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 reliability of 0.5-0.8 correlated with subjective assessment at 1.5 years postinjury 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. 2. Sullivan T et al. Rating the burn scar. Journal of Burn Care and Rehabilitation 1990, 11: 256-260. 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: 205212. 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.

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

A
Mexameter color

800 700 600 500 400 300 200 100 0 Melanin

C
Normal Donor

Erythema

1.2

Cutometer elasticity

1 0.8 0.6 0.4 0.2 0 r0 r2 r3 * * * Normal Mature HSc

B
TGF- (ng ng/ml) /ml) Serum TGF

300 250 200 150 100 50 0

0 2 4 6 8 10

Figure

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. 2. Davey RB et al. Computerized color: A technique for the assessment of burn scar hypertrophy. Burns 1999, 25: 207-213. 3. Li-Tsang CW et al. Validation of an objective scar pigmentation measurement by using a spectrocolorimeter. Burns 2003, 29:779-784. 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. 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?

Scar Maturation Collagen Fibril Crosslinking

REMODELING
Endothelium Epithelium Collagen Deposition Fibroblasts Proteoglycans

PROLIFERATION
Lymphocytes Macrophages Neutrophils

INFLAMMATION
Fibrin Platelets

Hypertrophic Scar

HEMOSTASIS WOUND

Time after Injury

What are the important features of fibroblasts in hypertrophic scar? Collagenase activity Collagenase production Decorin Apoptosis Fibroblast density Fibronectin production Type I / III collagen production TGF- 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-
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. 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. 3. Yang L et al. Identification of fibrocytes in post-burn hypertrophic scar. Wound Repair and Regeneration 2005, 13:398-404.

IL-2 IL-12 IFN- Native CD4+ T cell IL-4 IL-10 (TGF-) IL-2; IL-4

TH1
IFN-

IFN- IL-2

pTH

TH2

IL-4 IL-5 IL-6 IL-10

IL-4 Anti-IL-12 mAb IL-12 IFN-

?
TH3
TGF- IL-4 IL-10
Letterio JL et al, Ann Rev Imm, 1998

Percentage of Lymphocytes Producing IFN- and IL-4

IL-4 Percent positive cells(%)

20

15

10

0 0.25 0.5 0.75 1 2 6 12 12+

IFN Percent positive cells (%)

*p<0.05 vs normal mean

20

15

10

*
5

*
0.25 0.5

*
0.75

* *
1 2 6 12 12+ Time post-burn (months)

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 4 0 1 2 6 12 12+

* *
HSc noHSc

B
IL-12 pg / ml

* p<0.05 vs normal mean + p<0.05 HSc vs noHSc

16 12 HSc 8 4 0 noHSc

*
1

6 Time post-burn (months)

12

12+

Tredget et al, J Interferon Cytokine Res, 2006

CD4/TGF- Staining in Hypertrophic Scar

CD4 + TGF- + Double ++

Increased CD4+/TGF-+ T Lymphocytes in Burn Patients

60.00% 50.00%

% of CD4+TGF- T cells

40.00% 30.00%

% of CD4+TGF- T cells
20.00% 10.00% 0.00%

Normal

<6d

7-14d

15-21d

22d-1m

2-3m

3-5m

Increased CD4+/TGF-+ T Lymphocytes in Hypertrophic Scar

p<0.00001

35 30 Cells/0.05mm2 25 20 15 10 5 0 HTS

CD4 CD4TGF-

MS

Skin

1. Murphy TJ et al. CD4+CD25+ regulatory T cells control innate immune reactivity after injury. J Immunol 2005, 174: 29572963. 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

Morphology of BLM-Induced Lung Fibrosis

Hashimoto N et al, JCI, 2004

What is the role of bone marrow hematopoietic stem cells and mesenchymal stem cells in HTS?

Dunsmore et al, JCI 2004

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

Peripheral Blood Fibrocytes

h have

fibroblast-like properties:

spindle shaped cells produce types I and III collagen and fibronectin

hemotopoietic cell features: CD34+ h have antigen-presenting ability h can rapidly enter subcutaneously implanted wound chambers in mice h present in scar tissue
h display
Chesney J et al, Proc Natl Acad Sci USA 1997

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

Fibrocytes in HSc from Burn Patients treated with IFN -2b

LSP-1 Normal Skin Procollagen-1 DAPI Merge

HSc

INF-2b Treatment

Quantitation of Fibrocytes in HSc Tissue before and after IFN 2b Treatment

40 35 30 25 20 15 10 5 0 0 Months 2 Months 4 Months 6 Months 8 Months

HTS Normal Skin

What are the important inflammatory cytokines and receptors in HTS ?

Normal Burn

Gene Name MCP-3 #77 IL-1R2 #37 MPIF-2 #72 MCP-2 #78 ENA-78 #82 GCP-2 #83 CCR1 #2 IL-11Ra #19 HCC-1 #61 MIP-1 delta #62 PARC#65 MCP-1(SCYA2) #67 IFN-gamma #14 CCR4 #5 CXCR4 #13 IL21 #40 IL2R #43 TNF-/Lta #54 LT- #55 LTbR #56 I309 #58 SCYC2 #85 Fractalkine #86 SCYE1 #87

Description Ratio Homo Sapiens mRNA for monocyte chemotactic protein-3 Interleukin-1 receptor type II Small inducible cytokine subfamily A (Cy5-Cy5), member 24 Small inducible cytokine subfamily A (Cy5-Cy5), member 8 (monocyte chemotactic protein 2) Small inducible cytokine subfamily B (Cy50Cy5), member 5 Human chemokine alpha 3 (CKA-3) mRNA Chemokine (C-C motif) receptor 1 Interleukin-11 receptor, alpha Small inducible cytokine subfamily A (Cy5-Cy5), member 14 Small inducible cytokine subfamily A (Cy5-Cy5), member 15 Small inducible cytokine subfamily A (Cys-Cy5), member 18 Pulmonary and activation-regulated Small inducible cytokine A2 (monocyte chemotactic protein 1) Interferon gamma Chemokine (C-C motif) receptor 4 Chemokine (C-X-C motif) receptor 4 Homo sapiens interleukin 21 Interleukin2 receptor beta Lymphotoxin-alpha (TNF subfamily, member 1) Lymphotoxin-beta Homosapien lymphotoxin receptor Small inducible cytokine A1 Small inducible cytokine subfamily C member 2 Small inducible cytokine subfamily D member 1 Small inducible cytokine subfamily E member 1

Burn/Normal 0.9/0.3 0.9/0.3 1.0/0.4 1.3/0.6 0.0/0.7 0.0/0.2 1.0/0.7 0.1/0.1 0.3/0.2 0.6/0.5 0.2/0.2 10/0.8 1.4/1.9 1.1/1.7 1.0/1.6 1.2/1.8 0.5/0.9 0.3/0.5 0.2/0.5 0.2/0.6 1.4/2.1 0.8/1.4 0.5/0.9 0.2/0.8 2.9 2.7 2.7 2.2

1.4 1.5 1.4 1.3 1.4 1.3 0.6 0.6 0.6 0.6 0.6 0.5 0.4 0.3 0.6 0.6 0.6 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 have large spindle-shaped fibroblast-like cells and small round cells.

am h s

ligation of coronary artery

lls e c

dilated and scarred ventricle

S M

mesenchy mal stems cells home to the site of injury

regeneration of 75-80% of muscle function

BM-MSCs in the Wound Express Cytokeratins

20 50

20

What is the optimal form of treatment for HTS?

1. 2. 3. 4. 5. Mustoe TA et al. International clinical recommendations on scar management.Plastic and Reconstructive Surgery 2002, 110: 560-571 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. Kealy GP et al. Prospective randomized comparison of two types of pressure therapy garments. Journal of Burn Care and Rehabilitation 1990, 11: 334-336. Patino O et al. Massage in hypertrophic scars. Journal of Burn Care and Rehabilitation 1999, 20:268-271. Discussion 267. Costs AM et al. Mechanical forces induce scar remodeling: Study in non-pressure-treated versus pressure-treated hypertrophic scars. American Journal of Pathology 1999, 155: 1671-1679.

Classification of Level of Evidence

Level
Level I

Description

Strength
Good Fair Fair Fair Poor

Large randomized trials with clear cut results (and low risk of error) Level II Small randomized trials with uncertain results (and moderate to risk of error) Level III Non-randomized, contemporaneous controls Level IV Non-randomized, historical controls Level V No controls, case series only

Sacket DL, Can J Physiol & Pharmacol (1986)

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 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 Groce 2000b Tredget Moore 2000 Study design Mean burn size (%TBSA) Within patient Within patient Within patient Between patient Between patient Between patient 48.3 (11-99) 10.4 (6.9) Not reported Co-interventions Control Low pressure garment None Low pressure garment None None Low pressure garment

Not reported None Not reported

Chang 1995

21.1 (15.8) 11.2 (1-30)

Not reported Not reported

Groce 2000a Van den Kerckhove 2005

8.5 (1-30)

Not reported

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. Lee JP. et al. Antifibrogenic effects of liposome-encapsulated IFN-alpha2b cream on skin wounds in a fibrotic rabbit ear model. Journal of Interferon & Cytokine Research. 25(10):627-31, 2005. 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. 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] Burns. 29(7):649-64, 2003 Hillmer MP, et al. MacLeod SM. Experimental keloid scar models: a review of methodological issues. Journal of Cutaneous Medicine & Surgery. 6:354-9, 2002. Polo M, et al. An in vivo model of human proliferative scar. Journal of Surgical Research. 74:187-95, 1998.

2. 3. 4. 5. 6.

What are useful antifibrogenic agents for HTS?

antagonists of TGF- Interferon , , Bleomycin 5-fluorouracil others

Acknowledgements
Firefighters Burn Trust Fund Canadian Institute for Health Research Alberta Heritage Foundation for Medical Research

systemic circulation
T T T T T T T

cytokines

bone marrow

PBMC / fibrocytes myofibroblasts

TH1
_

IFN

-4 IL-10, IL
- TGF

+ +

wound

wound contraction

TH2

TH3
Figure

extracellular matrix synthesis / degradation 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. 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. 4. Spurr ED et al. Incidence of hypertrophic scarring in burninjured children. Burns 1990, 16: 179-181. 5. Bombaro KM et al. What is the prevalence of hypertrophic scarring following burns? Burns 2003, 29: 299-302.