Hypertrophic Scar Following Burn Injury - American Burn Association

Hypertrophic Scar following Burn Injury
Analysis of the Major Unanswered Questions
Edward E. Tredget, MD, MSc, FRCSC
Professor, Department of Surgery
Firefighters’ Burn Treatment Unit
Senior Scholar, Alberta Heritage Foundation for Medical Research
Plastic Surgery Research Laboratory
University of Alberta
Edmonton, Alberta, Canada

Unresolved Features of Hypertrophic Scar
following Thermal Injury
• definition of HTS vs normal skin, mature
scar and keloid
• diagnosis and scar severity
• prevalence and socioeconomic impact
• pathophysiology
• treatment
• future directions

How do you measure the severity of
HTS and response to treatment
• clinical observations
• Vancouver Burn Scar Scale (VBSS)
• scar volume
• photography
• scar color and vascularity
• scar pliability and viscoelastic properties
• ultrasound

• subjective ratings of
pigmentation, vascularity,
pliability, height
• inter-rater rater reliability of 0.5-0.8
0.8
• correlated with subjective
assessment at 1.5 years post-
injury but not at 3 months
Nedelec B et al. JBCR 2000

1. Baryza MJ et al. The Vancouver Scar Scale: An
administration tool and its interrater reliably.Journal of
Burn Care and Rehabilitation 1995, 16: 535-538.
538.
2. Sullivan T et al. Rating the burn scar. Journal of Burn
Care and Rehabilitation 1990, 11: 256-260.
260.
3. Nedelec et al. Rating the resolving bur hypertrophic scar:
Comparison of the Vancouver Scar Scale and scar volume.
Journal of Burn Care and Rehabilitation 2000, 21: 205-
212.
4. Draaijers LJ et al. The patient and observer scar
assessment scale: A reliable and feasible tool for scar
evaluation. Plastic and Reconstructive Surgery 2004, 113:
1960-1965.
1965.

Major Question regarding HTS
What is the most objective, sensitive, specific
and universally available measure to
quantitate the severity of HTS and its
response to treatment

Materials and Methods
Outcome measurements included:
– scar assessment using VBSS and rVBSS
– skin pliability and elasticity using the
Cutometer
– skin vascularity and melanin level using the
Mexameter
– standardized photography

2
4
6
8
10
A
Mexameter
color
800
700
600
500
400
300
200
100
0
Melanin
*
Erythema
Normal
Donor
C
Cutometer
elasticity
1.2
1
0.8
0.6
0.4
*
Normal
Mature
HSc
0.2
0
*
r0 r2 r3
*
B
300
250
Serum TGF-ß (ng ng/ml)
200
150
100
50
Figure
0
0
Pre Rx Early Rx Late Rx Post Rx Control

1. Cheng W et al. Ultrasound assessment of scald scars in
Asian children receiving pressure garment therapy.
Journal of Pediatric Surgery 2001, 36: 466-469.
469.
2. Davey RB et al. Computerized color: A technique for the
assessment of burn scar hypertrophy. Burns 1999, 25:
207-213.
213.
3. Li-Tsang CW et al. Validation of an objective scar
pigmentation measurement by using a
spectrocolorimeter. . Burns 2003, 29:779-784.
784.
4. Martin D et al. Changes in subjective vs objective burn
scar assessment: Does the patient agree with what we
think Journal of Burn Care and Rehabilitation 2003, 24:
239-244. 244. Discussion 238.

Is it possible to objectively assess the burn
wound to determine if HTS will develop
Riordon CL et al. JBCR 2003

Pathophysiology of HTS Formation
• What are the factors locally within the burn
wound that are important in the development
of HTS
• What role does the systemic response to
injury play in the development of HTS

HEMOSTASIS
WOUND
Macrophages
Neutrophils
Fibrin
Platelets
PROLIFERATION
Lymphocytes
INFLAMMATION
Fibroblasts
Proteoglycans
REMODELING
Endothelium
Epithelium
Collagen Deposition
Scar Maturation
Collagen Fibril Crosslinking
Hypertrophic Scar
Time after Injury

What are the important features of fibroblasts in hypertrophic scar s
Collagenase activity
Collagenase production
Decorin
Apoptosis
Fibroblast density
Fibronectin production
Type I / III collagen production
TGF-β
Versican
Biglycan
Synthesis
Degradation
ECM
Remodeling

Collagenase Expression in Fibroblasts from
Different Layers of the Dermis

What are the important fibrogenic growth
factors in HTS
• TGF- β
b1, b2, b3
• CTGF
• IL-4
• IL-13
• PDGF
• others

What is the role of T cells in HTS
and how do we control their effects
1. Dunsmore SE et al. The bone marrow leaves its scar: New
concepts in pulmonary fibrosis. The Journal of Clinical
Investigations 2004, 113: 180-182.
182.
2. Tredget EE et al. Polarized T helper cells Th2 cytokine
production in patients with hypertrophic scar following
thermal injury. Journal of Interferon & Cytokine Research
2006, 26:179-189.
189.
3. Yang L et al. Identification of fibrocytes in post-burn
hypertrophic scar. Wound Repair and Regeneration 2005,
13:398-404.

Native CD4+
T cell
IL-2
IL-12
IFN-γ
IL-2; IL-4
IL-4
IL-10
(TGF-β)
T H 1
T H 2
IFN-γ
IFN-γ
IL-2
IL-4
IL-5
IL-6
IL-10
pT H
T H 3
IL-4
Anti-IL
IL-12
mAb
IL-12
IFN-γ
TGF-β
IL-4
IL-10
Letterio JL et al, Ann Rev Imm, , 1998

Percentage of Lymphocytes Producing
IFN-γ and IL-4
A
IL-4 Percent positive cells(%)
20
15
10
5
* *
*
0
0.25 0.5 0.75 1 2 6 12 12+
B
IFN Percent positive cells (%)
20
15
10
5
0
*p

Tredget et al, J Interferon Cytokine Res, 2006
Time Course of IL-10 and IL-12 from PBMC
ex vivo in HSC and non-HSc Patients
A
IL-10 pg / ml
16
12
8
+
*
*
HSc
noHSc
4
B
0
16
1 2 6 12 12+
* p

CD4/TGF-β Staining in Hypertrophic Scar
→ CD4 +
→ TGF-β +
→ Double ++

Increased CD4+/TGF-β+ + T
Lymphocytes in Burn Patients
% of CD4+TGF-β T cells
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
∗
∗
∗
% of CD4+TGF-β T cells
∗
Normal

Increased CD4+/TGF-β+ + T Lymphocytes in
Hypertrophic Scar
35
∗
∗
p

1. Murphy TJ et al. CD4+CD25+ regulatory
T cells control innate immune reactivity
after injury. J Immunol 2005, 174: 2957-
2963.
2. Choileain NN et al. Enhanced regulatory
T cell activity is an element of the host
response to injury. The Journal of
Immunology 2006, 176: 225-236
236

Morphology of BLM-Induced
Lung Fibrosis
Hashimoto N et al, JCI, 2004

Dunsmore et al, JCI 2004
What is the role of bone marrow hematopoietic stem
cells and mesenchymal stem cells in HTS

Immunostaining for Type I Collagen
A B C
A. Fibroblasts B. Fibrocytes C. Fibrocytes with
non-immune IgG

Day 0
Femur
Day 3
Day 5

Chesney J et al, Proc Natl Acad Sci USA 1997
Peripheral Blood Fibrocytes
h have fibroblast-like like properties:
– spindle shaped cells
– produce types I and III collagen and fibronectin
h display hemotopoietic cell features: CD34 +
h have antigen-presenting ability
h can rapidly enter subcutaneously implanted
wound chambers in mice
h present in scar tissue

Identification of LSP-1 1 in Fibrocytes by 2D
SDS-PAGE of Cell Proteins

Fibrocytes in HSc from Burn Patients treated with IFN α-2b
LSP-1
Procollagen-1
DAPI
Merge
Normal Skin
HSc
INFα-2b Treatment

Quantitation of Fibrocytes in HSc Tissue
before and after IFN α−2b Treatment
40
35
30
HTS
Normal Skin
25
20
15
10
5
0
0 Months 2 Months 4 Months 6 Months 8 Months

What are the important inflammatory cytokines and receptors in HTS H
Normal
Burn
Gene Name Description Burn/Normal
Ratio
MCP-3 #77 Homo Sapiens mRNA for monocyte chemotactic protein-3 0.9/0.3 2.9
IL-1R2 #37 Interleukin-1 receptor type II 0.9/0.3 2.7
MPIF-2 #72 Small inducible cytokine subfamily A (Cy5-Cy5), member 24 1.0/0.4 2.7
MCP-2 #78 Small inducible cytokine subfamily A (Cy5-Cy5), member 8 1.3/0.6 2.2
(monocyte chemotactic protein 2)
ENA-78 #82 Small inducible cytokine subfamily B (Cy50Cy5), member 5 0.0/0.7
GCP-2 #83 Human chemokine alpha 3 (CKA-3) mRNA 0.0/0.2
CCR1 #2 Chemokine (C-C motif) receptor 1 1.0/0.7 1.4
IL-11Ra #19 Interleukin-11 receptor, alpha 0.1/0.1 1.5
HCC-1 #61 Small inducible cytokine subfamily A (Cy5-Cy5), member 14 0.3/0.2 1.4
MIP-1 delta #62 Small inducible cytokine subfamily A (Cy5-Cy5), member 15 0.6/0.5 1.3
PARC#65 Small inducible cytokine subfamily A (Cys-Cy5), member 18 0.2/0.2 1.4
Pulmonary and activation-regulated
MCP-1(SCYA2) #67 Small inducible cytokine A2 (monocyte chemotactic protein 1) 10/0.8 1.3
IFN-gamma #14 Interferon gamma 1.4/1.9 0.6
CCR4 #5 Chemokine (C-C motif) receptor 4 1.1/1.7 0.6
CXCR4 #13 Chemokine (C-X-C motif) receptor 4 1.0/1.6 0.6
IL21 #40 Homo sapiens interleukin 21 1.2/1.8 0.6
IL2Rβ #43 Interleukin2 receptor beta 0.5/0.9 0.6
TNF-β/Lta #54 Lymphotoxin-alpha (TNF subfamily, member 1) 0.3/0.5 0.5
LT-β #55 Lymphotoxin-beta 0.2/0.5 0.4
LTbR #56 Homosapien lymphotoxin β receptor 0.2/0.6 0.3
I309 #58 Small inducible cytokine A1 1.4/2.1 0.6
SCYC2 #85 Small inducible cytokine subfamily C member 2 0.8/1.4 0.6
Fractalkine #86 Small inducible cytokine subfamily D member 1 0.5/0.9 0.6
SCYE1 #87 Small inducible cytokine subfamily E member 1 0.2/0.8 0.3

What is the role of other potential
important cells in HTS
• mast cells
• mesenchymal stem cells
• endothelial stem cells
• transdifferentiating cells

What is the role of stem cells in injured tissue
and circulating stem cells in HTS
Korbling et al, NEJM 2003

Morphology of Early MSC
Passage 1 murine BM-MSCs
MSCs have large spindle-shaped
shaped
fibroblast-like like cells and small round cells.

sham
ligation of
coronary
artery
dilated and
scarred
ventricle
MS cells
mesenchy
mal stems
cells home
to the site
of injury
regeneration
of 75-80% of
muscle
function

BM-MSCs MSCs in the Wound
Express Cytokeratins
A
B
20
50
C
D
E
F
20

What is the optimal form of
treatment for HTS
1. Mustoe TA et al. International clinical recommendations on scar
management.Plastic and Reconstructive Surgery 2002, 110: 560-571
571
2. Chang P et al. Prospective randomized study of the efficacy of
pressure garment therapy in patients with burns. Journal of Burn
Care and Rehabilitation 1995, 16: 473-475.
475.
3. Kealy GP et al. Prospective randomized comparison of two types of
pressure therapy garments. Journal of Burn Care and Rehabilitation
ion
1990, 11: 334-336.
336.
4. Patino O et al. Massage in hypertrophic scars. Journal of Burn Care
and Rehabilitation 1999, 20:268-271. 271. Discussion 267.
5. Costs AM et al. Mechanical forces induce scar remodeling: Study in
non-pressure
pressure-treated versus pressure-treated hypertrophic scars.
American Journal of Pathology 1999, 155: 1671-1679.
1679.

Sacket DL, Can J Physiol & Pharmacol (1986)
Classification of Level of Evidence
Level
Description
Strength
Level I
Level II
Level III
Level IV
Level V
Large randomized trials with clear cut results
Good
(and low risk of error)
Small randomized trials with uncertain results Fair
(and moderate to risk of error)
Non-randomized, contemporaneous controls
Fair
Non-randomized, historical controls
Fair
No controls, case series only
Poor

Pressure Garment Therapy for
the Prevention of Abnormal
Scarring After Burn Injury: A
Meta-Analysis
A. Anzarut, MD, MSc (1,2,3) P. Singh, BSc (4) B. Rowe, MD, MSc, (2,5) E. Tredget, MD, MSc,
FRCS(C) (1) E. Van den Kerckove (6) J. Olson, MD, FRCS(C) (1)
From the Division of Plastic and Reconstructive Surgery (1), Department of Public Health Sciences (2),
EPICORE Centre (3), Faculty of Medicine and Dentistry (4), and Department D
of Emergency Medicine
(5), all at the Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta; and the
Department of Rehabilitation Sciences, Katholieke University, Leuven, Belgium (6).

Pressure Garment Therapy (PGT)
Morbidity of PGT:
– unattractive
– over-heating
– pruritis
– wound breakdown
– abnormal bone growth
(Johnson, Journal of Burn Care and Rehab, , 1994)
(Fricke, Journal of Burn Care and Rehab, , 1999)
Costs of PGT:
> $ 100,000 / year

Characteristics of included trials
Study
Study design
Mean burn size
(%TBSA)
Co-interventions
Control
Groce 2000b
Within
patient
48.3 (11-99)
Not reported
Low pressure
garment
Tredget
Within
patient
10.4 (6.9) None
None
Moore 2000
Within
patient
Not reported
Not reported
Low pressure
garment
Chang 1995
Between
patient
21.1 (15.8) Not reported
None
Groce 2000a
Between
patient
11.2 (1-30)
Not reported
None
Van den
Kerckhove
2005
Between
patient
8.5 (1-30)
Not reported
Low pressure
garment

Results – Global scar score
Pooled estimate: -0.46 (-1.07, 0.16)

What is the best animal models of
HTS to assess new treatments
• red duroc pig
• transgenic mice
– TGF-b, IL-4, IL-13,
Smad 7 KO, others
• animals with HTS grafts
• bleomycin model in mice
• Human controlled incisions (coma shaped
wounds)

1. Gallant-Behm
CL. et al. Cytokine and growth factor mRNA expression patterns
associated with the hypercontracted, hyperpigmented healing phenotype of red duroc
pigs: a model of abnormal human scar development Journal of Cutaneous Medicine
& Surgery. 9:165-77, 2005.
2. Lee JP. et al. Antifibrogenic effects of liposome-encapsulated encapsulated IFN-alpha2b cream on
skin wounds in a fibrotic rabbit ear model. Journal of Interferon n & Cytokine
Research. 25(10):627-31, 2005.
3. Liang Z. Engrav LH. Muangman P. Muffley LA. Zhu KQ. Carrougher GJ.
Underwood RA. Gibran NS. Nerve quantification in female red Duroc pig (FRDP)
scar compared to human hypertrophic scar. Burns. 30:57-64, 2004.
4. Zhu KQ. et al. The female, red Duroc pig as an animal model of hypertrophic
scarring and the potential role of the cones of skin. [Journal Article] A
Burns.
29(7):649-64, 64, 2003
5. Hillmer MP, et al. MacLeod SM. Experimental keloid scar models: a review of
methodological issues. Journal of Cutaneous Medicine & Surgery. 6:354-9, 2002.
6. Polo M, et al. An in vivo model of human proliferative scar. Journal of Surgical
Research. 74:187-95, 1998.

What are useful antifibrogenic
agents for HTS
• antagonists of TGF- β
• Interferon α, β, γ
• Bleomycin
• 5-fluorouracil
• others

Acknowledgements
Firefighter’s s Burn Trust Fund
Canadian Institute for Health Research
Alberta Heritage Foundation for Medical Research

systemic
circulation
bone marrow
T T
T
T T
T
T
cytokines
PBMC / fibrocytes
myofibroblasts
_
TH 2
TH 1
_
IFN γ
IL-10, IL-4
TGF-β
_
+
+
wound
wound contraction
TH 3
extracellular matrix
synthesis / degradation
Figure
fibroblasts

Prevalence of HTS
• What is the frequency of HTS following burn injury
• How large is the socioeconomic impact of HTS
• Who will is more likely to develop HTS given similar
severity of initial injury
• What is the role of the severity of injury in the
development of HTS in terms of depth of burn and total
body surface area involved
• How does age, sex, racial background, and treatment of the
acute injury affect the development of HTS
• What is the psychological impact of HTS to the surviving
burn patient

The most common
complication for burn
survivors is abnormal scarring
(Bombaro, Burns, , 2003)
30-70% of burn patients
develop abnormal scars
(Bombaro, Burns, , 2003)

1. Deitch EA et al. Hypertrophic burn scars: Analysis of
variables. Journal of Trauma 1983, 23: 895-898.
898.
2. Dedovic Z et al. Time trends in incidence of hypertrophic
scarring in children treated for burns. Acta chirurgica
plastique 1999, 41: 87-90.
3. McDonald WS et al. Hypertrophic skin grafts in burned
patients: A prospective analysis of variables. Journal of
Trauma 1987, 27: 147-150.
150.
4. Spurr ED et al. Incidence of hypertrophic scarring in burn-
injured children. Burns 1990, 16: 179-181.
181.
5. Bombaro KM et al. What is the prevalence of
hypertrophic scarring following burns Burns 2003, 29:
299-302.