury on the microvasculature
of the skin are discussed in pathophysiology of the Burn
Wound.
Anemia in Burn Patient
The term anemia generally pertains to reduction below
normal of the concentration of oxygen transporting capacity
of a designated volume of blood and is generally expressed
as the amount of hemoglobin in 100 ml of blood (Stedman
1976).2
Following extensive burn, plasma is rapidly lost from
intravascular compartment to extravascular compartment
and causes significant rise in hemoglobin and hematocrit
levels. This slows down the blood circulation particularly
in the zone of stasis leading to local tissue anoxia (Jackson
1953).10 Hence, blood transfusions are not advisable at this
stage. Hemoconcentration can have deleterious effect on
the zone of stasis where the circulation in the tissues has
already slowed down.
After initial rise of hemoglobin and hematocrit levels, a
patient with extensive burn injury becomes rapidly anemic.
Lowered hemoglobin can lead to tissue hypoxia, retard
wound healing and hamper the ‘take’ of skin grafts.
In a burn patient, anemia can develop due to various
causes/mechanisms (Davies 1982).11
1. Red cells destruction as a result of direct heat during
the process of burning.
2. The red cells which are not completely destroyed but
are exposed to heat are rendered unduly fragile. They
are sooner or later removed from the circulation earlier
than the expected balance life span of these RBCs.
3. Overzealous resuscitation leads to over-hydration and
consequent hemodilution gives rise to a false anemic
state.

Pathophysiology of Burn Shock
41
4. Bacterial toxins can cause hemolysis, particularly the
invasive infection by various hemolytic organisms
sometimes leading to massive red cells destruction.
5. Severe hemorrhage at the dressing changes (particularly
when the dry dressing is used) during removal of
adherent dressing at the stage when the granulation tissue
in the wound has started developing after the necrotic
tissue has come out.
6. Thrombocytopenia leading to increase in the bleeding
tendency all over the body which may result in bleeding
from the wound, mucous membrane, lungs,
gastrointestinal tract and in fact anywhere in the body.
7. Inadequate assessment of blood loss and resultant
inadequate blood transfusions during operative procedure
like escharectomy, debridement of granulation tissue,
skin grafting operation, primary or secondary excision
of the burn wound, etc.
8. Poor nutritional status of patients with extensive burns.
9. Biochemical and morphological alterations in red cells
and hemoglobin lead to their reduced efficiency in oxygen
transport.
REFERENCES
1. Kramer GC, Nguyen TT. Pathophysiology of Burn Shock and Burn
Edema In D.N. Herndon (Editor) Total Burn Care. WB Saunders
Co. Ltd 1996;44-52.
2. Stedman’s Medical Dictionary, 23rd Ed. The William and Wilkins
Company 1976.
3. Cotran RS. The delayed and prolonged vascular leakage in
inflammation II. An electron microscope study of the vascular
response after thermal injury. Am J Pathol 1965;46:589-620.
4. Arturson (1966) reference from “ Physiological responses to burn
injury. Ed. JWL. Davies, Academic press, 1982;47.
5. Arturson G. Cross reference from “Local Effects: Principles and
Practice of Burn Management Ed. JAD. Settle, Publishers Churchill
Livingstone 1st edition 1996”.
6. Baxter CR. Fluid therapy of Burns, J. Trauma 1967;7:69-73.
7. Baxter CR. Fluid volume and electrolyte changes in early post burn
period. Clin. Plast. Surg 1974;1:693-709
8. Muir, Barkley, Settle. Burns and their treatment 3rd Ed. Butterworth
1987.
9. Wilkinson. Cross reference from “Physiological responses to burn
Injury Ed. JWL Davies (1982) Academic Press” 1966; 262.
10. Jackson DM. The diagnosis of the depth of burning. Br J Surg
1953;40:588-96.
11. Davies JWL. Anemia and red cell Disorders, Physiological responses
to burning injury, Academic Press 1982.

42
Principles and Practice of Burn Care
Pathophysiology of Burn Wound
5
Arvind Vartak
Chapter
INTRODUCTION
The skin is the largest organ in the body. It is made of
epidermis and the dermis. In the dermis there is ingrowth
of epidermal elements like sweat glands, sebaceous glands
and hair follicles (Fig. 5.1).
The epidermis consists of stratified squamous epithelium.
The deepest layer consists of the tall columnar cells. As
they approach the surface they flatten. The outer layer is
continuously shed off as dead cells and forms a keratin
layer which is continuously replaced from underneath.
Dermis is formed mainly by the fibrous connective tissue
and ingrowths of various epidermal structures from out
side, namely, sweat glands, sebaceous glands and hair
follicles. Blood flow to the skin comes through capillaries
arising from subdermal plexus of blood vessels which bring
nutrients to the skin. It also contains nerve endings which
provide sensation to the skin.
The characteristics of the skin vary from place to place
depending upon the function of the particular part of the
body. Thus, the skin of upper eyelid is very thin while that
of the sole is very thick. It can be different even in the
same part of the body, e.g. the skin of the palm is thick
while that on the dorsal aspect of hand is thin and very
loose so that free movements of the fingers and the hand
are possible.
Heat destroys a greater or lesser depth of the skin,
depending upon the intensity and the duration of exposure.
When the burn injury causes partial thickness damage, the
epithelium from the epidermal appendages grows to the
surface of viable dermis underneath the devitalized layers.
The dead tissue separates and is shed off after the healing
underneath is complete.
Greater the depths of the burn, lesser are the epidermal
structures available to grow and develop a new epidermis.
Thus, the superficial burns heal rapidly while the deeper
partial thickness burns take a longer time to heal; may be
even 3-6 weeks. Full thickness burns, however, heal either
by contraction, if very small or with a contracture formation
if large and opposite joints. They may not heal at all over
rigid areas of bone, e.g. skull unless surgical intervention is
done in the form of skin grafting.
The burn wound, although it damages only the skin cover
of the body, is the cause for a variety of widespread
complications in the body involving almost all organs
systems, initially as a result of oligemia and later due to
septic complications.
FUNCTIONS OF THE SKIN
The skin acts as a protective barrier and prevents invasive
infection. When this barrier in broken, the microorganisms
from outside can invade into the body. When large areas of
Fig. 5.1: Diagrammatic representation of anatomy of the skin and
classification of burn injury in relation to the depth of burn-superficial
partial thickness, deep partial thickness and full thickness

Pathophysiology of Burn Wound
43
skin are damaged, they can lead to bacteremia or even
septicemia.
Skin also helps in retaining body fluids. When large areas
of skin are damaged, plasma leaks out. This can result in a
state of hypovolemic shock. This loss can sometimes be so
large that a correction by intravenous infusion becomes
necessary and the replacement of fluids by oral route alone
is not enough. This lost fluid contains various salts leading
to electrolyte imbalance.
Body temperature is regulated by dilatation or constriction
of blood vessels in the dermis. Vasoconstriction reduces
the heat loss while vasodilatation increases it. Sweating helps
in cooling the body in a hot climate.
The sebaceous glands secrete a fatty substance which
lubricates the skin, keeps it soft and protected from water.
The nerve endings are the defence mechanism of the
body through various sensations, which perceive touch,
pain, pressure and changes in temperature, etc.
Skin also helps in immune responses of the body. Rickatts
et al (1951)1 also postulated that the skin has self-sterilizing
ability.
The transfer of thermal energy to the skin occurs in
various ways like conduction-in a case of contact burn,
convection-when there are currents of hot air or liquid and
radiation-by transfer of heat without involving the medium
through which it occurs, like in case of sunburn or from
bonfire (Williams and Philips 1996).2
Mortiz and Henriquez (1947) 3demonstrated that the
transfer of heat from the surface to the deeper layers leads
to cellular necrosis depending upon the temperature and the
duration of exposure.
The Heat Required to Cause Skin Burns
Full thickness
At 44°C in 6 hours.
At 52°C in 6 minutes.
At 59°C in 1 minute.
At 62°C in 30 seconds.
At 67°C in 20 seconds.
At 71°C in 10 seconds
Partial thickness
At 70°C in 1 second.
Ofeigsson (1965) 4conclusively demonstrated that use
of water as a cooling agent for the burn wound is beneficial.
His animal experiments showed that the general condition
of the treated animals was far better than that of controls.
There are much less secondary infections. Water cooling
arrests and causes retrogation of the degenerative process
of the skin and underlying tissues, thus significantly
shortening the period of healing. He also showed that the
superficial temperature of the scalded skin of controls takes
several minutes to fall and thus causing longer exposure to
heat (longer than what we believe.) He also warned that
extensive burns treated with ice water can cause
hypothermia. This is likely to be true in cold countries and
in Northern India in winter.
Cotran (1965)5 carried out electron micrographic studies
and showed that a burn induces endothelial cell gaps in the
capillary microvasculature. This leads to increased capillary
permeability from intravascular compartment to
extravascular compartment leading to burn edema.
When injured, the skin repairs itself by:
1. The growth of the epidermis from the margins of
unburnt skin.
2. Epidermal growth from intradermal epidermal adnexal
elements, namely, sweats glands, sebaceous glands and
hair follicles.
HISTOPATHOLOGY OF BURN WOUNDS
Burn injury has been classified into partial thickness burn
and full thickness burn in relation to the anatomical
involvement of the layer of skin involved (Fig. 5.1).
In a partial thickness burn, whole of epidermis and part
of dermis are burnt (Figs 5.2 and 5.3). The epithelial elements
from the remaining part of dermis grow over the surface
between the viable dermis and the superficial necrotic layer
which finally leads to healing. The necrotic layer separates
and is finally shed off.
The partial thickness burn is further classified as
superficial partial and deep partial thickness burns. Superficial
partial thickness burns heal rapidly in up to 3 weeks due to
greater availability of epidermal adnexal elements in them.
In deep partial thickness burns, however, these
epidermal elements are much less in number and obviously
take a longer time to heal, may be up to 6 weeks (Figs 5.4
and 5.5).
In a full thickness burn, entire thickness of the skin
involving whole of epidermis and dermis is damaged. The
epithelial elements are completely destroyed. Healing cannot
take place at all. However, in small full thickness burn
wounds, healing can occur from peripheral unburnt skin
and wound contraction. Larger burns can heal only after
skin grafting (Fig. 5.6).

44
Principles and Practice of Burn Care
Fig. 5.3: Histological appearance (20X) of burn wound treated with
BPP (Boiled Potato Peel) bandage. 11th post burn day. Re-
epidermalization is quite evident between the slough on the surface
and viable dermis underneath
Figs 5.2A and B: Superficial burns. (A) Note the characteristic
large blisters. (B) Rapid healing in 10 days post burn
A
B
Fig. 5.5: Histological appearance (40X) of burnt skin on 2nd post burn
day shows thin sloughed off epidermis. There is marked dermal edema
with neutrophilic infiltration all over
Figs 5.4A and B: Deep burns. (A) Appearance 2 days post burn.
(B) Complete healing in three weeks
A
B

Pathophysiology of Burn Wound
45
Following a full thickness burn, the dead and necrotic
skin gradually gets separated from underlying viable tissues
and is shed off. The resulting raw area gets covered with
granulation tissue, a tissue developed from the capillary
network (Fig. 5.7), over which a skin graft can be applied
for complete healing of wound. Immediately underneath
the granulation tissue a thin fibrous layer is formed which
gradually undergoes thickening.
Figure 5.7 illustrates the character of granulation tissues
under microscope with resultant micro pockets (shown with
an arrow). These need to be cleared by debridement of the
granulation before the application of the skin graft to ensure
better ‘take’ of the graft (Fig. 5.8).
Jackson (1953) 6described 3 zones in the damaged burn
tissue (Fig. 5.9).
1. Zone of coagulation is the central zone with complete
coagulative necrosis of the tissue cells which become
completely nonviable and obviously cannot recover with
any treatment.
2. Zone of stasis is on the periphery of zone of coagulation
where a significant reduction in microcirculation occurs
following damage to the tissues and its arterioles, venules
and capillaries. The circulation is sluggish. This zone
can recover after adequate resuscitation and proper
wound care. This can result in healing of the burn
wound. With inadequate resuscitation initially and due
to invasive sepsis later, this zone can get converted into
a zone of coagulative necrosis.
3. Zone of hyperemia is the outermost zone, peripheral to
the zone of stasis, due to intense vasodilatation. This
eventually recovers following adequate resuscitation, with
passage of time.
Zawaki (1974) 7demonstrated that the zone of stasis
begins to recover in about 16 to 24 hours post burn. Oxygen
consumption returns to this zone as early as 4th post burn
day and exceeds normal by 7 to 14 days when resuscitation
is adequate and drying of the wound is prevented. When a
burn wound is exposed to air, dehydration of tissues occurs
and the process of reversal of stasis is minimum or absent.
Deep partial skin loss or whole skin loss occurs when the
dermis is allowed to dry. The retention of blister skin or
coverage with allograft or xenograft prevents dehydration
of the dermis and helps the healing process favorably (Fig.
5.10).
Zawaki (1987)8 showed that when the drying of a burn
wound is prevented, epithelium migrates towards the surface
around the necrotic hair follicles (Fig. 5.2) soon after the
circulation in the viable tissue returns. He also showed that
Figs 5.6A and B: Full thickness burns. (A) Appearance 1 week post
Burn. (B) Healing after skin grafting; skin graft marked by arrows
Fig. 5.7: Histological appearance of granulation tissue as seen under
Microscope (40X) Note the pockets within the granulation tissue
(shown by an arrow)
A
B

46
Principles and Practice of Burn Care
zone of stasis begins to recover in about 16 to 24 hours and
circulation returns in few days post burn. When drying is
prevented, oxygen consumption returns to this zone as early
as 4th post burn day.
Winter (1963)9 showed that the scab which forms over
the exposed wound decreases the possibility of migration
of keratinocytes ultimately causing impairment in the process
of wound healing. On the other hand Zawaki (1974)11
demonstrated that the occlusive dressings which do not
allow the wound to dry also allow the tissue to retain the
viability.
Figs 5.8A to D: Unhealthy granulation tissue. (A) Injection of saline-adrenaline 1:100,000 to prevent blood loss. (B) Debridement of
granulation tissue. (C) Wound ready for skin grafting after debridement. (D) After complete healing
Lawrence (1975)10stressed that this perinecrotic zone
(Zone of stasis) can be saved by applying cover of suitable
dressing. If left without proper covering, the epithelial
elements in the dermis are lost due to dehydration. Zawaki
(1974)11 supported this hypothesis. Janzekovic (1970)12
demonstrated that early excision of the necrotic tissue and
a cover with skin graft improved the outcome in burn
patients.
Groves and Lawrence (1971) demonstrated the
significant improvement of the quality and quantity of dermal
tissue in healed burns treated by tangential excision and

Pathophysiology of Burn Wound
47
epithelial regeneration and promoted rapid healing. Pain relief
after BPP Bandage was significant (Figs 5.11A to 5.12B).
The burn eschar is essentially a dried, devitalized skin
covering the burn wound. Eschar does not provide the
protection from infection. However, it is a good medium
for growth of bacteria in the necrotic tissues. The property
of the skin to prevent loss of fluids like plasma and its
component is absent in eschar due to its high degree of
permeability (Pruitt and Moncrief 1967).15
The necrotic tissue anywhere in the body allows bacterial
colonization. The extensive burn itself is a large quantum of
necrotic tissue on the body. The appreciation of this fact
has led to the idea of removal of this tissue as early as early
as possible (primary excision) with the hope that the septic
complications can be avoided after removal of burned skin.
This is expected to lead to a marked improvement in patient’s
condition and improved survival rate.
Hermans (1986)16 reported the use of occlusive
dressings using hydrocolloid material improved the quality
of healing due to rapid re-epithelization, low incidence of
infection and good cosmetic results. This led to better quality
of life of the burn patient after healing. Lawrence and Lilly
(1988) showed that hydrocolloid does act as a bacterial
barrier.
Hermans (1992)17 stressed that desiccation of the burn
wound leads to the formation of the scab which decreases
the migration of keratinocytes which eventually lead to
impaired wound healing. On the other hand, the occlusive
dressing with retention of moisture would not allow the
Fig. 5.9: Diagrammatic representation of three zones as described
by Dr Douglas Jackson
Fig. 5.10: Changes in the burn wound which can occur due to various pathological conditions and as a result of
response to adequate treatment
autografting in comparison with vapor permeable material.
The studies by Lawrence (1975)10 have also demonstrated
that a polyurethane film can prevent loss of epithelium in
experimental tangentially excised burn wound.
Hinman and Maiback (1963)13 showed that impermeable
and semipermeable materials prevent the desiccation of the
wound and indirectly help the process of healing (Fig. 5.10).
Keswani, Vartak and others (1990)14 demonstrated
conclusively that the use of moisture retaining dressing made
from Boiled Potato Peel (BPP) did improve the quality of
healing and shorten the period of healing. Compared with
the treatment with the plain gauze, the application of potato
peel dressing reduced or eliminated desiccation and hastened

48
Principles and Practice of Burn Care
wound to dry out and allows the tissues in the zone of
stasis to retain the viability.
Semipermeable and impermeable dressings like synthetic
films, ‘Boiled Potato Peels’, skin allografts or hydrocolloids
prevent the desiccation of the wound and provide protection
to the zone of stasis (Fig. 5.10).
Some dressings (like cotton gauze) adhere to wound
bed and cause damage to the newly growing epidermis and
the granulation tissue. When dressing is removed during
the dressing changes there is intense pain and bleeding from
the wound bed. This clearly indicates that such dressings
may cause a significant damage to the healing tissues (Figs
5.13A to 5.14D). Occlusive dressings, especially those which
have the ability to retain moisture, do not adhere to the
wound bed. There is no pain or bleeding during the dressing
changes and thus indirectly help in faster healing of the
burn wound.
Figs 5.11A and B: Histological appearance (40X) after complete healing 21st post burn day. (A) Treated with BPP shows orderly stratification
with basal layer of columnar epithelium. (B) Area treated with gauze and silver sulfadiazine cream shows complete healing with dysplastic
stratification and basal layer of cuboidal cells
Figs 5.12A and B: Histological appearance (40X) 7th post burn day. (A) Shows the encrustation on the surface regeneration of the epidermis
with dysplastic stratification of epidermis. (B) Section shows the moist slough on the surface following BPP dressing. Note more orderly
stratification of the epidermis
When the dressing becomes dry and firmly adheres to
the burn wound, destruction of growing neoepithelium
occurs during change of dressing and delays the process
of wound healing.
This epidermal damage is observed during the change
of dressing using gauze or similar materials. There is a
bleeding from the wound and also a severe pain indicating a
mechanical damage (Winter 1963).9
Hermans (1992)17 has shown that the infection rate under
the occlusive dressings is lower than that under the
conventional dressing. Pseudomonas aeruginosa disappears
in a moist wound environment and the wounds do heal in
spite of bacterial contamination.
Lawrence and Lilly (1988)18 have shown that dressings
provide a physical barrier for microorganisms and against
bacterial contamination. This helps reduce overall bacterial
load which the host defense mechanisms have to deal with.
A
B

Pathophysiology of Burn Wound
49
Figs 5.13A and B: (A) Burn injury treated with BPP bandage. Middle part of the wound treated with gauze. (B) Note areas of bleeding from the
wound treated with gauze and resultant hemorrhage when the gauze dressing is removed, other areas are undamaged due to moisture
retention and non-adherence
A
B
Figs 5.14A to D: (A) Burn injury on the chest right side treated with BPP dressing and left side treated with gauze. (B) Note that bleeding on
left side after the dressing change (Horizontal arrow). (C) Complete healing of the epidermis can be seen on the side treated with BPP bandage.
(D) Note the broken epidermis in the area treated with gauze
A
B
C
D

50
Principles and Practice of Burn Care
WOUND HEALING OF THE DONOR AREAS
3. Moritz AR., Henriquez FC. Studies of Thermal injury II: The relative
importance of time and surface temperature in the causation of
cutaneous burns. Am J Pathol 1947; 23:695-720.
4. Ofeigsson OJ. Water cooling: First-aid treatment for scald burns-
Surgery 1965; 57(3) 391-400.
5. Cotran R.S. The delayed and prolonged vascular leakage in
inflammation II. An electron microscope study of the vascular
response after thermal injury. Am J Pathol 1965; 46:589-620.
6. Jackson DM. The diagnosis of the depth of burning. Br J Surg 1953;
40:588-96.
7. Zawaki B E. Reversal of capillary stasis and prevention of necrosis
in burns. Ann Surg 1974; 180:98-102.
8. Zawaki B E. The local effects of burn injury. The Art and Science
of Burn Care. Editor J.A Boswick Jr. Aspen, 1987:27.
9. Winter G.D. Effect of air drying and dressings on surface of a
wound. Nature 1996;197,91.
10. Lawrence JC. The perinecrotic zone in burns and its influence on
healing burns 1975;1;197-206.
11a. Zawaki BE. The natural history of reversible burn injury. Surg.
Gynecol. Obstet 1974a;139, 867.
11b. Zawaki BE. Reversal of capillary stasis and prevention of necrosis
in burns. Ann. Surg 1974b;139, 98.
12. Janzekovic Z. A new concept in early excision and grafting of
burns. J. Trauma 1970;10;1103-8.
13. Hinman CD, Maiback H. Effect of air exposure and occlusion on
experimental human skin wounds. Nature 1963;200,377.
14. MH Keswani, AM Vartak, A Patil, JWL Davies. Histological and
bacteriological studies of burn wounds treated with boiled potato
peel dressings. Burns 1990;16, (2),137-143
15. Pruitt BA, Moncrief JA. Current trends in burn research. J Surg Res
1967;7:280-93.
16. Hermans MHE, Hermans RP. Duoderm, an alternative for the
treatment of smaller burns. Burns 1986;12,214.
17. Hermans MHE. Treatment of burns with occlusive dressings: some
pathophysiological and quality of life aspects. Burns (1992) 18,
Supplement 2, S15-S18.
18. Lawrence JC, Lilly HA. Bacterial barrier properties of hydrocolloid
dressing in vitro. In: Ryan T.J. (Ed). Beyond occlusion, wound care
proceedings. R. Soc. Med. 1988;136,75.
19. Vartak AM, Keswani MH, Patil AR, Savitri Sr, Fernandis Sr B.
Cellophane-a dressing for split-thickness skin graft donor sites.
Burns 1991;17,(3),239-242.
BIBLIOGRAPHY
1. DN Herndon. Total Burn Care. W B Saunders Co Ltd 1996.
2. GJ Carrougher. Burn Care and Therapy. Mosby 1998.
3. John A Boswick; Jr ASPN. The Art and Science of Burn Care 1987.
4. JAD Settle. Principles and Practice of Burns Management. Churchill
Livingstone 1996.
5. JWL Davies Academic Press. Physiological Responses to Burning
Injury 1982.
6. ML Hanumadas, M Ramkrishnan. Handbook on Burn management.
Jaypee Brothers Medical Publishers Pvt. Ltd. India 1991.
A
B
Figs 5.15A and B: (A) Donor area of a split skin graft treated with
cellophane paper. (B) Complete healing on 10th day
Donor areas also heal in the same manner as the burn
wounds, i.e. by epithelial migration from the lining of
epidermal appendages. Donor sites of the split skin graft
are very painful, in the same way as superficial burns because
of exposed nerve endings. When these donor sites are treated
with occlusive dressings, they heal satisfactorily and do
not cause any problems (Figs 5.15A and B). Occlusive
dressing on the other hand, help rapid healing and significant
relief of pain Hermans (1992)17, Vartak (1991).19
REFERENCES
1. Rickatts C R, Squire J R, Topley E. Human skin lipids with particular
reference to the self-sterilizing power of the skin. Clin sci Mol Med
1951; 10:89-111.
2. W.G.Williams and Lindagi Philips. Pathophysiology of the burn
wound from Total Burn care, Editor D.N. Herndon - WB Saunders
Company Ltd. London 1996;64

Wound Healing in Burns
51
Wound Healing in Burns
6
Mary Babu, N Shanmuganathan, B Karthikeya Prabhu
Chapter
You were advised his flesh was capable of wounds and scars.
– William Shakespeare, Henry IV
INTRODUCTION
A burn injury is caused by damage to skin and other deeper
tissues by extreme heat, flame and contact with heated
objects or chemicals. Burn depth is generally categorized
as first, second, or third degree. The treatment of burn
wounds depends on the depth, extent and location of the
burn as well as some additional factors. Treatment options
may range from simply applying a cold pack immediately
to skin grafts.
A first degree burn is superficial and has characteristics
similar to a typical sun burn. The skin is red in color and
sensation is intact. In fact, it is usually painful and damage
is limited to the epidermis characterized by erythema and
hyperemia. Second degree burns extend into the dermis to
varying depths, involving papillary dermis alone or along
with reticular dermis. In third degree burns, the damage
progresses to the point of death of full thickness of skin.
They are painless due to damage to nerve endings, exposing
subcutaneous fat layer. Regardless of the type of burn, the
result is fluid accumulation and inflammation in and around
the wound. Moreover, it should be noted that the skin is the
body’s first defense against infection by microorganisms.
Any damage to the skin predisposes a burn victim to
infections not only at the site of the wound but also
systemically. In addition to depth, the extent of the burn,
i.e. the total body surface area (TBSA) burnt is also
significant. This is measured in terms of percentage of TBSA
burnt. Burns that cover more than 15% of the total body
surface in adults can lead to hypovolemic shock requiring
hospitalization for intravenous fluid resuscitation and wound
care.
Location of burn on the body is even more important
than the above factors as far as wound healing is concerned.
In acute stage, burns of the face or signs of burns to the
nose or mouth may require urgent guarding of the patient’s
airway, as edema can result in life-threatening obstruction
of the airway. Burned tissue shrinks and can cause damage
to underlying structures. Burns that extend circumferentially
around limbs require their surgical release, escharotomy, to
prevent gangrene of distal limb. Deep dermal burns heal
with hypertrophic scarring while full thickness burns, if
not split skin grafted lead to contractures.
FACTORS AFFECTING WOUND HEALING
There are several factors that affect the wound repair either
beneficially or adversely. Any attempt to improve healing or
prevent complications of healing should concentrate on the
causes of the impairment.
Age
Age affects many aspects of wound repair. Several
investigations have shown that rate of healing appears to
slow down with increasing age. The fibroblast proliferation
is at a slower rate in older individuals and they also have a
decreased synthetic capability. The soluble collagen
decreases and insoluble cross-linked collagen increases with
age. The amount of elastin increases but the numbers of

52
Principles and Practice of Burn Care
elastic fibers decrease to make skin less elastic. Possibly, in
the earlier stages of life, wound healing is faster as compared
to older individuals as it more closely resembles tissue
regeneration seen in simpler animals.
Infection
The effects of infection on tissue repair can be divided into
local or systemic effects. Several bacterial strains
(Pseudomonas, Staph. aureus) have been found to impair
wound healing. Bacterial contamination leads to an increased
inflammatory response with release of more cytokines to
attract more of the cells needed for wound repair. However,
the wound becomes overwhelmed with virulent
microorganisms and the bacterial proteases and the products
of inflammatory cells lead to local tissue damage.
Nutrition
One of the major breakthroughs in the survival of burn
patients has been made possible through aggressive
nutritional support. Starting nutritional support as soon as
possible after injury helps in preventing wound healing
problems.
Vitamins and Trace Elements
Some vitamins are clearly important for normal tissue repair.
Vitamin C is essential for normal collagen synthesis and
deficiency leads to defects in connective tissue and impaired
wound healing. Vitamin A induces fibroblasts to differentiate
and to enhance collagen synthesis. Topical application of
vitamin A may also improve healing. Vitamin E is a potent
antioxidant and is likely to improve healing.
Deficiencies in trace elements can also lead to problems
in wound healing. These trace elements are actually metals
that are required for enzyme functions. Iron, Zinc, Copper,
Manganese and Selenium are the most important trace metals
during the process of healing as they are involved from the
initial days of healing at different stages imparting specific
functions.
Oxygen
Oxygen is so essential to healing and it is found that high
levels of oxygen may be even more beneficial. The treatment
of wounds with hyperbaric oxygen is based on this
hypothesis. High levels of oxygen may lead to the formation
of oxygen free radicals that have potential to damage tissues.
Steroids
The most notorious drugs associated with altered healing
are steroids. Steroids are powerful inhibitors of the
inflammatory response and therefore, have the potential to
inhibit wound healing. Collagen synthesis and fibrosis are
inhibited with steroid use.
Chemotherapeutic Agents
They act by killing rapidly growing cells with the hope of
eliminating proliferating cancer cells. However, the healing
wound also requires cellular proliferation. It is reported that
most of the chemotherapeutic agents given, locally or
systematically, impair healing.
Radiation
The impairment in wound healing seen after acute radiation
exposure is also due to inhibition of proliferating cells.
Radiation may cause chronic vasculitis that impairs local
blood supply and predisposes the patient to impaired healing.
Local factors pertain mainly to persistence of debris
within the wound, for example, devitalized tissues, clots,
foreign material including sutures and bacterial
contamination. These can act as a physical barrier to the
ordered development of granulation tissues and collagen
deposition or may exaggerate the evoked inflammatory
response. Ultimately, this can change the way the tissues
heal, changing primary to secondary union or resolution to
organization. Local tissue hypoxia, either chronic (e.g.
caused by radiation enteritis, atherosclerosis or diabetic
microvascular disease) or more acute (e.g. caused by
vascular damage or tight sutures) may compromise healing.
Mobile wounds do heal but more slowly than those held
together by sutures.
Patients with uncontrolled diabetes suffer from a variety
of problems in healing. High blood sugar levels decrease
both neutrophil chemotaxis and phagocytic ability.
Additionally, a sugar-rich environment favors microbial
growth, further interfering with healing.
WOUND HEALING: GENERAL
A wound is a break in the epithelial integrity of the skin and
may be accompanied by disruption of the structure and
function of underlying normal tissues. A wound may result
from precise disruption of tissue by the surgeon’s knife

Wound Healing in Burns
53
(incision) or there may be widespread damage of tissues
(e.g. after major musculoskeletal trauma and burns). A
wound may also result from a contusion, laceration or an
abrasion. The continuity of the skin must be restored
expeditiously as it plays a crucial role in maintaining
homeostasis.
Wound healing is a highly complex physiological process.
It involves the interplay of various cellular and biochemical
components, which drive the repair cascade. In addition,
several enzymatic pathways also become active during repair
and help the tissues to heal. Following tissue injury, blood
vessels are disrupted resulting in extravasation of blood
components. This is followed by platelet aggregation and
blood coagulation resulting in the generation of a fibrin-rich
clot that fills the discontinuity in the injured tissue and
re-establishes hemostasis. Subsequently, the migration of
inflammatory cells to the wound site begins and these cells
debride the wound. This marks the beginning of the
inflammatory phase of wound healing. This is followed by
the formation of new blood vessels or angiogenesis within
the newly forming granulation tissue. Fibroblasts deposit
and remodel the granulation tissue. Granulation tissue is
then transformed to the final product of the healing process,
a scar.
Although the desirable final result of coordinated healing
would be the formation of tissues with a similar structure
and comparable functions as of an intact skin, regeneration
is uncommon (with notable exceptions such as early fetal
healing). Healing, however, does result in a structurally and
functionally satisfactory but not identical outcome. Wound
healing processes seem to be strictly regulated by multiple
growth factors and cytokines released at the wound site.
Alterations that disrupt controlled timely healing processes
extend tissue damage and prolong repair.
WOUND HEALING: TYPES
Primary healing (healing by first intention) occurs when
a wound is closed within 12-24 hours of its creation (e.g.
clean surgical incision, clean laceration). The wound edges
are approximated directly using sutures, tissue glue, tapes
or a mechanical device. Because of an appropriate balance
between all phases of the healing process (including cellular
proliferation, collagen metabolism, activity of matrix
metalloproteinase and degradation of the extracellular
matrix), the wounds heal well and proceed rapidly towards
complete closure.
Secondary healing (healing by second intention) occurs
in a wound with extensive loss of soft tissues, as seen in
major trauma, severe burns and after some surgical
procedures (e.g. laparotomy). Regeneration of epithelial cells
alone cannot restore the original architecture. There is
ingrowth of granulation tissue from the wound margin,
followed by accumulation of extracellular matrix with the
laying down of collagen (Fig. 6.1). These open, full-
thickness wounds thus close by subsequent wound
contraction and epithelialization. For example, large defects
Fig. 6.1: Primary and secondary healing. Adapted from: Pathophysiology of wound healing. Surgery (Oxford), Volume 20, Issue 5, 2002,
Pages i-v CM Halloran and JP Slavin

54
Principles and Practice of Burn Care
of skin may reduce to 5-10% of their original size within
six weeks, largely by contraction. Myofibroblasts, having
structural properties between those of a fibroblast and a
smooth muscle cell, are thought to play a key role in this
type of healing. They appear in the wound about three days
after wounding and increase in number to a maximal level
between 10 and 21days. Healing by secondary intention is
slower, may lead to contractures (particularly over joints)
and may lead to functional restriction.
Healing of partial-thickness wounds is seen in injuries
such as superficial dermal burns, donor sites of split-
thickness skin grafts and abrasions where the injury involves
the epidermis and the papillary part of the dermis. Healing
occurs purely by epithelialization from the epidermal
appendages and anatomical and physiological restoration is
virtually complete. Healing of some very deep dermal and
full thickness burns require skin grafting or else a contracture
forms.
PHASES IN WOUND HEALING
Inflammatory Phase
Inflammation is a highly effective component of the innate
initial reaction of the body to injury. It is an important
consequence of injury and one that normally leads to tissue
repair and restoration of function. The inflammatory
response can be subdivided into vascular and cellular
responses. Early in the wounding process, local
vasodilatation, blood and fluid extravasation into the
extravascular space and blocking of lymphatic drainage can
produce cardinal signs of inflammation, including redness,
swelling and heat. This acute inflammatory response usually
lasts between 24 and 48 hours and may persist for up to 2
weeks in some cases. Tissue injury causes blood vessel
disruption and bleeding. Platelets adhere, aggregate and
release many mediators to facilitate coagulation. Although
hemostasis is the major function of blood coagulation, a
secondary but equally important function of platelets is to
initiate the healing cascade via release of chemo-attractants
and growth factors. At the same time, the clot provides a
matrix scaffold for the recruitment of cells to an injured
area. In response to these important mediators, leukocytes,
including neutrophils and macrophages infiltrate the
wounded area and assist in cleaning and removing damaged
tissue debris and foreign particles. Once in the wound site,
activated macrophages release several important growth
factors and cytokines, initiating granulation tissue formation
(Fig 6.2).
Fig. 6.2: ECM: Extracellular matrix; MMP: Metalloproteinases; TIMP: Tissue inhibitors of metalloproteinases.
Adapted from: Clark RA. In: Goldsmith LA, (ed.) Physiology, biochemistry, and molecular biology of the skin, 2nd edn,
Vol. I. New York: Oxford University Press, 1991, p 577

Wound Healing in Burns
55
The Cellular Response and Inflammation
The cellular response of the inflammatory phase is
characterized by the influx of leukocytes into the area of
injury. In the early inflammatory state, neutrophils and
monocytes are the predominant cells at the site of injury.
Shortly after injury, neutrophils and monocytes begin to
emigrate from capillaries into the wounded tissue with
neutrophils being the first to arrive in great numbers. Later
in inflammation, the number of neutrophils decline and
macrophages (tissue-derived monocytes) predominate.
Neutrophils and monocytes are recruited to the wound by
chemotactic factors released during hemostasis and by mast
cells. Chemotactic factors generated during the coagulation
process, such as kallikrein, fibrinopeptides released from
fibrinogen and fibrin degradation products, also serve to
up-regulate the expression of important intercellular adhesion
molecules. Substances released by mast cells, such as tumor
necrosis factor, histamine, proteases, leukotrienes (LTs),
and cytokines (interleukins), represent additional sources
of chemotactic signals for the recruitment of leukocytes.
Growth factors like PDGF and Transforming Growth
Factor- (TGF-) are also potent chemotactic factors for
leukocytes. Once in the wound site, integrin receptors found
on the cell surface of neutrophils enhance cell-matrix
interactions. This allows neutrophils to perform their function
of killing and phagocytosing bacteria and damaged matrix
proteins within the wound bed. Neutrophil infiltration
normally lasts for only a few days but the presence of wound
contamination prolongs the presence of neutrophils within
the wound and may delay healing. Monocytes immigrate
into tissue spaces and transform into larger phagocytic
macrophages that soon become the predominant cell type
during the latter part of the inflammatory phase. Monocytes
are initially attracted to the wound site by some of the same
chemo-attractants that attract neutrophils and their
recruitment continues through signals released by monocyte-
specific chemo-attractants such as monocyte chemo-
attractant protein-1 and macrophage inflammatory protein-
1. The extracellular matrix degradation products, collagen
fragments, fibronectin fragments and thrombin are also
specific chemo-attractants for monocytes. Macrophages
are considered to be the most important regulatory cells in
the inflammatory reaction. Macrophages phagocytose, digest
and kill pathogenic organisms, scavenge tissue debris and
destroy any remaining neutrophils. After binding to the
extracellular membrane, bacterial, cellular and tissue
phagocytoses and subsequent destruction are accomplished
through the release of biologically active oxygen
intermediates and enzymatic proteins.
Chemical Mediators of Inflammation
A number of chemical substances are involved in the initiation
and control of inflammation. These chemicals work in
concert: some are protagonists and others are antagonists
of inflammation. They can be grouped as vasoactive amines
of histamine and serotonin, plasma proteases of kinins and
complements, plasma proteins of the coagulation system,
arachidonic acid metabolites of prostaglandins (PGs) and
LTs, cytokines and growth factors and free radicals of nitric
oxide and oxygen-derived free radicals (Fig. 6.3).
Proliferative Phase
The initial inflammatory responses to injury provide the
necessary framework to the subsequent production of a
new functional barrier. In this phase of healing, cellular
activity predominates. The major events during this phase
are the creation of a permeability barrier (i.e., re-
epithelialization), the establishment of appropriate blood
supply (i.e. angiogenesis), and reinforcement of the injured
dermal tissue (i.e., fibroplasia).
Reepithelialization (Fig. 6.4)
Reepithelialization is the process of restoring an intact
epidermis after cutaneous injury. It generally involves several
processes, including the migration of adjacent epidermal
keratinocytes into the wound, the proliferation of
keratinocytes used for the supplement of the advancing and
migrating epithelial tongue, the differentiation of the
neoepithelium into a stratified epidermis, and the restoration
of an intact basement membrane zone (BMZ) that connects
the epidermis and the underlying dermis.
Keratinocyte Migration
Keratinocyte migration is an early event in wound
reepithelialization. The keratinocytes initially respond to an
epidermal defect by migrating from the free edges of the
wound within 24 hours. The keratinocyte migration in
partial-thickness wounds also occurs from remaining skin
appendages and wound edges. Epidermal stem cells from
the hair follicle are now thought to originate from the hair
bulb that is believed to be the germinative portion of the hair
and serve as a reservoir for keratinocytes in wound healing.

56
Principles and Practice of Burn Care
projections of lamellipodia, loss of cell-cell and cell-matrix
contacts, retraction of intracellular tonofilaments and
formation of actin filaments at the edge of their cytoplasm,
occurs. Although keratinocytes are migrating, their
proliferative potential is inhibited. Migrating basal
keratinocytes may express selective cell surface markers
such as CD44 and some markers usually expressed by
squamous cells.
Several elements have been implicated in keratinocyte
migration, including the extracellular matrix, integrin
receptors, matrix metalloproteinases (MMPs) and growth
factors. An early provisional matrix formed by fibrin,
fibronectin, and type V collagen enables keratinocytes to
migrate and dissect under eschar and debris that may be
covering the wound. Keratinocytes use their surface integrin
receptors to interact with a fibronectin-rich provisional
matrix. The direction of migration is also regulated by the
binding of keratinocytes to integrin receptors on the newly
formed collagen molecules in the wound bed. Dissociation
of this binding allows the keratinocytes to migrate forward.
Matrix metalloproteinases also play an important role in
keratinocyte migration by their involvement in this
Fig. 6.3: Cytokines and healing. Adapted from: Pathophysiology of wound healing. Surgery (Oxford), Volume 20, Issue 5, 2002, Pages i-v
CM Halloran and JP Slavin
Fig. 6.4: Superficial dermal burns on lower leg showing reepitheliali-
zation. The epithelial buds can be seen over the surface
A number of processes must occur within keratinocytes in
preparation for migration. Approximately 12 hours after
wounding, a series of events, such as flattening and
elongation of keratinocytes, development of pseudopod-like

Wound Healing in Burns
57
dissociation. Migrating keratinocytes produce MMPs, such
as MMP-9, which specifically degrades type IV collagen
and laminins in the basement membrane. This allows cells
to leave the basement membrane and migrate into the
wound. Matrix metalloproteinase-1 disrupts any attachment
to a fibrillar collagen and facilitates the continued migration
of keratinocytes in the wound.
Keratinocyte Proliferation
Reepithelialization also involves increased proliferation of
keratinocytes located near the cells of the migrating front
tongue. This proliferating source of keratinocytes ensures
an adequate supply of cells to migrate and cover the wound.
When migration ceases, possibly as a result of contact
inhibition, keratinocytes reattach themselves to the
underlying substratum, reconstitute the basement membrane
and then resume the process of terminal differentiation to
generate a stratified epidermis. One can observe that there
are single-layered keratinocytes toward the wound center
whereas there are multiple-layered or stratified keratinocytes
near the wound edges. Differentiation then follows
proliferation as the proliferative index is found significantly
increased at the wound center, whereas the differentiation of
the neoepidermis (keratins 1/10, filaggrin, and loricrin) and
regeneration of the dermoepidermal junction (laminin 5 and
collagen IV) are more advanced toward the wound margin.
Epidermal growth factor (EGF), keratinocyte growth factor
and Transforming Growth Factor- (TGF-) have been
shown to be among the important stimuli for keratinocyte
migration, proliferation, and reepithelialization.
Restoration of the Basement Membrane
Zone (BMZ)
The formation of an intact BMZ between the epidermis and
the dermis is essential for the reestablishment of skin integrity
and function. Within 7 to 9 days after reepithelialization, the
BMZ reforms. The BMZ forms an adhesive structure
because its superior aspects serve as an attachment site for
basal keratinocytes through the formation of a
hemidesmosome-anchoring filament complex whereas the
inferior or lower portion stabilizes the attachment to the
underlying dermis by anchoring fibrils. The importance of
BMZ is evidenced by a group of inherited blistering diseases
known as epidermolysis bullosa. In these conditions,
mutations of one of a variety of BMZ proteins, such as
hemidesmosome collagen XVII in atrophic epidermolysis
bullosa, defects of laminin 5 of a major anchoring filament
in junctional epidermolysis bullosa and deficiency of collagen
VII anchoring fibrils in dystrophic epidermolysis bullosa
lead to blistering and ulcer formation.
The skin BMZ consists of many extracellular matrix
proteins; among these, collagens and laminins are the major
components. Collagen IV is the most abundant component
and forms a 3-dimensional lattice network within the
electron-dense or lamina densa portion of skin BMZ.
Collagen VII proteins, also called anchoring fibrils, span
from the lamina densa to the upper papillary dermis, where
they form a structure known as an anchoring plaque. Another
important collagen is collagen XVII, also known as bullous
pemphigoid antigen (bullous pemphigoid antigen 2 or bullous
pemphigoid antigen 180), which is a 180-kDa transmembrane
protein located on the hemidesmosome complex of basal
keratinocytes. Laminins are the major noncollagenous
extracellular matrix components in a wide range of BMZs
within human tissues. Several laminins are present in the
BMZ of the dermoepidermal junction. Composed of a, b
and c subunits, laminin 5 (a3b3c2) and laminin 10 (a5b1c1)
are the major laminins in skin BMZ and found to be actively
involved in wound repair. In response to wounding,
keratinocytes in the migrating front edge deposit laminin 5;
this serves as a track to allow subsequent keratinocytes to
migrate. Recently, a new laminin member, laminin 10, has
been described; it is located within the lamina densa. Laminin
10 was also found to be a major laminin of dermal
microvascular blood vessels. Laminin 10 knockout mouse
skin was found to exhibit discontinuity in BMZ lamina densa
and defects in hair development.
Reconstitution of the Dermis
Dermal reconstitution begins approximately 3 to 4 days after
injury, characterized clinically by granulation tissue
formation, which includes new blood vessel formation or
angiogenesis and the accumulation of fibroblasts and ground
matrices, named fibroplasia. The provisional extracellular
matrix that is formed in part by the fibrin clot, which is rich
in fibronectin, promotes granulation tissue formation by
providing scaffolding and contact guidance for cells to
migrate into wound spaces and for angiogenesis and
fibroplasia to occur in an effort to replace the wounded
dermal tissue.
Fibroplasia
Fibroplasia describes a process of fibroblast proliferation,
migration into wound fibrin clot and production of new

58
Principles and Practice of Burn Care
collagen and other matrix proteins, which contribute to the
formation of granulation tissue. As an early response to
injury, fibroblasts in the wound edges begin to proliferate
and by approximately day 4 start to migrate into the
provisional matrix of the wound clot, where they lay down
a collagen-rich matrix, including collagens, proteoglycans,
and elastin. Once the fibroblasts have migrated into the
wound, they gradually change to profibrotic phenotypes
and switch their major function to protein synthesis.
Fibroblasts are also modulated into phenotypes of
myofibroblasts and participate in wound contraction.
Structural molecules of the early extracellular matrix,
such as fibronectin and collagen, contribute to granulation
tissue formation by providing a scaffold for contact guidance
and a reservoir for cytokines and growth factors.
Fibronectin, a glycoprotein, is a major component of the
gel like substance initially secreted and provides for enhanced
fibroblast activity. Fibronectin allows fibroblasts to bind to
the extracellular matrix and provides an adherent base for
cell migration. The fibronectin matrix also provides a scaffold
for collagen fibrils and mediates wound contraction. Major
fibroblastic chemotactic factors are, in part, derived from
macrophages present in the wound. Both PDGF and TGF-
 can stimulate fibroblast migration and up-regulate the
expression of integrin receptors. Epidermal growth factor
and fibroblast growth factor, among others, modulate
fibroblast proliferation and migration. Fibroblast proliferation
is also stimulated by an acidic low-oxygen condition found
in the center of the wound. As angiogenesis proceeds with
the formation of new vessels and increased oxygen carrying
capacity, this stimulus diminishes.
Angiogenesis
Angiogenesis refers to new vessel growth by the sprouting
of preexisting vessels adjacent to the wound. As in most
normal adult tissues, dermal blood vasculatures remain
quiescent. In response to the injury, microvascular
endothelial cells initiate an angiogenic process consisting of
activation of endothelial cells. Newly formed vessels
participate in granulation tissue formation and provide
nutrition and oxygen to growing tissues. In addition,
inflammatory cells require the interaction with blood vessels
to enter the site of injury. During angiogenesis, endothelial
cells also produce and secrete biologically active substances
or cytokines.
Angiogenesis involves a phenotypic alteration of
endothelial cells, directed migration and various mitogenic
stimuli. Cytokines released by macrophages stimulate
angiogenesis during wound healing, as do low-oxygen
tension, lactic acid and biogenic amines produced in the
wound. Several growth factors have been shown to play
critical roles in wound angiogenesis, including vascular
endothelial growth factor, angiopoietins, fibroblast growth
factor and TGF-. Vascular endothelial growth factor, also
known as vascular permeability factor, which exerts its
biologic activity predominantly on endothelial cells, is a key
mediator of angiogenesis.
Formation of the Extracellular Matrix (ECM)
Besides providing turgor to soft tissues and rigidity to bone,
extracellular matrix supplies a substratum for cell adhesion
and critically regulates the growth, movement and
differentiation of the cells within it. The extracellular matrix
consists of fibrous structural proteins (collagens, elastin)
and an interstitial matrix composed of adhesive glycoproteins
embedded in a proteoglycan and glycosaminoglycan gel.
The seemingly random array of interstitial matrix in
connective tissues soon becomes highly organized around
epithelial cells, endothelial cells and smooth muscle cells,
forming the specialized basement membrane (which directs
cell polarity and is required for the orderly renewal of the
epithelial tissue).
Collagens are synthesized by fibroblasts and are the most
abundant family of proteins in the body. They provide
strength and integrity to all tissues and so play a vital role in
wound repair. Platelet-derived growth factor, basic fibroblast
growth factor, transforming growth factor-, interleukin-
1, tumor necrosis factor (TNF) induce collagen synthesis
during the proliferative and remodeling phases. Collagens
are composed of three protein -chains braided into a rope-
like triple helix; the individual chains are able to intertwine
tightly because each  polypeptide has one glycine molecule
at every third position. More than 30 distinct -chains form
about 20 different collagen types (some of which may be
unique to specific cells and tissues). Some collagen types
(e.g. I, III, V) form fibrils due to lateral crosslinking of the
triple helices and form most of the connective tissue in
healing wounds; other collagens (e.g. type IV) are non-
fibrillar and become components of the basement membrane.
Adhesive glycoproteins are structurally diverse proteins
that link the components of the extracellular matrix to one
another and to cells and include fibronectin, laminin and
thrombospondin.

Wound Healing in Burns
59
•
Fibronectin, a large (400-kD) disulphide-linked
heterodimer, is associated with cell surfaces, basement
membranes and the pericellular matrix. It has specific
domains that bind to a wide spectrum of components
of the extracellular matrix (e.g. collagen, fibrin and
proteoglycans) and can also attach to cell integrins.
Adhesive matrix proteins such as fibronectin can directly
mediate the attachment, spreading and migration of cells.
Fibronectin also enhances the sensitivity of certain cells
(e.g. endothelial) to the proliferative effect of growth
factors by activating intracellular signaling pathways.
•
Integrins are a family of / heterodimeric glycoproteins
that mediate cell-cell and cell-matrix adhesion. They are
important in the integration of function between the
extracellular matrix and the cytoskeleton.
Proteoglycans consist of glycosaminoglycans (e.g.
dermatan sulfate, heparan sulfate) linked to a protein
backbone; they help to regulate the structure and
permeability of the extracellular matrix. Proteoglycans can
modulate the growth and differentiation of cells (e.g.
syndecan binds extracellular matrix collagen, fibronectin,
thrombospondin and basic fibroblast growth factor and
associates with the intracellular actin cytoskeleton to maintain
the normal morphology of epithelial sheets).
Glycosaminoglycans without a protein core (e.g. hyaluronan)
are important constituents of the extracellular matrix.
Formation of granulation tissue in 3-5 days (indicative
of optimal healing) is well established. Granulation tissue
has a pink, soft, granular gross appearance (such as that
seen beneath the scab of a skin wound). Histologically, it is
denoted by proliferating fibroblasts and loops of capillaries
in a loose extracellular matrix. This phase is characterized
by angiogenesis or formation of new blood vessels from
pre-existing vessels at the site of injury (neovascularization).
Remodeling Phase
Remodeling consists of the deposition of the matrix and its
subsequent changes over time. It occurs throughout the
entire wound repair process as fibrin clot formed in the
early inflammatory phase is replaced by the granulation tissue
that is rich in type III collagen and blood vessels during the
proliferative phase and subsequently replaced by a
collagenous scar predominantly of type I collagen with much
less mature blood vessels. One of the characteristics of
wound remodeling is the change of extracellular matrix
composition. Collagen fibers constitute approximately 80%
of the dry weight of normal human dermis and are the
principal proteins providing structure, strength and stiffness
to dermal tissue. In healthy adults, type I collagen accounts
for approximately 80% of collagens and type III collagen
constitutes 10% of collagens in the dermis. During early
wound healing, however, similar to the fetal dermis, type
III collagen is the predominant collagen synthesized by
fibroblasts in granulation tissue. Type III collagen first
appears after 48 to 72 hours and is maximally secreted
between 5 and 7 days.
The total amount of collagen increases early in repair,
reaching a maximum between 2 and 3 weeks after injury.
Over a period of 1 year or longer, the dermis gradually
returns to the stable preinjury phenotype, consisting largely
of type I collagen. Tensile strength, a functional assessment
of collagen, increases to 40% of strength before the injury
at 1 month and may continue to increase for 1 year, reaching
up to 70% of its preinjury strength. With wound closure, a
gradual turnover of collagen occurs as type III collagen
undergoes degradation and type I collagen synthesis
increases. The process of this conversion of the dermis is
accomplished through a tightly controlled synthesis of new
collagen and lysis of old collagen, mainly carried out by the
actions of MMPs. The stimulus for this conversion may be
the biomechanical stress and strain placed across a closed
wound. Matrix metalloproteinases are usually not detectable
or at very low levels in healthy resting tissue and are instead
induced during wound repair, in response to cytokines,
growth factors, and/or cell contact with the extracellular
matrix. The catalytic activity of MMPs is also controlled in
part by a family of tissue inhibitors of metalloproteinases.
Tissue inhibitors of metalloproteinases specifically bind
MMPs and are their natural inhibitors. The balance between
the activities of MMPs and tissue inhibitors of
metalloproteinases is also critical to wound repair and
remodeling.
The final phase of wound healing is resolution. It involves
remodeling of ECM and contraction of the wound.
Contraction of the wound occurs after formation of the
granulation tissue and requires the fibroblasts to assume a
myofibroblast phenotype. Remodeling of the ECM, which
leads to the conversion of granulation tissue to scar tissue,
follows this and is dependent on the continual synthesis
and degradation of collagen fibrils. Metalloproteinases
participate in regulating mechanisms in all of these repair
processes. For example, inflammation is shaped by cytokines
and chemokines, which arise largely from resident cells
(epithelium, endothelium, fibroblasts, etc.). MMPs can

60
Principles and Practice of Burn Care
activate these mediators, by cleaving them from the cell
surface or processing them to increase their activity or
degrade them, thereby inhibiting inflammatory signals. Also
the MMPs are able to cleave components of cell–cell
junctions and cell–matrix contacts within the epithelium to
promote reepithelialization. Furthermore, MMPs are involved
in remodeling the scar ECM either directly by proteolytic
degradation of proteins, such as collagens or indirectly via
their ability to affect cell behavior. Alteration of the ECM is
integral to the resolution of wound healing but also has
implications in regulation of inflammation. Thus, MMPs
are key regulators of multiple aspects of tissue repair and
further study of these enzymes and their interaction with
their substrates will not only advance our basic knowledge
of wound healing, but also provide insight into possible
therapies.
FETAL VS ADULT
Wound healing is a complex process involving cellular
migration, proliferation and differentiation with extracellular
matrix (ECM) synthesis and matrix remodeling. Many of
these mechanisms are common to development and wound
healing. Recent studies have demonstrated significant
differences between adult and fetal wound healing. In a
fetus, healing occurs rapidly by a regenerative process
without scar formation while in an adult it leads to scar
formation, often limiting tissue function and restricting
further growth. The fetal response to a wound may be
related to its developmental stage, the sterile aqueous fetal
environment or a combination of both factors. For the
interstitial collagens (types I, III, V, and VI), the critical
difference between fetus and adult is in the supramolecular
organization of the collagen fibrils deposited in the wound.
In the fetus, a reticular collagen pattern is restored and this
is indistinguishable from the surrounding normal tissue. In
the adult, large, parallel bundles of collagen, typical of scar
tissue, are seen running across the wound site approximately
at right angles to the basement membrane, thus disrupting
normal tissue architecture. One fundamental difference
between fetal and adult wound healing is, therefore, the
control and patterning of collagen fibrillogenesis. Collagen
fibril formation is a complex process which is only partially
understood. Collagen types I, III and V are present within
the same fibrils and interactions between different collagens
will alter fibril size in vitro. Interactions with other
macromolecules are also likely to influence collagen
fibrillogenesis. Proteoglycans will alter fibril size as will
fibronectin. Finally the role of the cell has to be considered.
Fibroblasts separate stages of fibril formation in distinct
cellular compartments allowing the cell to control the final
fiber orientation. Several of the differences in the ECM of
fetal and adult wounds, shown in the studies, potentially
may be altering collagen fibril formation. Collagen types I,
III, V and VI are present concurrently in both fetal and
adult wounds but the relative proportions of these collagen
types are unknown. Fetal tissues contain a higher proportion
of type III collagen to type I collagen and this may influence
collagen fibril size. The role of glycosaminoglycans is
intriguing as studies have shown high levels of hyaluronic
acid in fetal wounds whilst addition of hyaluronic acid to an
adult tympanic membrane wound decreased scar formation
and altered the degree of organization of collagen within the
wound. The interactions of sulfated GAGs with collagen
may alter fibril formation within the fetal wound. During
development, fetal bovine and chick collagen fibrils were
small in diameter at a time when hyaluronic acid was
abundant and a rapid increase in collagen fibril size during
mid gestation coincided with a decrease in hyaluronic acid
and increase in chondroitin sulfate content. The different
patterning of collagen fibrils in healing fetal (regeneration
of normal dermal pattern) and adult (scar consisting of
parallel, densely packed collagen fibrils at right angles to
the epithelial surface) wounds may therefore relate to
differences in the cellular and extracellular milieu influencing
the process of fibrillar differentiation in the primary wound
‘scaffolding’ into which fibroblasts migrate and lay down
collagen fibrils. The fetal wound shows rapid epithelial and
mesenchymal migration, the latter into a loose honeycomb
lattice, rich in (and presumably held open by)
glycosaminoglyeans, proteoglycans and glycoproteins.
Thus, fetal fibroblasts can rapidly and easily migrate into a
loose wound lattice and rapidly deposit collagen fibers. The
transmission of mechanical forces to these fibrils by
fibroblast tractioning, within a compliant matrix, may
orientate the fibrils in a reticular pattern similar or identical
to that of the normal dermis. By contrast, adult wounds
show slow fibroblast migration into a denser, more resistant,
wound matrix where fibroblasts seem to migrate more easily
up and down the wound margins rather than into the body
of the wound scaffold itself. Within the adult wound the
‘stiffer’ matrix and the forces of wound contracture
generated by myofibroblasts may alter the orientation of
collagen fibrils leading to the early establishment of an
abnormal, scar-like, orientation. Moreover, the extracellular

Wound Healing in Burns
61
matrix of the fetal (and neonatal) dermis is less highly
ordered, has a more rapid turnover and consists of less
differentiated cells compared to the adult; these may enhance
the scar-less healing pattern of the fetus. Interestingly, studies
of wound healing in adult hair follicles have shown scar
free healing of the damaged dermal papilla and the fibroblast-
like cells of the papilla retain characteristics associated with
embryonic cell lines, such as cell aggregation in tissue
culture. It has been observed that wound healing is always
faster and more fetal like on the oral mucosal side of the
incision than on the skin side even in an adult. The adult
oral mucosa possesses many embryonic characteristics and
its structure is relatively simple by comparison with the
complex, heavily keratinized, structure of adult epidermis.
The epithelium of the adult oral mucosa is more fetal-like in
its state of differentiation as reflected in the expression of
cytokeratins. Human adult oral mucosal fibroblasts exhibit
numerous embryonic characteristics and additionally, the
oral mucosa is continuously bathed in fluid (saliva) rich in
growth factors and antimicrobial agents, just like fetal skin
and amniotic fluid.
However, the state of epithelial differentiation and
potential epithelial-mesenchymal interactions during wound
healing requires further investigation and a study of
cytokeratins in fetal and adult wounds has recently been
completed. Such a slow transition is indicative of the
multifactorial differences between fetal and adult wound
healing: rapidity of wound closure, degree of inflammatory
response, maturity and competence of the immune system,
degree of organization and differentiation of the wounded
tissues, composition and organization of the extracellular
matrix, presence or absence of an aqueous sterile
environment (amniotic fluid) rich in growth (and other)
factors, differences in tissue oxygenation, wound metabolism
and pH, degree of tissue tension from function, presence
or absence of acute inflammation, etc. Moreover, whether
or not fetal wounds scar also depends upon the type and
size of the wound (large, deep excisional wounds scar), the
tissue wounded (internal tissues e.g. diaphragm, scar) and
the species used—rabbit fetuses do not heal their wounds
properly probably due to the large amounts of maternal
immunoglobulin in rabbit amniotic fluid as a result of the
size of the zona chorion during placentation. The differences
between fetal and adult wound healing are multiple and
subtle. There are studies to suggest a number of ways in
which the healing adult wound might be experimentally
manipulated to reduce scarring: enhance cell migration by
addition of exogenous tenascin, develop a more open early
wound scaffold to facilitate fibroblast migration and
regeneration of the normal reticular collagen fibril orientation
by addition of exogenous glycosaminoglycans and
proteoglycans, clonally expand or transplant (e.g. from the
oral mucosa) fibroblasts with embryonic characteristics at
the wound site. It may be possible to manipulate adult wound
healing to show a more fetal-like, scar-less, pattern by
altering only one or two of the (major) components that are
different between fetal and adult healing.
PIGMENT CHANGES IN SKIN FOLLOWING
BURNS
The nature and extent of pigmentary changes in burned
areas are not always predictable. The usual observation is
that hyperpigmentation (Fig. 6.5) occurs after superficial
burns whereas hypopigmentation/depigmentation are seen
following deeper burns (Figs 6.6A to 6.7). Similarly, donor
sites, where thin skin grafts are harvested, tend to
hyperpigment later on. Whereas, if thick grafts are
harvested, the donor area becomes hypopigmented. These
changes are more noticeable in individuals with dark skin.
There is enough literature on this subject as it has been
extensively researched because the post burn pigmentary
changes are a cause of considerable concern to the patients.
The pathophysiology for these pigmentary changes can
be explained if the anatomy of normal skin is understood.
The color of normal skin is determined by the number of
melanocytes, the amount of melanin synthesized and its
distribution and transfer to a keratinocyte. The melanocytes
Fig. 6.5: Hyperpigmentation starting to appear in the scar

62
Principles and Practice of Burn Care
melanocytes in the deeper layers of epidermis and dermis
are still intact. They become overactive in response to the
trauma of burns and stimulus of healing and probably
synthesize more melanin. This leads to hyperpigmentation.
This is also affected by the innate skin color, sun
responsiveness of the individual’s skin and amount of
ultraviolet light exposure.
In case of deeper burns there are only few melanocytes
left in the deeper layers of dermis. Therefore, there is a
deficiency of melanin causing hypopigmentation or
depigmentation in these areas after they heal. Only the
residual melanocytes lining the intact hair follicles in the
depth of the dermis migrate upwards to the basal layer of
surface regenerating epidermis and give color to the healed
skin which is deficient compared to surrounding healthy
skin. Following skin grafting of raw areas, the re-innervation
of the graft alters the melanocyte control and also increases
the number of melanosomes in the epidermal-melanin unit
leading to hyperpigmentation of the grafted areas.
HYPERTROPHIC SCARS AND KELOIDS
The word scar was derived from the Greek word eschara,
meaning place of fire (fireplace). Scars are areas of fibrous
tissue that replace normal skin or other tissues after injury.
A scar results from the biological process of wound repair
in the skin and other tissues of the body. Thus, scarring is
a natural part of the healing process. With the exception of
very superficial lesions, every wound (e.g. after accident,
disease or surgery) results in some degree of scarring. Scar
contain organelles, melanosomes, in which melanin is
synthesized and then transferred to keratinocyte via cell to
cell contact between melanocyte and keratinocyte. This is
called the epidermal-melanin unit.
In case of superficial burns, the superficial layer of
keratinocytes and melanocytes are destroyed but the
Figs 6.6A to C: (A) Hypopigmented area immediately after wound
healed with reduced number of hair. (B and C) Hypertrophic scar
showing hypopigmentation in the center where the wound was deeper
and hyperpigmentation in the periphery where it was comparatively
superficial
B
C
Fig. 6.7: Post burn contracture of elbow with hypopigmentaion in
the scar
A

Wound Healing in Burns
63
tissue is not identical to the tissue which it replaces and is
usually of inferior functional quality. For example, the sweat
glands and hair follicles do not grow back in the scarred
skin (Fig. 6.8).
Examples of disfiguring scars include hypertrophied
scars and keloids. In both keloid as well as hypertrophic
scars, the wounds heal overzealously above the skin surface.
The difference between a keloid and a hypertrophied scar
is that whereas the former continues to enlarge beyond the
original size and shape of the wound, the latter enlarges
within the confines of the original wound (Figs 6.9 and
6.10). Although both can be red and raised, keloids continue
to grow while hypertrophic scars tend to regress over time.
Both can recur after surgical excision; however, keloids
recur more commonly. Hypertrophic scars are far more
common than keloids and may occur in persons of any age
or at any site and tend to regress spontaneously. In general,
they are more responsive to treatment. Keloids occur more
frequently in black persons, being 5-15 times more common
in black as compared to whites. They may occur in persons
of any race with a proven tendency to keloid formation.
This predilection for keloid formation in persons of certain
races is not observed with hypertrophic scars. Keloids are
more prevalent in persons aged 10-30 years while
hypertrophic scars occur in individuals of any age. In
general, the risk for either type of abnormal scar diminishes
with age. Furthermore, the propensity for keloid formation
can be familial, genetically transmitted as an autosomal
dominant or recessive trait. For example, ear piercing custom
prevalent in many cultures of the world, including India,
can give rise to keloids (Fig. 6.11).
Fig. 6.8: Post burn scarring of chest with contracture of axilla. The
scar is showing areas of both hypo- and hyperpigmentation
Fig. 6.9: Post burn keloid in arm with severe dorsal
contracture of hand
Fig. 6.10: Severe hypertrophic scarring over right side of face
following scalds
Fig. 6.11: Keloid over helical rim following burns

64
Principles and Practice of Burn Care
Etiology
Abnormal scar formation is unique to humans and as such,
animal model research has not produced much significant
information to further the understanding and treatment of
keloids and hypertrophic scars. Despite numerous studies,
no uniformly accepted theory or explanation indicates as to
what initiates keloid or hypertrophic scar formation.
Several genetic and environmental causes have been
implicated in the etiology of keloid and hypertrophic scars.
In both, an excessive accumulation of collagen occurs either
from increased collagen synthesis or decreased collagen
degradation. Proposed causes for abnormal scar formation
include foreign body reaction and bacterial infections. Almost
all abnormal scars are associated with tattoos, burns,
injections, bites, vaccinations, trauma, surgery or infection.
Skin tension is frequently implicated in hypertrophic scar
formation. Abnormal scar healing commonly involves areas
of high skin tension, such as the anterior chest, shoulders
and upper back. Other factors implicated in the etiology of
these scars include wound infection or anoxia, a prolonged
inflammatory response and wound orientation different from
the relaxed skin tension lines.
Immunologic alterations have been demonstrated in
abnormal scars. Specifically, irregular immunoglobulin and
complement levels, increased transforming growth factor-
 and mast cells have been found in them. Additionally,
decreased tumor necrosis factor and interleukin-1 levels
have been found in these scars.
Pathophysiology
Although multiple factors are involved in their formation,
studies indicate that keloid and hypertrophied scars result
from increased collagen production and decreased collagen
degradation. It was earlier demonstrated that levels of the
collagen-related enzyme prolyl hydroxylase are elevated in
keloid-affected skin compared to normal skin. Prolyl
hydroxylase is required for the hydroxylation of proline
during collagen synthesis suggesting that collagen
overproduction occurs with keloids.
Collagen production is elevated in keloid biopsy samples
and in cultured fibroblasts derived from keloids. Additional
studies show that increased collagen production by cultured
fibroblasts derived from keloids persists throughout their
in vitro life span. No significant differences in DNA content
or cellularity are found when keloid dermis is compared
with normal dermis. This suggests that each fibroblast is
producing more collagen rather than an increase occurring
in the number of fibroblasts producing a normal amount of
collagen. In keloid formation, excessive collagen production
by fibroblasts may be due to the wound environment.
Abnormal wound healing in susceptible individuals can
result in the formation of keloids that have an elevated content
of extracellular matrix material compared to normal scars.
Keloid-derived fibroblasts exhibit as much as a four-fold
increase in the rate of fibronectin biosynthesis compared to
fibroblasts from normal dermis and normal scars. Altered
biosynthesis is due to an increase in the steady-state level
of fibronectin mRNA. The level of fibronectin gene
expression that is responsible for this increase has been
identified. Thus, altered degradation of fibronectin mRNA
is unlikely to contribute to overproduction of fibronectin in
keloids.
The overproduction of fibronectin and type I collagen
in keloids and hypertrophic scars implicates altered
regulation of extracellular matrix components as an important
aspect of these wound healing pathology.
Contrary to the increased collagen production theory,
keloids and hypertrophic scars may also result from a
decrease in collagen degradation. Levels of the collagenase
inhibitor alpha-2 macroglobulin have been shown to be
decreased in keloids.
The hypertrophic scars also have within them a large
number of fibroblasts with contractile filaments in the
cytoplasmic matrix and a deeply indented nucleus, indicating
contraction of the cells. These fibroblasts with contractile
properties are called myofibroblasts because they are
structurally similar to smooth muscle cells. There is a marked
increase in the number of these myofibroblasts in the
granulation tissue and immature scars especially in the deep
dermal portion of the hypertrophic scars. As these
myofibroblasts contract, the stress on the adjoining collagen
fibrils is released causing them to form compact collagen
nodules which are seen in the depth of the dermis. The
contracting myofibroblasts in the deeper dermis also cause
the ‘piling up ‘of the scar as it is forced outwards. The
increased number of myofibroblasts with their contractile
properties also exerts sufficient force to cause severe
distortion of facial structures and joint contractures as the
new collagen fibers in the wound may fuse together in the
position of comfort.
Clinical
On clinical examination, keloids and hypertrophic scars
are raised above the skin level. Hypertrophic scars are

Wound Healing in Burns
65
self-limited; they hypertrophy within the confines of the
wound (Fig. 6.12). Initially, hypertrophied scars can be
raised, red, pruritic, and even painful; however, over time,
they become pale and flat. Hypertrophied scars appear worst
at 2 weeks to 2 months.
Medical Management of Hypertrophic
Scars and Keloids
No single therapeutic modality is best for all hypertrophic
scars and keloids. The location, size and depth of the lesion,
the age of the patient and the past response to treatment
determine the type of therapy used. Prevention is the key,
but therapeutic treatment of hypertrophic scars and keloids
includes occlusive dressings, compression therapy,
intralesional corticosteroid injections, cryosurgery, excision,
radiation therapy, laser therapy, interferon therapy, 5-
fluorouracil (5-FU), doxorubicin, bleomycin, verapamil,
retinoic acid, imiquimod 5% cream, tamoxifen, tacrolimus,
botulinum toxin and other promising therapies such as
transforming growth factor (TGF)–3, and recombinant
human (rh) interleukin (IL)–10 (rhIL-10), which are directed
at decreasing collagen synthesis.
Prevention
Avoid performing nonessential cosmetic surgery in patients
known to form keloids; however, the risk is lower among
patients who have only earlobe lesions. Close all surgical
wounds with minimal tension. Incisions should not cross
joint spaces. Avoid making mid-chest incisions and ensure
that incisions follow skin creases whenever possible.
Standard Treatments
These include occlusive dressings, compression therapy
and intralesional corticosteroid injections.
Occlusive dressings include silicone gel sheets and
dressings, non-silicone occlusive sheets and Cordran tape.
These measures have been used with varied success. Anti-
hypertrophic effect of silicone gel sheets appear to result
from a combination of occlusion and pressure (especially
when associated with pressure garments) and hydration.
Studies to determine whether silicone compound itself
affects the maturation of the scar have been inconclusive.
Compression therapy involves pressure, which has long
been known to have thinning effects on skin. Reduction in
the cohesiveness of collagen fibers in pressure-treated
hypertrophic scars has been demonstrated by electron
microscopy. Studies have shown that pressures of 15-40
mm Hg can be applied by custom made pressure garments
and this reduces the reddish discoloration in the scar and
also flattens and softens them after 6-12 months of continued
application.
Corticosteroids, specifically intralesional corticosteroid
injections, have been the mainstay of treatment.
Corticosteroids reduce excessive scarring by reducing
collagen synthesis, altering glucosaminoglycan synthesis and
reducing production of inflammatory mediators and
fibroblast proliferation during wound healing. The most
commonly used corticosteroid is triamcinolone acetonide
in concentrations of 10-40 mg/ml administered
intralesionally with a 25- to 27-gauge needle at 4 to 6 weeks
intervals.
Recent Innovations
Newer treatments for keloids and hypertrophic scars include
intralesional interferon, 5-fluorouracil, doxorubicin,
bleomycin, verapamil, retinoic acid, imiquimod 5% cream,
tacrolimus, tamoxifen, botulinum toxin, TGF-3, and
rhIL-10.
Radiation Therapy
Using radiotherapy to treat keloids remains controversial.
Although many studies have demonstrated efficacy and
decreased recurrence rates, the safety of radiotherapy has
been questioned. In one retrospective study of superficial
X-ray therapy of 24 excised keloids, the author reported a
recurrence rate of 53%. Use of iridium Ir192 interstitial
irradiation after excisional surgery resulted in a 21%
Fig. 6.12: Raised, red hypertrophic scar over chest following burns

66
Principles and Practice of Burn Care
recurrence rate after 1 year. Excisional surgery and
preoperative hyaluronidase solution (150 U/ml sodium
chloride) followed by external radiation (7.2-10.8 Gy) had
a 0% recurrence rate. Adjunctive high dose rate
brachytherapy (192Ir) used after excision and closure resulted
in a 12% recurrence rate after 26 months. When excisional
surgery is followed by postoperative radiation therapy, the
total fractionated dose should be a minimum of 12 Gy,
according to a comparative study showing a higher
recurrence rate for patients treated with total doses less
than 12 Gy.
Other Potential Therapies
Additional potential therapeutic options for treating
hypertrophic and keloidal scarring that have been shown in
vitro to affect collagen synthesis include the use of proline-
cis-hydroxyproline and azetidine carboxylic acid, tranilast
(anti-allergic drug shown to decrease collagen and GAG
synthesis) and pentoxifylline (inhibits DNA replication).
In addition, wounds treated with anti-TGF compounds
healed with minimal scar tissue formation without affecting
wound tensile strength. A possible candidate for affecting
wounds via the neutralizing effect of TGF  is the
proteoglycan termed decorin.
Surgical Management of Hypertrophic
Scars and Keloids
Cryotherapy
Cryosurgical media (e.g., liquid nitrogen) affects the
microvasculature and causes cell damage via intracellular
crystals, leading to tissue anoxia. Generally, 1, 2, or 3 freeze-
thaw cycles lasting 10-30 seconds each are used for the
desired effect. Treatment may need to be repeated every
20-30 days. Care is taken to administer liquid nitrogen in
short application periods because of the possibility of
reversible hypopigmentation. Cryotherapy can cause pain
and permanent depigmentation in selected patients. As a
single modality, cryosurgery led to total resolution with no
recurrences in 51-74% of patients after 30 months of follow-
up observation.
Excision
For excision of small hypertrophic scars, closure with
minimal tension, paralleling the relaxed skin tension lines is
carefully planned.
Use buried sutures when necessary for layered closure
and to reduce tension. Whenever feasible, apply pressure
dressings and garments during the immediate postoperative
period to wounds of patients in whom hypertrophic scars
and keloid formation occur. Decreased recurrence rates have
been reported with excision in combination with other
postoperative modalities, such as radiotherapy, injected
interferon, or corticosteroid therapy. Excisional surgery alone
has been shown to yield a 45-100% recurrence rate and
should very rarely be used as a solitary modality, although
excision in combination with adjunct measures can be
curative. Most studies in which excisional surgery was
combined with injected steroids indicated less than 50%
recurrence.
Shaving and over grafting has been used for severe
hypertrophic scarring which causes functional restriction
also. This involves shaving the hypertrophic scar by using
a manual skin grafting knife till the entire fibrous tissue is
removed and healthy reticular dermis is reached, over which
a thin skin graft is applied. This usually helps in release of
minor contractures as well.
Laser Therapy
Ablation of keloids and hypertrophic scars using a carbon
dioxide laser (10,600 nm) can cut and cauterize the lesion,
creating a dry surgical environment with minimal tissue
trauma. When used as a single modality, the carbon dioxide
laser was associated with recurrence rates of 39-92% and
when it was combined with postoperative steroids injection,
it was associated with recurrence rates of 25-74%.
The argon laser (488 nm), similar to the carbon dioxide
laser, can induce collagen shrinkage via generation of
excessive localized heat. The argon laser has demonstrated
recurrence rates of 45-93%. The pulsed dye laser (585 nm)
provides photothermolysis, resulting in microvascular
thrombosis. Beginning in the 1980s, authors noted that scars
became less erythematous, more pliable, and less
hypertrophic after treatment with the 585 nm pulsed dye
laser. The findings were later confirmed using objective
measurements of erythema by reflectance spectrometry
readings, scar height, and pliability measurements. Because
of its efficacy, safety, and relatively low cost, the pulsed
dye laser remains the laser treatment of choice for
hypertrophic scars.
CONTRACTURES
A contracture (Figs 6.13 and 6.14) is a tightening of skin
that may affect the underlying muscles and tendons limiting
the joint mobility. Contractures develop when normal elastic

Wound Healing in Burns
67
connective tissues are replaced with inelastic fibrous tissue.
This makes the tissues resistant to stretching and prevents
normal movements of the affected area. Physical therapeutic
measures can aid in controlling burn scar contractures. If
these treatments do not control the effects of contractures,
surgical intervention is required. After release of contracture,
a skin graft or a flap cover is provided. The management of
this post burn complication is discussed elsewhere in detail
in this volume. Conservatively managed deep dermal burn
wounds and delayed skin grafting of full thickness burns
commonly end up in various kinds of scar deformities and
contractures. This significantly restricts physical and social
rehabilitation of a burn victim.
Skin scar contractures due to destruction of skin are
very frequent and when present over joints lead to
contractures of joint. Secondary contractures cause
shortening of musculo-tendinous units and neurovascular
bundles and of tissues around a joint leading to decreased
range of movement. Primary arthro-osseous contractures
result from direct deep burns in a joint, leading to severe
and irreversible processes. Contractures can occur at any
joint of the body. This joint dysfunction can also be a result
of immobilization from burn injury. A contracture is
different from spasticity, but they are often related. Spasticity
is an abnormal increase in muscle tone. This can worsen
the development of contractures.
SUMMARY
Skin is the largest organ in the human body forming its
outer protective covering like a shield. When it is damaged
in the case of a burn patient, it alters even the internal milieu.
Therefore, it is important to restore its structure and function
as soon as possible by conservative and surgical means. In
order to achieve this goal, it is essential to understand the
various biological processes which tend to heal the skin
wounds and how they can be hastened. The wound healing
response is by and large predictable with some degree of
variability. Sometimes, the healing processes go haywire as
is evident from development of hypertrophic scars and
keloids. Newer modalities are being explored to maximize
the rate of wound healing and minimizing the scar.
Webster states “The great ignominy of the plastic
surgeon is his inability to remove a scar without leaving
another one. The best we can do is to occasionally improve
on another surgeon’s scar while indeed he may be improving
on several of ours.”
BIBLIOGRAPHY
1. Aarabi S, Longaker MT, Gurtner GC. Hypertrophic scar formation
following burns and trauma: new approaches to treatment. PLoS
Med. 2007;4(9):e234.
2. B Latha, Mary Babu. The involvement of free radicals in burn
injury: A review. Burns: 2001;27,309-17
3. Badylak SF. Regenerative medicine and developmental biology:
the role of the extracellular matrix.Anat Rec B New Anat.
2005;287(1):36-41.
4. Chalain TMB, Tang C, Thonson HG. Burn area color changes after
superficial burns in childhood: can they be predicted? J Burn Care
Rehab 1998;19:39-49.
5. Clarke J. Burns. Br Med Bull. 1999;55(4):885-94.
6. De-Souza DA, Greene LJ. Pharmacological nutrition after burn
injury. J Nutr. 1998;128(5):797-803.
Fig. 6.13: Post burn hypertrophic scar over dorsum of hand and distal
forearm leading to contracture at wrist and fingers with deformity of
fingers
Fig. 6.14: Hypertrophic scarring over chest causing contracture
of neck and both axilla

68
Principles and Practice of Burn Care
7. Ferguson MW, O’Kane S. Scar-free healing: from embryonic
mechanisms to adult therapeutic intervention. Philos Trans R Soc
Lond B Biol Sci 2004;359(1445):839-50.
8. Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing. J
Leukoc Biol. 2001;69(4):513-21.
9. Greaves MW, Vella Briffa D. UV–A and the skin. Br J Dermatol1985;
105:477-82.
10. Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair
and regeneration. Nature 2008; 453(7193):314-21.
11. Hantash BM, Zhao L, Knowles JA, Lorenz HP. Adult and fetal
wound healing. Front Biosci. 2008;13:51-61.
12. J. Meenakshi, V Jayaraman, KM Ramakrishnan and Mary Babu.
Ultrastructural differentiation of abnormal scars. Annals of Burn
and Fire Disaster 2005;28(2):83-88.
13. J.Meenakshi, V Jayaraman, KMR amakrishnan, M Babu. Keloids and
hypertrophic scars: a review. Indian J Plast Surg 2005; 38(2):175-79.
14. Kahn AM, Cohen MJ, Kaplan L. Treatment of depigmentation
resulting from burn injuries. J. Burn care Rehabil 1991;12:468-73.
15. Leventhal D, Furr M, Reiter D. Treatment of keloids and
hypertrophic scars: a meta-analysis and review of the literature.
Arch Facial Plast Surg. 2006;8(6):362-8.
16. Martin P. Wound healing—aiming for perfect skin regeneration.
Science. 1997;276(5309):75-81.
17. Mary Babu, Alan Wells. Dermal-epidermal communication in wound
healing. Wounds 2001;13(5):183-9.
18. Mathangi Ramakrishnan, Prathiba V, Sridhar Rao K, Jayaraman, V
Mary Babu, Gupta PD. Expression of Keratin in Postburn Scars and
Keloids. Annals of Mediterrenean Burns Club-Burns and Fire
Disasters 1995; Vol.III:220-3.
19. Morgan JE, Gilchrest B, Golwyn RM. Skin pigmentation; current
concepts and relevance to Plastic surgery. Plast Reconst Surg1975;
56:617-28.
20. Nurden AT, Nurden P, Sanchez M, Andia I, Anitua E. Platelets and
wound healing. Front Biosci. 2008;1(5);13:3532-48.
21. Papini R. Management of burn injuries of various depths. BMJ.
2004 Jul 17;329(7458):158-60.
22. Prathiba V Kumaresan, R Mary Babu, Gupta PD. Ultrastructural
studies of keloids. Biomedical letters 1998;58:41-50.
23. Quinn K J. Silicone gel in scar treatment. Burns 1987;13:533-40.
24. Tsukada S. The melanocytes and melanin in human skin autografts.
Plast Reconst Surg 1974;53:200-7.
25. Tyack ZF, PeggS, ZirvaniJ. Post burn dyspigmentation: its
assessment, management and relationship to scarring. A review of
the literature. J burn care Rehab 1997;18:435-40.
26. Werner S, Kreig T, Smola H. Keratinocyte-fibroblast interactions
in wound healing. J Invest Dermatol. 2007;127(5):998-1008.

Immunological Responses to Burn Injury
69
Immunological
Responses to Burn Injury
7
Shankar Sriniwasan
Chapter
Infection continues to be the leading cause of death in burn
patients. Increased susceptibility to infection arises from
the large open wounds, increased metabolic requirements,
decreased nutritional intake, loss of proteins into the burned
areas and suppression of host immune response.
Alteration of host defense mechanisms in thermal injury
is a complex interaction of nutrition, hypermetabolism and
immunological alteration leading to a complex tango of
events which, to date, is an unresolved problem.
Nonspecific Immunity: Skin is an important defense
mechanism against microbiological invasion. Any breach in
this barrier is a harbinger for infection and its attendant
complications.
Any organism which gains entry must first contend with
local conditions at the point of entry. Vasoactive amines like
histamine and serotonin, kinin polypeptides and various other
mediators initiate a local inflammatory response. They cause
constriction of venular sphincters and dilatation of capillaries,
resulting in increased capillary permeability. Thus, fluid and
fibrinogen leave the dilated vessels and create a fibrin
network which localizes bacteria. Leucotaxis is also aided
by the kinin system.
Serum has two classes of protein:
1. Beta lysine which is lethal to gram-positive organisms.
2. A group of proteins called complement which acts
against gram-negative organisms.
If local mechanisms do not stem the infection, the
microorganisms are transported to the lymph nodes by the
lymphatics where macrophages phagocytose the organisms.
The specific immune responses are now called into play at
this location by the lymphocytes.
Specific Immunity: It can be discussed under two sub-
divisions: cell mediated and humoral.
The lymphocyte is the key cell in the initiation of specific
immune response. It is the only cell capable of recognizing
an antigen as foreign and initiating specific mechanisms for
its disposal.
Lymphocytes form two response systems:
1. T cell, associated with cell mediated immunity.
2. B cell, associated with humoral or antibody mediated
immunity.
Both T and B lymphocytes arise from a common bone
marrow stem cell. On maturity, the T and B lymphocytes
seed the peripheral lymphoid organs. It must be emphasized
here that there is a complex interaction between these
systems and that any simplified description is only to aid in
the understanding of this complex process.
Cell mediated immunity (CMI): Briefly, the cell mediated
immunity when stimulated, causes the T-lymphocytes to
undergo blast transformation and proliferate. T cell co-
operation is needed for B cell responses to occur. T cells
also secrete a variety of lymphokines which regulate immune
responses. Lymphokines also activate macrophages to
increase microbicidal activity.
Altered CMI following burns has been well documented
and researched. This is evidenced by prolonged survival of
allografts in extensively burnt patients. There is a decrease
in total counts and increase in suppressor T cells
(lymphocytes also have a depressed responsiveness owing
to generation of a subpopulation of T cells called suppressor
T cells).

70
Principles and Practice of Burn Care
Humoral immunity: The hallmark of humoral immunity is
the production of antibodies by the B lymphocytes.
Antibodies which have significant activity against
microorganisms are IgG, IgM and IgA.
All immunoglobulins are decreased in quantity during
the first week post burn and return to normal levels during
the second week after the injury. The decrease in IgG levels
is more than IgM or IgA. The initial decrease is followed by
higher than normal concentrations owing to antigenization
which burn patients are subjected to during their clinical
course. Serum IgM level is elevated owing to widespread
fungal infection in these patients.
Complement system: It is a complex integrated network
of serum proteins that bridges both specific and non-specific
immunity. On activation, they mediate inflammation and
tissue damage and facilitate phagocytosis. Edema formation
in injured tissue is complement dependant. Complement
components C3a and C5a facilitate serotonin and histamine
release which cause vasodilatation and thus edema.
A co-ordinated immuno-inflammatory response requires
the regulation of many cell types achieved by the actions of
soluble protein (cytokine) and lipid or peptide (autacoid)
mediators released by macrophages, neutrophils,
lymphocytes, platelets and endothelium. Some cytokines
like tumor necrosis factor alpha, interleukin-1 (IL-1) and
IL-8 have pro inflammatory actions while IL-4, IL-10 and
IL-13 have counter regulatory activities. Some cytokines
(e.g., IL-1) are released early while IL-6 and IL-8 appear
later in the pathological continuum.
Cytokines and autacoids regulate the amplitude and
duration of the immune response. They have multiple effects
on cell differentiations and there is considerable overlap in
their actions. To attempt to control and localize an
inflammatory process, both pro- and anti-inflammatory
mediators are released simultaneously. A balance between
these two competing mechanisms is critical if homeostasis
is to be maintained. Their often compartmentalized
production results in serum concentrations which do not
reflect accurately their local physiological levels. Quite aptly,
Cavaillon et al, have described serum cytokine
concentrations as being tip of the iceberg.
IMMUNOMODULATORY THERAPIES
The treatment of severe sepsis and septic shock has centered
around the eradication of established infection with surgery
and antibiotics along with intensive organ support. With the
realization that the pathological effects of sepsis can result
from the direct actions of micro-organisms and their toxins
and indirectly from the host response, interest has been
sparked off in newer immuno modulatory therapies. These
therapies have attempted to attenuate the actions of bacterial
toxins, limit host inflammatory response or to reduce tissue
damage resulting from established inflammatory processes.
Anticytokine therapies have attempted to inhibit pro-
inflammatory or enhance anti-inflammatory responses. In
all this, a delicate balance needs to be achieved between
inhibition of an excessive response and ablation of essential
host defenses. Derangement of either can lead to systemic
sepsis and death.
Endotoxin
Endotoxin or lipopolysaccharide (LPS) is found within the
external membrane of the cell wall of gram-negative bacteria
and is released from growing and damaged microorganisms.
LPS binds to many serum carrier molecules, the most
important of which is lipopolysaccharide binding protein
(LBP). The LPS-LBP complex interacts with macrophages
via the CD14 cell-surface receptor. LPS is a potent
stimulator of cytokine production. Administration of
endotoxin to healthy human volunteers produced clinical
features of sepsis.
Anti-endotoxin therapies: In early studies, polyclonal
human antisera with high levels of anti-LPS antibodies were
shown to reduce the mortality arising from gram-negative
sepsis. With technological advances came anti-LPS
monoclonal antibodies (e.g., HA-1A and E5). Despite
encouraging animal study reports, large, random, controlled
trials in humans have been disappointing.
Tumor Necrosis Factor Alpha (TNFα)
It is a polypeptide produced predominantly by the cells of
the reticuloendothelial system. It has a short half life and a
pivotal role in the expression of sepsis. It acts directly on
cells and stimulates the release of other mediators of
inflammation.
Anti-TNF alpha therapies: Anti-TNF alpha monoclonal
antibodies are some of the most promising therapeutic
modalities available. Unfortunately, this did not bear out in
some large scale studies.
The Interleukins (IL)
The interleukins are a class of cytokines produced by many
cell types. IL-1 and IL-8 have pro-inflammatory action
while IL-4 and IL-10 have anti-inflammatory actions. IL-
6 is regarded as the best available serum marker of the
severity of inflammation. IL-1 is a key mediator in the

Immunological Responses to Burn Injury
71
immuno-inflammatory cascade, interacting closely with
other cytokines, particularly TNF alpha.
Anti-IL-1 therapies: IL-1Ra (IL-1 receptor antagonist)
is the most extensively investigated therapy. Unfortunately,
as with other therapies, it has not been found to be effective
clinically.
Other Potential Immunomodulatory Therapies
The autacoids are important mediators in the immuno-
inflammatory cascade and include arachidonic acid
derivatives-prostacyclin, thromboxane A2, the
prostaglandins (particularly PGE2) and the leukotrienes.
1. Cyclooxygenase (COX) is an enzyme central to the
production of these derivatives and COX inhibitors
appear to have some benefits in septic patients.
Ibuprofen was tried but conferred no survival
advantage.
2. Selective TXA2 inhibition may be preferable to non-
selective COX inhibition as prostacyclin synthesis
would be maintained. The azole anti-fungal agent
ketoconazole is a selective thromboxane synthetase
inhibitor and a reduction in mortality has been observed
in a small placebo controlled trial.
3. Platelet activating factor (PAF) is a pro-inflammatory
mediator produced by the endothelium, neutrophils and
platelets. Treatment with the naturally occurring PAF
antagonist, BN 52021 has produced encouraging
results.
4. Bradykinin is a potent pro-inflammatory mediator that
is a potent stimulator of other autacoids and nitric
oxide. The bradykinin antagonist, CP 0127 has been
tried and shown to be useful in a small population of
patients with gram-negative infection.
5. Nitric oxide synthetase inhibition.
6. Fungal glycans: They increase macrophage
microbicidal activity and induce monokine release, e.g.
glucan.
7. Thymic extracts, e.g. thymopentin(TP-5)is licensed
for use and works by its ability to activate T cell
population. It decreases prostaglandin E2 production.
PGE2 is an immunosuppressant and inhibits T cell
activation.
8. Granulocyte colony stimulating factor has been shown
to have benefit.
9. Decreasing bacterial translocation following burns can
be achieved by giving prostaglandin analogues, e.g.
misoprostol, enisoprost.
10. Interferon gamma
11. Lipopolysaccharide antagonist: polymyxin B. It absorbs
LPS and prevents endotoxemia.
12. Free radical scavengers like superoxide dismutase
improve survival in animal models.
The complexities of the immunoinflammatory cascade
are such that pharmacological intervention has proved
difficult and cumbersome, not to mention prohibitively
expensive and beyond the reach of the common man. Despite
encouraging results from animal studies, no therapy has
shown to produce an unequivocal reduction in mortality in
clinical sepsis trials. It is unlikely that any single agent will
do the trick and the early use of immunostimulants followed
by the use of immunosuppressants later may prove to be
the most useful approach. It is nevertheless certain that the
benefits are likely to be small and unlikely to be realized in
the absence of optimal resuscitation, surgery, antibiotics
and intensive care therapy.
NUTRITIONAL IMMUNOMODULATION
Three categories of products have shown promising results
in chronic burn patient:
1. Long chain fatty acids
2. Arginine, glutamine and branched chain amino acids.
3. Nucleotides.
Amongst all above, glutamine is the best studied. It is
the most common amino acid in the body and is used by
rapidly dividing cells. Its administration supports the gut
barrier and minimizes gut translocation of bacteria and thus
the catastrophic complications arising from breakdown of
the gut barrier is mitigated.
IMMUNOTHERAPY
In 1964, Kefalides noted a decrease in serum levels of gamma
globulin in burned children. The levels returned to normal
in 7 to 9 days.
The general consensus was that there was a marked
drop in IgG levels immediately post burn which gradually
returned to normal approximately two weeks after the insult.
The severity of suppression of IgG was related to burn
size and the degree of depletion of IgG could be correlated
to mortality from sepsis.
IgG concentrations under 300 mg% within a day or
two of admission carry a very poor prognosis.
As an adequate concentration of IgG is critical to the
outcome, it is reasonable to:
1. Stimulate production by vaccination.
2. Increase serum concentration by passive immunization.
3. Combination of the above.

72
Principles and Practice of Burn Care
Early attempts centered on the development of vaccines
against Pseudomonas aeruginosa. This is not in widespread
use today because the incidence of life threatening infections
due to Pseudomonas infection have fallen and for the vaccine
to be effective,it is necessary to vaccinate the patient several
days before infection becomes a threat to get sufficient
antibody levels in the serum. This luxury of time is often
not available.
Passive immunization: It holds a lot of promise. It is
attractive because the product is immediately available and
effective at the time of administration. Cutter® Iv Ig was
first tried in the US and was successful. It was 90% IgG
fractionated from a pool of donors. It had excellent antibody
titres against all types of Pseudomonas aeruginosa along
with useful titres against streptococci, E. coli, Staph. aureus
and some viruses too.
Pentaglobin®, i.e. IgM enriched intravenous Immuno-
globulins have an anti-bacterial, anti-toxic, anti-inflammatory
and immune modulating activity.
Pentaglobin embodies natural principles of physiological
defense mechanisms and presents a rational basis for the
therapy of sepsis. Hence, it reduces rate of metabolism and
energy requirement.
CONCLUSION
Active vaccination is not practical because of changing
spectrum of pathogenic microorganisms in burn patients.
Passive immunization holds a lot of promise. A high titre,
polyvalent immunoglobulin product that offers protection
against gram-negative organisms (other than Pseudomonas)
is desirable.
A combination of topical and systemic antibiotics, better
nutrition and immunomodulatory methods will play a decisive
role in the management of burn patients in the foreseeable
future.
BIBLIOGRAPHY
1. Abbas AK. Diseases of Immunity in Robbins and Cotran Pathologic
Basis of Disease. V Kumar, AK Abbas, N Fausto (Eds) 7th edition
Saunders Philadelphia, 2007;Chapter 6, 193-267.
2. Munster AM. The immunological response and strategies for
intervention in Total Burn Care. DN Herndon (Editor) WB Saunders
Philadelphia 1996;Chapter 27, 279-91.
3. Warden GD. Immunoglogic Response to Burn Injury in the Art and
Science of Burn Care JA Boswick (Editor). Aspen Publishers Inc.
Maryland 1987;113-21.
4. Recent advances in Surgery. No. 23, CD Johnson, I. Taylor.
Fig. 7.1: Rationale for immunotherapy with IV immunoglobulins

The Immunobiology of Skin Transplant Rejection and Acceptance
73
The Immunobiology of Skin
Transplant Rejection and Acceptance
8
Rakesh Kain
Chapter
INTRODUCTION
Attempts at skin transplantation have been recorded over
the centuries, often in graphic detail. The earliest reports of
transplantation in humans included several miracles. One
of best-known examples is the thirteenth century story of
Cosmos and Damian. They were Christian Arab saints who
were martyred around 300AD and were reputed to have
successfully replaced the diseased leg of sexton with that
from a moor who had died several days earlier. The practice
originated in India and was modified to great effect in
Europe.1
The immune system identifies and attempts to destroy
all cells and tissue recognized as foreign. Only the tissues
from genetically identical donors are exempt as they do not
express foreign antigen that can initiate a response. Even
the smallest disparity, a single amino acid in a major
histocompatibility (MHC) molecule, can bring about the skin
transplant rejection in mouse. The transplantation barrier
can be defined in terms of genetic difference(s) between
the donor and the recipient and this is discussed later. In
general terms, there are both antigen specific and non-
specific responses including antibody mediated, T-cell -
mediated and inflammatory reactions.
To reduce transplant rejection, one either avoids
transplantation into a sensitized recipient or minimizes the
antigenic disparity between the donor and the recipient or
else, treats the patient with various immunosuppressive
agents. Knowledge of the immune response to transplanted
tissue provides a rationale for the use of currently available
agents and may help in the design of new products/ protocols
for future use.
THE SCIENTIFIC BASIS OF TRANSPLANTATION
The modern era of transplantation began in 1950s. However,
the preceding decade was a defining period for
transplantation because it was during this period when Peter
Medawar and colleagues showed that the loss of a tissue
allograft was brought about by the immune response it
provoked in the recipient and not as a result of some kind
of non-specific inflammatory reaction(s).2
With the aid of grant from the Medical Research Council,
Medawar travelled to Glasgow to study skin grafting in the
burns unit at Glasgow Royal Infirmary. There, he worked
with Tom Gibson, a gifted plastic surgeon, who was
interested in the study of skin graft rejection. Shortly after
Medawar’s arrival, a young lady was admitted to hospital
with severe burns after falling on to an open gas fire. Gibson
grafted the women’s wounds with a series of small pinch
skin grafts taken from her brother and Medawar proceeded
to study the fate of the skin grafts by taking their biopsies
for histological examinations.3 As expected, the grafts were
destroyed after some days and when a second set of graft
from the same donor was applied 2 weeks later, they were
destroyed even more quickly. This so called “second set”
phenomenon was taken as clear evidence that the rejection
response was due to actively acquired immunity and not to
a non-specific inflammatory reaction. Medawar and Gibson
published these findings in the journal of anatomy (1944).4
They concluded that an, as yet, unidentified antibody is
responsible for rejection.
Regardless of whether antibody or cellular immunity
was responsible, there was now widespread acceptance
that immunological mechanisms were the cause of graft

74
Principles and Practice of Burn Care
rejection. Immunological rejection was viewed as an
inevitable consequence of organ transplantation and
unfortunately no effective way of preventing rejection was
known. Attempts to surpass graft rejection had met with,
at best, minimal success.
Behavior of Transplanted Tissue
One important point to bear in mind is that the graft responds
to its environment in the recipient. The organ is not inert. It
changes its expression of cell surface and produces a range
of cytokines and other factors. It does so in response to
physiological stress and immune attack. These normal tissue
responses of graft influence the outcome of transplantation,
by increasing graft antigenicity and immunogenicity.
TRANSPLANTATION AND GENETIC BARRIERS
The degree of antigenic disparity between the donor and
recipient is important and can be defined in terms of genetic
differences between individuals. Donor tissue carries a large
number of different molecules that may be recognized as
foreign by the immune system of the recipient. These include
the blood group antigens to which there are preformed
antibodies. Then there are major histocompatibility (MHC)
molecules that may be recognized directly by the receptors
on recipient T cells. In addition, there is all the protein of
the tissue that can be degraded to peptides and can activate
cellular responses in the recipient (Table 8.1).
THE IMMUNOGENIC ELEMENTS
Donor tissue carries a large number of different
immunogenic molecules which may be the functional cells
of the graft. These molecules are recognized as a foreign
substance by the immune system of the recipient. Together
they carry out one or more different functions. These cells
usually express only low level of immunogenic response.
These include the blood group antigen to which there are
preformed antibodies. Other is MHC molecule that may be
recognized directly by the receptors on recipient T cells.
The endothelial cells form the lining of all the vasculature
present in whole of the body. The endothelium is usually
quiescent but can be activated in a variety of ways by
cytokines and by cell-cell contact. The endothelium contains
a number of adhesion molecules called selectin and integrins.
IFN-γ a cytokine and TNF-α alter endothelial cell expression
of adhesion molecule and increase the expression of both
MHC 1 and MHC 2 antigens on endothelial cells. Hence
endothelium, especially after activation, is highly antigenic.5
THE MECHANISM OF GRAFT REJECTION
The two immune systems operate together. One is an innate
immune system which combines cells of monocyte and
granulocyte origins, macrophages, neutrophils and
eosinophils, along with complement components, to provide
a simple phagocytic and inflammatory defense against
bacteria and other organisms. Another immune system is
“adaptive immune” system which involves the activation of
two classes of antigen-specific lymphocyte. The B cells
are derived directly from bone marrow and form antibodies
against antigens.6 The T cells originate in the bone marrow
but pass through the thymus and go on to recognize peptide
fragments of antigen present on the surface of strongly
MHC positive antigen presenting cells. Once activated, the
B and T cells can be more readily activated so that second
or subsequent exposure to the same antigen elicits a bigger
and more rapid response.1
The immune system operates by importing a vast
armamentarium to the site of inflammation. The number of
possible reactions and combinations of immunological
components that come into play to destroy a transplant has
led to some difficulty in understanding the process fully.
The Recipient in the Graft
During rejection process, cells have to migrate from the
blood stream in to the transplanted tissue. Endothelium
Table 8.1: Genetic relation affecting outcome of transplant
Transplant
Donor –patient relationship
Outcome of transplant
Autograft
Same individual
No foreign antigen, therefore, no rejection
Isograft
Genetically identical donor and recipient
No foreign antigen, therefore, no rejection
(monozygotic twin)
Allograft
Genetically different individuals of same species
Recognition mainly of mismatched donor MHC antigens.
(the usual clinical situation)
Vigorous rejection without MHC matching and immunosuppression.
Xenograft
Different species (e.g., pig to human)
In some species combination there are preformed antibodies,leading to
hyperacute rejection, the greater the phylogenetic gap the more vigorous the
cellular rejection response.

The Immunobiology of Skin Transplant Rejection and Acceptance
75
expresses the adhesion molecules called E-selectin,
P-selectin, etc. which appear on surface within seconds of
stimulation with thrombin, histamine and other agonists.
This causes leukocytes to slow down and roll along the
endothelial cell surface followed by activation of integrin
adhesion molecules. This and other mechanisms lead to
firm adhesion of leukocyte to endothelial cells and their
flattening against the vessel wall.
SKIN ALLOGRAFT REJECTION AND
ACCEPTANCE
Skin is a strongly antigenic organ due to ‘skin specific
antigen’, an antigen presented by the skin graft after it is
transplanted. Hence, it is subject to rejection.
The obvious alternative to split skin autograft and the
problem of limited availability of donor sites is skin allograft.
Though cultured keratinocytes can be used as substitutes
for skin allografts, the facility and infrastructure for skin
culture is not available in many of the developing countries.
For such places, the only skin substitute that can be used in
extensive burns where enough donor areas are not available,
is allograft, taken from the burned patients’ close relatives,
particularly mother, father or siblings. However, all allografts
from random donors start getting rejected in about 2 week’s
time. Even those obtained from parents start getting rejected
in about 3 weeks if patient is not severely immuno-
compromised. Rejection of full thickness skin allograft by
immunocompetent individuals is such a consistent and
reproducible phenomenon that it has been the central model
for the study of immunologically mediated tissue destruction
in vivo since the work done by Medawar (Gibson and
Medawar7, 1943). The only documented permanent
coverage with skin allograft in humans has been in identical
siblings (Converse and Duchet8, 1947; Stranc9, 1966;
Caruso et al10, 1996).
Skin graft revascularization is the basis for the beginning
of graft acceptance or rejection or both. The vascularization
of graft begins immediately after a graft is applied to the
host bed, be it an autograft or an allograft. By 6 hours, only
the plasmatic imbibition nourishes the graft, although the
host endothelium starts to proliferate. By 9 hours,
inflammatory cells invade the graft. By 12 hours, the host
endothelium penetrates the fibrin interface between graft
and host and small communications start forming between
graft and host vessels. This is called inosculation. By 24
hours, the host vessels grow into the graft and vascular
communications are well established by 48 hours. By
4 days the arterial end of the capillaries are open and slow
arterial flow from the host to the graft begins. The circulatory
outflow from the graft to the host (from the venous end of
capillaries) starts around the 7th day and this brings the
donors antigens into the host circulation in case of allograft.
The host then produces antibodies against the graft. By
10th day, this starts the rejection phenomenon and the
process of rejection becomes clinically evident by the 14th
day in the form of maceration of the graft. In fact, there are
2 types of reactions which take place following allograft
transplant. The first is cellular reaction which is local and
the evidence for the failure of homograft is found within
the graft themselves or their immediate surroundings. The
second reaction is humoral reaction which is systemic in
nature and follows a general pattern of antigen –antibody
reaction. Corollary to this is that if homograft is applied and
removed before venous return is established, i.e. 7th day
and then reapplied to the same or different area, it can survive
for another 14 days till it is rejected again.
White graft rejection: when homograft is applied to a
burn patient for a second time from the same donor, it has
been found that it stays white and does not turn pink as no
circulation is established in this graft and therefore, it is not
taken up as in normal circumstances. The explanation for
this is that once a homograft is applied to the host for the
first time, antibodies are formed against the homograft and
they stay in the host circulation from that time onwards.
On second application of the same homograft, it is
immediately rejected by the host and not taken up at all. In
other words, the graft remains white. This is called “White
rejection”. This is also known as ‘Second set rejection’.
In case of major burns, the immunocompromised state
of the patient can delay rejection of allografts for several
weeks.
However, allograft skin is used as a biological dressing
in the management of severely burned patient. Thus widely
meshed (>1:6) split skin autograft is interleaved with allograft
either by overlaying (Alexander et al11,1981; Phipps and
Clarke12, 1991) or insertion of allograft in holes cut in
autograft sheets(Yang et al13, 1979). Except for one isolated
case report (Takiuchi et al14, 1982) allografts do not survive
in the long term, the rejection always occurring before 3
months and often within the first 3 weeks of transplantation.
Attempts to modify this outcome with general
immunosuppression have been made (Burke et al15,16, 1974,
1976) but encountered a serious problem with sepsis as a
result of immunodeficiency.17

76
Principles and Practice of Burn Care
Experimental studies in animal model showed that skin
allograft survival was prolonged by cyclosporine (Borel
and Meszaros18 1976; Black et al19 1988). However, the
actual fate of the allograft in many of these studies is not
clear. In 1989, Frame et al20 treated patients with cyclosporin
after allografting .The allograft survived during drug
treatment but were rejected in two cases after cessation of
therapy.1
TYPES OF REJECTION
According to time interval and host response against graft,
the rejection can be classified into four categories:
1. Hyperacute rejection (minutes to hours): Occurs
within minutes to hours of transplantation. Preformed
anti-donor antibodies cause complement fixation and
thrombosis in the vasculature of graft.
2. Accelerated acute rejection (1-4 days): Refers to the
situation where the T cells of the recipient have been
immunized against antigens of the donor by prior
exposure to foreign cells and tissues (presensitized T
cells also known as “second set” rejection).
3. Acute rejection (days to weeks): Acute rejection
occurs within days to months after transplantation. In
fact the period of greatest risk of acute rejection extends
to about 3 months after grafting. The principle immune
response is activation of T- lymphocytes. These cells
are of two subtypes—CD-4 positive cells or helper cells
(Th) and CD-8 positive cells or cytotoxic cells (Tc).
The Cd-4 cells may have cytotoxic activity and CD-8
cell makes a range of cytokines.
4. Chronic rejection: Chronic rejection generally occurs
months to years after transplantation. Defining chronic
rejection is difficult against a background of other
processes that are going on at the same time.
Immunological mechanisms of chronic graft dysfunction
include the binding of antidonor antibodies and sub-acute
cell mediated damage. The sites of the damage are the
vasculature and the parenchyma. On-going immuno-
logical mechanisms of graft vasculopathy include
hypertension, hyperlipidemia, and cytomegalovirus
infection, etc. probably mediated by release of various
growth factors within the transplant.5
GRAFT VERSUS HOST DISEASE (GVHD)
Transplantation between genetically non-identical individuals
is, at present constrained by two major immunological
responses. First is graft rejection and second is graft versus
host disease (GVHD).21
In a burn patient whose immunity is very low, e.g. if the
patient is in sepsis, even if homografts are applied repeatedly
to the host’s raw areas, no antibodies are formed against
the graft by the host because of low immunological
response. Therefore, the graft is not rejected.22 Instead,
gradually the homografts antigens start becoming stronger
and they mount an immune response against the host. The
host’s antigens become foreign to the homograft and the
homograft starts forming antibodies which destroy the host
cells. This is called ‘Graft Versus Host disease’ (GVHD) or
‘Runting syndrome’. Patient may manifest with anemia,
weight loss, fever, edema, ulceration, loss of hair and
spleenomegaly.23
Acute GVHD is characterized by a distinctive syndrome
comprising dermatitis, hepatitis and enteritis. The severity
of disease is defined by a grading scheme from 0 to 5.
0 and 1are mild disease
2 is moderate
3 and 4 are severe and/or life threatening
5 is life threatening
Even with optimal immunosuppression, patient shows
significant morbidity and mortality. In long term after skin
transplantation, after 100 days, a different form of GVHD
may emerge even though initially it may have been
considered as successful graft ‘take’. These are considered
as autoimmune phenomenon with relative immunodeficiency
(Herman Waldman22, 1995). Experimental work in animal
models shows that both forms of GVHD can be caused by
both CD4 and CD8 cells.
Treatment for Graft-versus-Host Disease (GVHD):
1. UVA-1 Phototherapy: Clinical responses of sclerotic skin
to UV-A1 phototherapy were modest cause of UV-A1–
induced skin darkening, which is photo-protective and
attenuates antifibrotic responses.
2. Low dose Methotrexate: Methotrexate (MTX) appears
to be a well-tolerated, inexpensive and possibly steroid-
sparing agent that is worthy of further evaluation in
GVHD.
3. Extracorporeal Photo-chemotherapy (ECP), considered
as an effective treatment for patients with erythrodermic
cutaneous T-cell lymphoma, has recently been used
successfully in the treatment of GVHD and prospective
trials for treatment of chronic GVHD.
4. Anti-inflammatory proteins in combination with steroids.

The Immunobiology of Skin Transplant Rejection and Acceptance
77
CULTURED KERATINOCYTES ALLOGRAFT
Autogenous cultured keratinocytes normally takes 3-4 week
to grow a thin sheet of skin. Cultured keratinocytes would
overcome this problem and raise the alluring prospect of
banks of frozen skin readily available for transplantation. A
consideration of how alloantigens are recognized in the skin
suggests that cultured keratinocytes could survive without
rejection.24
In the skin, the Langerhans cell (LC) is the dendritic cell
and the immunogenicity of skin allograft correlate directly
with the density of the LCs25, which they contain
functionally. However, LCs are complex because the
efficiency with which they process and present antigen and
activate T cell depends upon their location and
differentiation.25,26 LCs in the epidermis encounter antigen,
which is than processed and displayed in association with
major histocompatibility complex(MHC) class 1 or 2.27 LCs
then migrate to lymph node and mature to a cocktail of
cytokine signals. In the lymph node, the MHC antigen
complex displayed at the LC plasma membrane is presented
to any T cell reactive to the original antigen. In skin allograft
responses, the evidence is that host T cells are activated by
donor LCs which migrates from the graft to the draining
lymph node. Thus if the afferent lymphatics are severed,
graft rejection is either abrogated (Barker and Billingham28,
1968) or delayed (Tilney and Gowans29, 1970).
Langerhans cells are nonadherent in vitro and are
eliminated from primary keratinocyte culture within 7 days
as a consequence of medium changing (Morhenn et al30,
1982; Hammond et al31, 1987). Cultured keratinocyte do
not constitutively express MHC class 2 glycoproteins or
the co-stimulatory molecules CD80 CD86 (Nickoloff and
Turka32, 1994) and fail to stimulate autologous lymphocyte
in mixed lymphocyte reactions.
There is very little clinical or histological evidence for
acute rejection of cultured keratinocyte allografts on ulcers
or wounds.33-35 Aubock et al36 (1988) used cultured allograft
to partially cover tangentially excised third degree burns
and split thickness donor site. These grafts were rejected
after 14 days, about 4-5 days longer than that reported for
split thickness allograft. Histologically, rejection was
accompanied by a mononuclear cell infiltration and
vacuolation and necrosis of grafted epithelium.24,27,35,37,38
THE PRESENT AND FUTURE OF SKIN ALLOGRAFT
There is now a good understanding of the multiple
mechanisms of allograft rejection. The hope of inducing
graft acceptance persists and seems more likely in the future.
Unlike transplantation of other organs, skin is readily
available in the patient himself/herself and has lowest
morbidity in the live donor.9,14,33,34 For some other organs,
only dead can donate.
The use of chronic immunosuppression, with associated
toxicity, for prolonged survival of skin allografts is not
justified. Instead some form of site specific immuno-
suppression is being investigated, where the immuno-
suppressant is delivered to the site of allograft application
without any systemic administration.
Skin transplant, where the outcome is complete
regeneration of the morphologically normal organ, remains
both an experimental and a clinical challenge. Our
understanding of the role of the skin in the immune responses
and the modulation of these responses has advanced
significantly.
REFERENCES
1. Hutchinson Ian V. The immunobiology of transplant rejection and
acceptance. Transplantation Surgery, Springer Specialist Surgery
Series, Editor: John SE Lumley 2001;55-72.
2. Waldmann H, Polliak A, Hale G, et al. Elimination of graft versus
host disease by in-vitro depletion alloreactive lymphocytes
monoclonal rat antihuman lymphocyte antibody (CAMPATH-
1).Lancet 1984;2:483-86.
3. Bradley Andrew J, Hamilton David N. Organ transplantation; an
historical perspective. Transplantation Surgery 1990:1-5.
4. Medawar PB. The behavior and fate of skin autografts and skin
homografts in rabbits. Journal of Anatomy 1944;78:176-99.
5. Bradley J Andrew, Hamilton David NH. Organ transplantation: an
historical perspective. Transplantation surgery 1990;1-6.
6. Pedersen NC, Moris B. The role of the lymphatic system in the
rejection of homograft: a study of lymph from renal transplant. J
Exp Med 1970:131; 936.
7. Gibson T and Medawar PB. The fate of skin homograft in man. J.
Anat. 1943;77:299.
8. Converse JM, Duchet G. Successful homologous skin grafting in a
war burn using an identical twin as a donor. Plast Reconst Surgery
1947;2:342-44.
9. Stranc ML. Skin Allograft survival in a severely burned triplet;
study of triplet zygotic type. Plast.Reconstr.Surg 1966,37:280-90
10. Caruso DM, Gregory MW, Schiller WR. The use of skin from a
monozygotic twin combined with cultured epithelial autografts as
coverage for a large surface area burns: a case report and review of
the literature. Journal of Burn care and rehabilitation 1996;17:
432-4.
11. Alexander JW, MacMillan BG, Law E, Kittur DS. Treatment of
severe burns with widely meshed skin autografts and meshed skin
allograft overlay. Journal of Trauma 1981;21,433-8.
12. Phipps AR, Clarke JA. The use of intermingled auto graft and
parental allograft skin in the treatment of major burns in children.
British J of Plastic Surgery 1991;44,608-11.

78
Principles and Practice of Burn Care
13. Yang CC, Shih TS, Chu TA, et al. The intermingled transplantation
of autografts and homografts in severe burns. Burns 1979;6,141-
45.
14. Takiuchi L, Higuchi D, Sei Y, Nakajima T. Histological identification
of prolonged survival of a skin Allograft on an extensively burned
patient. Burns 1982;8,164-67.
15. Burke JF, May JW, Albright B, et al. Temporary skin transplantation
and immunosuppression for extensive burns. N Eng J Med
1974;290:269.
16. Burke JF, Quinby WC JR, Bondoc CC. Primary excision and prompt
grafting as routine therapy for the treatment of thermal burns in
children . Surg. Clin. North Am 1976;56:477.
17. Yang, CC, Shih TS, Chu TA, et al. The intermingled transplantation
of Auto-and homografts in sever burns, Burns 1979;6:141-45
18. Borel JF, Feurer C, Gubler HU, Stahelin H. Biological effects of
cyclosporine A: A new antilymphocytic agent. Agents Actions
1976;6:468.
19. Black KS , Hewit CW, Smelser S, et al. Cyclosporine and skin allografts
for the treatment of thermal injury. Transplantation 1988;45:13-16.
20. Frame JD, Sanders R, Goodacre TEE, Morgan BDG.. The fate of
meshed allograft skin in burned patients using cyclosporine
immunosuppression. British Journal of Plastic Surgery 1989;42,27-
34.
21. Codner MA, Shuster BA, Steinman RM, et al. Migration of donor
leukocytes from limb allografts into host lymphoid tissues. Ann
Plastic surg 1990;25:353.
22. Delves Peter J, Roitt Ivan M. Graft Vs Host Diesease; in
Encyclopedia of Immnology. 2nd edition, vol 2, Academic Press
Ltd., Harcourt Brace and Co. Publishers, 1998;1015-20.
23. Lapidot T, Singer TS, Solomon O, et al. Booster irradiation to the
spleen following total body irradiation. A new immunosuppressive
approach for allogenic bone marrow transplantation. J of
Immunology 1988;141: 2619-24.
24. Kries RW, Hoekstra MJ, Mackie DP, Vloemans AF, Hermans R P
Historical appraisal of the use of skin Allograft in the treatment of
extensive full skin thickness burns at the red cross hospital burn
center, Beverwijk, Netherlands. Burns 1992;18:S19-S22.
25. Larsen CP, Barker H Morris PJ, et al. failure of mature dendritic
cells of the host to migrate from the blood in to cardiac or skin
allografts. Transplantation 1990;50: 294.
26. Larsen CP, Steinman RM, Witmer-pack M, et al. Migration and
maturation of Langerhans cells in skin transplants and explants
Exp Med 1990;172:1483.
27. Hefton JM, Madden MR, Finkelstein J, Shires GT. Grafting of burn
patients with Allograft of cultured epidermal cells. Lancet
1983;2:428-32.
28. Barker CF, Billingham RE. The role of afferent lymphatics in the
rejection of skin allografts. Journal of experimental medicine
1968;128:197-220.
29. Tilney NL, Gowans JL. The sensitization of rats by allografts
transplanted to a lymphatic pedicle. Journal of experimental
medicine 1970;133:951-62.
30. Morhenn VB, Benike CJ, Cox AJ, Charron DJ, Engle man EG.
Cultured human epidermal cells do not synthetize HLA–DR. Journal
of investigative Dermatology 1982;78:32-7.
31. Hammond EJ, Stanley MA, Munro AJ. Prolonged survival of
cultured keratinocyte allografts in the immunosupressed mouse.
Transplantation 1987;44:106-12.
32. Nickoloff BJ, Nestle FO, Zheng XG, Turka LA. T lymphocytes in
skin lesions of psoriasis and mycosis fungoides express B7-1: a
ligand for CD28. Blood 1994;83:2580-86.
33. Sakabu SA, Hansborough, JE, Cooper ML, Greenleaf,G. Cyclosporine
A for prolonging Allograft survival in patients with massive burns.
Journal of Burn care and Rehabilitation, 1990;11:410-18.
34. Sawada, Y. Survival of an extensively burned child following use of
fragments of Autograft skin overlain with meshed Autograft skin.
Burns 1985;11:429.
35. Yang CC, Shih TS, Chu T-A, et al. The intermingled transplantation
of Auto-and homografts in sever burns, Burns 1979;6:141-45.
36. Aubock J, Irschick E, Romani N, et al. Rejection after a slightly
prolonged survival time of Langerhans cells free allogenic cultured
epidermis used for wound coverage in humans. Transplantation
1988;45:730-32.
37. Teepe RGC, Kreis RW, Koeburgge EJ, et al. The use of cultured
autologous epidermis in the treatment of extensive burn wounds.
Journal of Trauma 1990b; 30:269-75.
38. Wu J, Barisoni D, Armato U. An investigation in to the mechanism
by which human dermis does not significantly contribute to the
rejection of allo-skin graft. Burns 1995;21:11-16.

Microbiology in Burns
79
Microbiology in Burns
9
Deepthi Nair
Chapter
INTRODUCTION
Burns wound injuries continue to be a prevalent and
devastating form of trauma. These patients require
immediate and effective care in order to minimize morbidity
and mortality. The survival rates for burn patients have
improved substantially in the past few decades due to
advances in modern medical care. The improved outcome
has been attributed to advances in fluid therapy, nutrition,
pulmonary care, burn wound care and infection control
practices. As a result, burn-related deaths have been halved
within the past 40 years.1-3 In patients with severe burns
with more than 40% of the total body surface area (TBSA)
involvement, 75% of all deaths continue to be due to sepsis
from burn wound infection and other infectious
complications which are potentiated immensely by inhalation
injury.4-6
Burn wound infections have been enigmatic in terms of
diagnosis. Multiple factors contribute towards this fact,
which include recognition of colonization versus true
infection, numerous/multiple pathogens/colonizers,
confounding symptoms and signs, poor sampling methods
and difficult microbiological techniques.7 Several workers
have published innumerable papers on evaluating the most
accurate and reproducible methods for diagnosing wound
infections. Chronic burn wounds contain a large number of
bacteria inherent within them. The numbers of bacteria
which constitute infection in an ordinary acute surgical
wound (i.e., > or = 105 microorganisms/gm of tissue) are
not valid in burn wounds. Direct correlation with quantitative
count is now seen not to be a fool proof method. Deep fluid
culture correlated with histopathology examination and
clinical signs and symptoms, in immunocompromised
patients like burns has been found to be more useful than
quantitative counts alone. Despite levels of microorganisms
as great as 108/gm of burned tissue, these burn wounds go
on to heal adequately.8-10 Burn wounds often contain three
or more microorganisms, both Gram-positive and -negative
bacteria, as well as aerobic and anaerobic species. They
can often contain fungal flora especially in long standing
wounds where multiple broad spectrum antibiotics have
been given. This cohabitation of flora with the resultant
competition for nutrients and space is thought to decrease
their virulence and ability to harm tissues.8,11 Due to
differences in the way acute and chronic wounds respond
to diverse numbers of microorganisms, emphasis is
currently being placed on clinical signs and symptoms as
compared to actual numbers of microorganisms in a wound.
Microorganisms invade the burn eschar over time
leading to burn wound infection and progressing to burn
wound sepsis. The use of systemic antibiotics, availability
of effective topical treatments and early burn wound
excision with closure have all decreased the incidence of
burn wound sepsis. Along with the classical signs of infection
(erythema, edema, heat, purulent exudates and pain) there
are a few more criteria which have been included, viz.,
serous exudates with concurrent inflammation, delayed
healing, discoloration of granulation tissue, friable granulation
tissue, pocketing at the base of the wound, foul odor and
wound breakdown. As the patient is being monitored the
symptom of increasing pain and dehiscence of wound is a
good predictor of infection.11,12 There are several sequels
of burn wound infections which include cellulitis, septicemia,
gas gangrene, graft failure, delayed healing, scarring,

80
Principles and Practice of Burn Care
conversion of a second-degree burn to full thickness third-
degree burn, etc. 13
PATHOGENESIS OF WOUND INFECTION AND
IMMUNOLOGY OF SKIN
The thickness of the epidermis and dermis vary with the
site of the body, sex, age of patient, etc. Infants, young
children, and elderly adults have a thinner dermal layer of
their skin, resulting in deeper burn injury. The dermis is
capable of producing new epithelial cells to replace those
lost from the epidermis by the shafts of appendages which
are lined with epithelial cells. Nerve endings occur throughout
both skin layers and the connective tissue of the dermis
also provides a firm structural base for the skin to renovate.3
Immunologically, thermal injuries cause an immuno-
compromised state. This was evident as homografts are
seen to survive well in these patients.14, 15 Host defenses
can be divided into innate and adaptive immune responses.
The innate immune response is the first line of defense acting
promptly after the breach of skin by the burn wound.16
When the wound covers large regions or critical organ
systems or is deep, the systemic component of immunity
also is evoked. The response results in a varied spectrum
of presentation, with serious complications like systemic
inflammatory response syndrome (SIRS), severe sepsis and
multiple-organ dysfunction syndrome (MODS) which result
in increased morbidity and mortality.17
The body tries to maintain homeostasis by a process of
contraction, retraction, and coagulation of blood vessels
immediately after a burn. Three zones have been described
within the wound:
i. Zone of coagulation, which comprises the dead tissues
that form the burn eschar that is located at the center
of the wound
ii. Zone of stasis, which comprises tissues adjacent to
the area of burn necrosis that is still viable but at risk
for ongoing ischemic damage due to decreased
perfusion, and
iii. Zone of hyperemia, which comprises normal skin with
minimal cellular injury that has predominant
vasodilatation and increased blood flow as a response
to injury.
The burn wound surface (in deep partial-thickness and
in full-thickness burns) is a protein-rich environment
consisting of avascular necrotic tissue (eschar) that provides
a favorable niche for microbial colonization and proliferation.
The lack of vascularity leads to an impaired migration of
host immune cells and a restriction in delivery of systemically
administered antimicrobial agents, while toxic substances
released by eschar tissue further impair the local host immune
responses. Although burn wound surfaces are sterile
immediately following thermal injury, these wounds
eventually become colonized with microorganisms.18-22
MICROBIAL ETIOLOGY
Microorganisms colonizing the burn wound originate from
the patient’s endogenous skin, gastrointestinal and upper
respiratory tracts. The normal microflora of the gut, oral
cavity and vagina are both diverse and abundant.
Microorganisms may also be transferred to a patient’s skin
surface via contact with contaminated external environmental
surfaces, water, fomites, air and the soiled hands of health
care workers.23 It is the slow healing wounds which have
continued exposure of devitalized tissue which allow for
colonization.
Immediately following injury, Gram-positive bacteria
from the patient’s endogenous skin flora or the external
environment are the initial culprits.22 Endogenous Gram-
negative bacteria from the patient’s gastrointestinal flora
also rapidly colonize the burn wound surface in the first
few days after injury.24 Wounds with a sufficiently hypoxic
environment are susceptible to colonization by a wide variety
of endogenous anaerobic bacteria. In most published studies,
the cultures for anaerobic bacteria are minimal or omitted.
However, when wounds are investigated by appropriate
microbiological techniques, anaerobes are found in large
numbers. Anaerobes have been termed “the secret pathogens”
and “invisible villains”. Wound colonization by yeasts and
fungi usually occurs later due to the use of broad-spectrum
antibiotic therapy.25 Microorganisms transmitted from the
hospital environment tend to be more resistant to
antimicrobial agents than those originating from the patient’s
normal flora.26 Tables 9.1 and 9.2 list the most common
microorganisms colonizing and infecting burn wounds. Prior
to the antibiotic era, Streptococcus pyogenes (Group A beta-
hemolytic streptococci) was the predominant pathogen
implicated in burn wound infections and a major cause of
death. In 1950’s, with the introduction of Penicillin G,
Streptococci were ‘wiped off’ and replaced by
Staphylococcus aureus. Even today, Staphylococcus aureus
remains a common cause of early burn wound infection.
The role of Pseudomonas aeruginosa from the patient’s
endogenous gastrointestinal flora and/or an environmental
source is one of the foremost causes of burn wound

Microbiology in Burns
81
Table 9.1: Bacteria and fungi that constituted burn wound infection:
National Nosocomial Infections Study of the Center for Disease
Control, USA
Species
Percentage of isolates
(1970-74)
(1980-98)
Staphylococcus aureus
22.9
23
Pseudomonas aeruginosa
20.9
19.3
Pseudomonas species
7.2
Escherichia coli
6.7
7.2
Group D streptococci
5.0
11
Streptococcus faecalis
4.2
Klebsiella pneumoniae
3.7
2.6
Serratia marcescens
3.1
3.5
Enterobacter cloacae
3.0
9.6
Proteus mirabilis
2.8
Enterobacter species
2.5
Klebsiella species
2.2
Staphylococcus epidermidis
1.4
4.3
Group A streptococci
1.1
Enterobacter aerogenes
1.0
Candida albicans
1.3
3.5
Others
16
infections in many centers.27 The incidence of rare infective
pathogens has also increased steadily28 (Table 9.2). While
less common, infections due to anaerobic bacteria typically
occur secondary to electrical burns or when open wound
dressings are used in place of occlusive dressings.29
The worldwide emergence of antimicrobial resistance
among a wide variety of bacterial and fungal pathogens,
particularly nosocomial isolates, limits the available
therapeutic options for effective treatment.27 MRSA,
methicillin-resistant Staphylococcus aureus, Vancomycin-
resistant enterococci and multiple resistant gram-negative
bacteria that possess several types of beta-lactamases,
including extended spectrum beta-lactamases, AmpC beta-
lactamases and metallobeta-lactamases have been emerging
as serious pathogens in hospitalized patients.26 Fungal
pathogens, particularly Candida spp., have increasingly
become important opportunistic pathogens due to the use
of broad-spectrum topical and systemic agents when
infection occurs in the burned patient and have demonstrated
increasing degrees of antifungal drug resistance. 30
QUANTITATIVE MICROBIOLOGY: SIGNIFICANCE
OF MICROBIAL NUMBERS
The clinical significance of the microbial load in delaying
wound healing was described in 1964 by Bendy et al31 who
swabbed decubitus ulcers and discovered that bacterial load
of 106 CFU/ml of wound fluid delayed healing. Later tissue
biopsy specimens were demonstrated to be superior
Table 9.2: Microflora of wounds
Aerobic and facultative microorganisms found and the Type of
wound (A-Acute, C- Chronic)
Gram-positive:
• Coagulase-negative staphylococci A, C
• Staphylococcus aureus A, C
• Beta-hemolytic Streptococcus (group C) A,
Beta-hemolytic Streptococcus (group G) C
• Streptococcus spp. (fecal) A,
• Streptococcus spp. (viridans) A, C
• Streptococcus intermedius C
• Corynebacterium xerosis C, Corynebacterium sp. A, C
• Micrococcus sp. C
• Bacillus sp. A
Gram-Negative:
• Escherichia coli A, C Escherichia hermanii A
• Serratia liquefaciens C
• Klebsiella pneumoniae A, C, Klebsiella oxytoca A, C
• Enterobacter cloacae A, C, Enterobacter aerogenes C
• Citrobacter freundii C
• Proteus mirabilis A, C,Proteus vulgaris C
• Providencia stuartii A
• Morganella morganii C
• Acinetobacter calcoaceticus A, C
• Pseudomonas aeruginosa A, C
• Stenotrophomonas maltophilia A
Anaerobic and facultative anaerobic microorganisms isolated from
acute and chronic wounds of varied etiology
• Clostridium limosum A Clostridium bifermentans A Clostridium
difficile C Clostridium sporogenes A, C Clostridium ramosum C
Clostridium tertium A Clostridium histolyticum A, C Clostridium
septicum A Clostridium baratii C Clostridium clostridioforme A, C
Clostridium perfringens A, C Clostridium cadaveris A, C
• Peptostreptococcus asaccharolyticus A, C Peptostreptococcus
anaerobius A, C Peptostreptococcus sp. A, C Peptostreptococcus
micros A, C Peptostreptococcus prevotii A, C Peptostreptococcus
magnus A, C C Peptostreptococcus indolicus C
• Bacteroides thetaiotaomicron C Bacteroides caccae C Bacteroides
capillosus C Bacteroides stercoris C Bacteroides fragilis A, C
Bacteroides uniformis A, C Bacteroides ovatus A Bacteroides
ureolyticus A, C
• Prevotella oralis A, C Prevotella oris A, CPrevotella disiens
APrevotella bivia C Prevotella buccae CPrevotella sp. A Prevotella
corporis A, CPrevotella intermedia A Prevotella melaninogenica C
• Porphyromonas asaccharolytica A, C
• Propionibacterium acnes A, C
• Sphingobacterium multivorum C
• Eubacterium limosum C
• Fusobacterium necrophorum C
• Veillonella spp. A
Fungi
• Candida parapsilosis A,
• Candida krusei A
samples.32 Quantitative culture of tissue biopsy specimens
is demanding on the microbiology laboratory and of minimal
value in facilitating prompt burn wound management. It
has been observed that a rapid Gram stain can reliably predict
a microbial load of 105 CFU/g of tissue if a single

82
Principles and Practice of Burn Care
microorganism was seen on the slide preparation.33,34 More
recently it has been35 demonstrated that a critical level of
bacteria of >104 CFU/g of tissue must be reached to cause
infection in wounds and that quantitative tissue cultures
can predict the likelihood of wound infection more effectively
than swab cultures. Some authors prioritize histopathology
over all other methods as it demonstrates true invasion. 36
Another technique which has been utilized is the velvet pad
surface imprint technique. Dermabrasion as a sampling
method has also been used.
The two most popular methods have been quantitative
studies on the basis of surface sampling techniques and
deep-tissue biopsy techniques. The value of superficial
cultures in wound assessment has been questioned and it is
stated that purulent wound fluid may fail to yield growth
whereas biopsied tissue may yield significant numbers of
bacteria.37 It has also been stated that superficial tissue is
likely to harbor a diversity of aerobic and anaerobic
microorganisms, one or more of which may invade deeper
tissue. A situation where the superficial tissue is “sterile”
while deeper tissue is “infected” is very improbable. Most
wounds are colonized with microorganisms and a failure to
isolate them is more likely to be a consequence of poor
microbiological technique. Some workers have stated that38
quantitative bacteriology for burn wounds provides little
information beyond that obtained from a surface swab and
swabs that yield more than 30 CFU reliably indicate a tissue
count of 105 CFU/g. The need to quantify bacteria by
performing tissue biopsy, which is invasive, potentially
traumatic to the patient and non-routine, requiring expert
manipulation and creates an increased workload for the
microbiology laboratory, is debatable. The role of tissue
biopsies has retained significance in detection of fungi,
anaerobes and other relatively rare pathogens.
SIGNIFICANCE OF SPECIFIC
MICROORGANISMS
The role of specific types of microorganisms on progression
and healing of burn wounds has been studied extensively.
Presence of microorganisms and their identity as the
etiological agent in infection or delayed healing can be
confirmed only if they are present as a pure mono-microbial
flora. Burn wounds tend to be of polymicrobial etiology
involving both aerobes and anaerobes. It is not possible to
differentiate between pathogenic and nonpathogenic species
in polymicrobially infected wounds and the presence of a
pathogen in a mixed-culture infection does not necessarily
establish that particular microorganism as the etiologic agent.
The diagnosis of infection in polymicrobially infected wounds
should be based primarily on clinical signs, such as heat,
pain, erythema, edema, suppuration and fever; microbio-
logical results may be helpful but can often be misleading.
However, when clinical signs of infection are less evident,
greater emphasis may have to be placed on microbiological
results. Aerobic pathogens such as S. aureus, P. aeruginosa
and Gram-negative bacteria etc. have been frequently
implicated as the cause of delayed wound healing and
infection. S. aureus is considered to be the most problematic
bacterium in burn wound infections, primarily based on the
knowledge that its incidence is high and complications tend
to be higher.39, 40 A consensus was reached in a meeting of
the European Tissue Repair Society and the European Wound
Management Association that the presence of beta-hemolytic
(group A) streptococci or P. aeruginosa in a chronic wound
was an urgent indicator for initiating antimicrobial
therapy.41,42
A symbiotic environment develops in long standing burn
wounds as observed in the association of aerobic and
anaerobic microorganisms. It has been observed that (i)
oxygen consumption by aerobic bacteria induces tissue
hypoxia and a lowering of the redox potential, which favors
the growth of anaerobic bacteria, (ii) specific nutrients
produced by one bacterium may encourage the growth of
fastidious and potentially pathogenic cohabiting
microorganisms. Thus, S. aureus promotes the growth of a
vitamin K-dependent strain of Prevotella melaninogenica
in vitro43 and the pathogenicity of P. melaninogenica has
been related to the presence of vitamin K. K. pneumoniae
was reported to be instrumental in enhancing virulence in
P. melaninogenica by providing succinate as an essential
growth factor, and (iii) some anaerobes are able to impair
host immune cell function and thus provide a competitive
advantage to themselves and other cohabiting
microorganisms. Antiphagocytic activity in P.
melaninogenica was expressed only in the presence of hemin
and vitamin K provided by microbes like S.aureus. Succinate
also inhibits the chemotaxis and phagocytosis of E. coli
and S.aureus.
In theory, the presence of S. aureus and a pigmenting
gram-negative anaerobe in a wound is likely to be more
detrimental than the presence of a similar quantity of S.
aureus alone. Therefore, the composition of the
polymicrobial wound flora is likely to be more important
than the presence of specific pathogens, since this will

Microbiology in Burns
83
determine whether pathogenic interactions are likely to
occur. Another critical factor in wound healing which cannot
be underestimated is immune response in dealing with
wound micro flora. Factors such as tissue necrosis, hypoxia
and ischemia impair immune cell activity in a wound, as do
debilitating conditions like diabetes mellitus, chronic
granulomatous disease, other immune deficiencies and
microorganisms can also participate in compromising the
immune response. Only by assessing the host and microbial
factors collectively, along with symptomatology and clinical
signs of the patient can the problem of burn wound infection
be addressed.
BIOLOGICAL ANALYSIS OF WOUNDS
There is little consensus whether sampling is relevant, when
and how a wound should be sampled, how a specimen
should be transported to the laboratory and what analyses
should be requested. Confusion also exists in view of the
fact that health care practitioners often consider a
microbiological report to provide definitive information on
whether a wound is infected 44 and the provision of an
antibiogram for a particular pathogen can often be misleading
and prompt unnecessary treatment. However, having stated
this it still remains critical that the burn wounds be assessed
clinically, microbiologically and histopathologically as stated
earlier in this chapter. It can predict worsening infection,
internal spread and the data generated is extremely vital in
terms of hospital infection control.
Wound-sampling Methods
In generalized terms, the quantitative and qualitative
microbiology of wounds can be investigated by sampling
either wound tissue or wound fluid. The fluid is usually
swabbed and the tissue biopsies are performed.
Wound Tissue Sampling
The acquisition of deep tissue biopsy following initial
debridement and cleansing of superficial debris is the most
useful method for determining the microbial load and the
presence of invasive pathogens. Tissue is obtained aseptically
and processed by weighing, homogenizing, serially diluting
and plating onto selective and nonselective media. These
are then incubated under aerobic and anaerobic conditions
to provide quantitative and qualitative information.
Superficial, devitalized tissue removed by curettage, may
also be investigated for microbial content. Another technique
involving dermabrasion is less invasive as compared to the
biopsy.45 Multiple samples from several areas of the burn
wound should be obtained and sent for workup. After
cleaning the wound with isopropyl alcohol, 2 parallel incisions
1-2 cm in length and 1.5 cm apart with a depth to obtain a
portion of the underlying fat are made in the skin. These are
similar to split skin smears. Biopsies 0.02-0.5 g may be
obtained with a 3 mm punch-biopsy technique. This is a
technically demanding technique.
Histopathological evaluation can detect bacteria, fungus
(periodic acid-Schiff [PAS] and Gomori methenamine silver
[GMS]), although cultures must be obtained to definitively
identify and give sensitivity of the pathogen. Viruses like
the Herpes simplex virus (Giemsa) can be associated with
wounds and are isolated via identification of inclusions on
light or electron microscopy. Histology also guides in the
staging of the burn wounds.46,47 The various stages used
to diagnose burn wound infections are as follows:
•
Stage I—Colonization
a. Superficial—Microorganisms present only on burn
wound surface
b. Penetrating—Variable depth of microbial penetration
of eschar
c. Proliferating—Variable level of microbial proliferation
at nonviable–viable tissue interface (subeschar space)
•
Stage II—Invasion
a. Micro-invasion—Microorganisms present in viable
tissue immediately subjacent to subeschar space
•
Stage III—Deep invasion
a. Penetration of microorganisms to variable depth and
expanse within viable subcutaneous tissues.
b. Micro-vascular involvement: Microorganisms within
small blood vessels and lymphatics (thrombosis of
vessels is common).
Wound Fluid Sampling
Wound swabs are taken using cotton-tipped swabs to
sample superficial wound fluid and tissue-debris. When a
copious volume of burn wound fluid exists, sampling by
needle aspiration can be employed. This is superior to
swabbing. The syringe technique may be used to sample
deeper pockets of fluid. If the technique is performed with
strict asepsis samples can be retrieved without any
significant exogenous contamination. In pus containing
cavity, irrigation with sterile saline and gentle massaging
may be performed to provide fluid for aspiration.

84
Principles and Practice of Burn Care
An alginate-tipped swab can also be used to perform a
fully quantitative analysis, since the swab will dissolve and
release all associated microorganisms when transferred to
an appropriate diluent. Despite its widespread use, there is
debate over the value of the swab sampling technique and
the value of cleansing a wound before swabbing is
performed. A variety of other techniques, including the dry
and presoaked velvet pad, filter paper disks and cylinder
scrubbing, have also been used to sample superficial wound
fluid for microbiological analysis. The immense number of
sampling methods available creates a problem since all are
reputed to have benefits and there is no single, universally
accepted method. Thus, the debate and controversy
continues regarding the type of sample required to provide
the most meaningful data.41,45,48
The results generated by the microbiology laboratory
may be misinterpreted and encourage inappropriate
treatment. Without question, tissue biopsy or excision is
the gold standard for microbial conclusive evidence. This
method is most beneficial in determining the optimal time
for skin grafting and surgical wound closure.49 However,
the value of a single biopsy specimen, particularly in chronic
wounds, is debatable. Particularly in slow-healing chronic
wounds that require long-term care, tissue biopsy is of
minimal value since it requires expert surgical technique, is
non-routine and is potentially traumatic to the patient.
Aspirates of purulent fluid are excellent representatives of
the wound infection. The role of superficial swab samples
has been intensely debated but the procedure is simple,
inexpensive, noninvasive and convenient for the majority
of wounds. Swab sampling has been challenged on the basis
that the superficial colonizers do not reflect the organisms
of deeper tissue and that subsequent cultures do not correlate
with the presence of pathogenic bacteria.48 The collection
of the swab should be meticulous, if taken prior to wound
cleansing or the removal of devitalized superficial debris,
the resulting culture will only have superficial colonizers
and provide misleading or useless information. Vital tissue
must be sampled or a specimen should not be collected at
all and the patient should be treated empirically.50
Another theory has it that since the majority of wounds
are contaminated with endogenous microorganisms, any
flora present in deeper tissues are also likely to be present in
the superficial debris. Thus, there are arguments to support
the use of the swab sample as a useful method. For routine
management, perhaps it can be suggested that only wounds
that are clinically infected or those that have no clinical
signs of infection but are deteriorating or have a long history
of failure to heal should be sampled for microbiological
analysis. However, indiscriminate and inappropriate
swabbing of wounds causes an unnecessary drain on labor
and financial resources. An important factor to consider
when sampling a wound for microorganisms is the
administration and route of antimicrobial therapy. If a patient
is receiving treatment, microbial isolation from swab samples
is likely to be significantly influenced by topical antimicrobial
agents whereas the microflora of deep tissues is more likely
to be influenced by systemic antibiotic therapy.
In summary, superficial swabbing can be justified as a
simple procedure for assessing the microflora of wounds
that are clinically infected or failing to heal. Semi-quantitative
analysis will provide a good indication of the microbial load
and the qualitative analysis, although complex will provide
an indication of the diversity of microorganisms and potential
for microbial synergy. A request for both aerobic and
anaerobic microbiological investigation is an important
aspect of obtaining the information required to guide
appropriate antibiotic therapy.
Sample Transport
The prompt delivery of the specimen to the laboratory is
considered to be of utmost importance. This is particularly
true for swabs which are susceptible to drying up. Aspirates
of purulent fluid and tissue samples are preferable to swabs
in these circumstances because they maintain the conditions
required to sustain microbial viability, i.e. a moist and reduced
environment. There are prereduced commercially available
transport media which can be used if culture is delayed
beyond 1 to 2 hour after sampling. This offers a cheap and
effective method to enable the culture of both aerobic and
anaerobic microorganisms. Since swab samples are
susceptible to desiccation and oxygen exposure, a
prereduced, nonnutritive transport medium is essential to
maintain the viability of microorganisms on cotton swabs.
Storage at room temperature, 25oC, is considered to be
appropriate for the maintenance of most microorganisms;
temperatures above or below this may cause inhibition or
death of some flora.
Processing of Wound Specimens
On arrival at the laboratory, a specimen must be accompanied
by a clinical description of the burn wound. Information
regarding the type/degree of wound, the position of the

Microbiology in Burns
85
wound, clinical signs of infection, presence of necrosis,
associated malodor, and antimicrobial therapy. All these
greatly assist the microbiologist in appropriate selection of
tests and accurate interpretation of the results of the
laboratory data. Also, the provision of information regarding
current antibiotic treatment may aid microbiologist in
determining which antimicrobials have become less useful
for the microorganisms. Microbial culture and antibiotic
sensitivity results are usually generated in about 48-72 hours
(and may, on occasion, take considerably longer) when using
manual methods.
A number of rapid investigations must be considered.
Direct Gram stain of the swab/pus/biopsy is one of the
most reasonable and effective rapid tests in the microbiology
department which has stood the test of time, for over a
century, Gram’s stain is still the most important stain in
microbiology.52 It is widely used as a rapid technique for
guiding antibiotic therapy in life-threatening infections (Figs
9.1 and 9.2). Also, the presence of microorganisms in a
Gram stained smear prepared from a wound swab has been
shown to consistently reflect a microbial load of >106
organisms isolated by a quantitative swab technique from
open burn wounds. Speciation on the basis of Gram’s stain
is possible only for gram-positive spore-forming anaerobes
such as Clostridium perfringens. However, the value of the
Gram stain as a final diagnostic tool is debatable as
contaminant, colonizer and true pathogen cannot be
differentiated. Its use in the evaluation of samples with risk
of contamination with skin/mucosal micro flora has been
reported as insufficiently reliable to guide antibiotic selection.
Gram staining of tissue biopsy specimen homogenate has
often been used to rapidly estimate the microbial load.53
The combined presence of leukocytes and bacteria is likely
to be a good indicator of infection.55 Having said that, Gram
stain remains a reliable, cheap, easily available and
standardized rapid method to diagnose wound infections,
particularly with single pathogen.
Other stains which have potential in assessing burn
wounds are the fungal stains. Potassium Hydroxide (KOH)
is used to dissolve keratin and highlight fungal elements in
wet smears. It has a great role to play in nail, hair and skin
samples, although the role in burn wounds is limited.
Lactophenol Cotton blue preparation is an excellent and cheap
stain used for staining and fixing fungal elements, which
are very clearly delineated using this method.
Histopathological tissue can be fixed and stained with
Periodic acid Schiff’s base (PAS) which specifically stains
and demarcates the only fungal tissue. More expensive stains
as the fluorescent Calcuofluor white stain, immuno-
peroxidase and other immunohistochemical stains are used
in specialized set-up. Giemsa has been used successfully to
demonstrate inclusion bodies of viruses like herpes and also
in mycology.
Culture and Sensitivity of Wound Specimens
Routine isolation analysis of wound specimens normally
involves the use of selective and nonselective media to culture
aerobic, anaerobic bacteria and yeasts (Fig. 9.3). If a
Fig. 9.1: Gram stain of pus from wound showing polymorphs and
gram-positive and negative bacteria
Fig. 9.2: Pus cells with streptococci in chains