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Analysis for CTGF and TGF-Beta in Deep Partial Thickness Thermal Burn Injury Tissue versus Normal Tissue

University of Florida Institutional Repository
Permanent Link: http://ufdc.ufl.edu/UFE0021464/00001

Material Information

Title: Analysis for CTGF and TGF-Beta in Deep Partial Thickness Thermal Burn Injury Tissue versus Normal Tissue
Physical Description: 1 online resource (139 p.)
Language: english
Creator: Mccurry, Donald E II
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: We know from previous studies that TGF-beta is found in several different cell types and that these cells all possess TGF-beta receptors. We also know that CTGF is not expressed unless induced as in the case of wound healing. The presence of estrogen receptors on all of the major cells involved in wound healing has been shown to stimulate these cells to produce TGF-beta in the presence of estrogen. Pre menopausal women having a higher level of estrogen versus post menopausal women and men, show a faster rate of wound healing compared with the other two groups: however, an increase in hypertrophic scar formation is also noted. We hypothesize that the release of TGF-beta and the subsequent production of CTGF during the initial phases of wound healing following a thermal burn injury will decline over time as the wound heals. We further hypothesize that immunohistochemical staining of tissue samples will show higher levels of these growth factors in pre menopausal women, due to higher estrogen levels, versus men and post menopausal women with lower levels of estrogen. Based on our research we were able to conclude the following: estradiol levels increase in response to the trauma of thermal burn injury; all of the cells involved in wound healing have estrogen receptors and that there are two isoforms of these receptors, ERalpha and ERbeta; and that males and pre-menopausal females have increased intensity of staining of TGF-beta and CTGF over post menopausal females.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Donald E II Mccurry.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Schultz, Gregory S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021464:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021464/00001

Material Information

Title: Analysis for CTGF and TGF-Beta in Deep Partial Thickness Thermal Burn Injury Tissue versus Normal Tissue
Physical Description: 1 online resource (139 p.)
Language: english
Creator: Mccurry, Donald E II
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: We know from previous studies that TGF-beta is found in several different cell types and that these cells all possess TGF-beta receptors. We also know that CTGF is not expressed unless induced as in the case of wound healing. The presence of estrogen receptors on all of the major cells involved in wound healing has been shown to stimulate these cells to produce TGF-beta in the presence of estrogen. Pre menopausal women having a higher level of estrogen versus post menopausal women and men, show a faster rate of wound healing compared with the other two groups: however, an increase in hypertrophic scar formation is also noted. We hypothesize that the release of TGF-beta and the subsequent production of CTGF during the initial phases of wound healing following a thermal burn injury will decline over time as the wound heals. We further hypothesize that immunohistochemical staining of tissue samples will show higher levels of these growth factors in pre menopausal women, due to higher estrogen levels, versus men and post menopausal women with lower levels of estrogen. Based on our research we were able to conclude the following: estradiol levels increase in response to the trauma of thermal burn injury; all of the cells involved in wound healing have estrogen receptors and that there are two isoforms of these receptors, ERalpha and ERbeta; and that males and pre-menopausal females have increased intensity of staining of TGF-beta and CTGF over post menopausal females.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Donald E II Mccurry.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Schultz, Gregory S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021464:00001


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2681fa83238c55a42868dead1382cef2
a03e8c7f2213cf4fe9a2562be814c1d12e9403af







ANALYSIS FOR CTGF AND TGF-BETA LEVELS IN DEEP PARTIAL THICKNESS
THERMAL BURN INJURY TISSUE VERSUS NORMAL TISSUE




















By

DONALD E. McCURRY II


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007

































2007 Donald E. McCurry II



























To all of the people who have sustained a burn injury, who at a time of crisis in their own lives
gave in order to help future burn victims.









ACKNOWLEDGMENTS

I would like to thank all the people that have guided me along my career path. First, the

professors who have taught me along the way, without the excellent education that I received at

South Carolina State University and the Medical University of South Carolina, I would not have

been prepared to enter the University of Florida. I would also like to recognize my professors at

the University of Florida whose commitment to excellence in education and research is an

inspiration. In addition to my class professors, I have to give a special thanks to my committee

members, Dr. Gregory Schultz, Dr. David Mozingo, and Dr. Lyle Moldawer, I truly could not

have had a better group of guys to guide me through this process. There are also several key

people, from the University of Florida that I would like to recognize for all of their assistance

during my time here: Karen Perrin, ARNP; Robin Masterson, RN; Angel Sampson; Dr. Jompo

Moloye; Dr. John Azeke; Paulette Sanders; Dr. Beverly Vidaurreta and all the staff on the burn

unit. Special thanks go out to all of the burn victims who generously donated tissue for my study.

There have been many people that have made an impact on my life both personally and

professionally. Dr. John Samies and Marie Gehling, RN the passion that you show for patient

care and your good moral character are worth emulating. I am proud to be their colleague and

friend. To my best friends, Big Luke and Vic, whose support and love has helped me through

this stressful time. MIBs bringing da heat! And the rest of my family and friends thanks!










TABLE OF CONTENTS

page

A CK N O W LED G M EN T S .................................................................. ........... .............. .....

L IS T O F T A B L E S .............. .... ............. ............................................ ............... 7

LIST OF FIGURES .................................. .. .... ... ...............8

A B S T R A C T ................................. ............................................................ 10

CHAPTER

1 IN TR O D U CTION ............... .............................. ..................... ........ .. 12

B u rn s ............ ...................................................................................12
H y p o th e sis .............. ... ................................................................12

2 B A CK G R O U N D .......................................... .............................................. 17

Skin .................... ....................................... 17
E p id erm is ...................................................... 17
B asem ent M em b ran e ................................................................ ...............................19
Dermis ........................................ 20
A ppendages..................................................................22
S tem C ells ......................................................2 4
S k in F u n ctio n .............................................................................................................. 2 5
Phases of W found H dealing ........................................................................................... .... ........ 27
H e m o sta sis ................................. ....................................................2 7
In flam m ato ry P h ase .................................................................................................... 3 2
P ro liferativ e P h a se ...................................................................................................... 3 7
R em o d elin g P h a se ...................................................................................................... 4 0
G row th F actors ........................................................................4 1
Transforming Growth Factor-Beta .........................................................................42
Connective Tissue Growth Factor ................................. .......................... ...43
H o rm o n es .. ............ ................. .......................................................................................... 4 4
E stro g en ................... ...................4...................5..........

3 M A TER IA L S A N D M ETH O D S ...................................................................................... 62

T issue e C collection ................................................................62
Im m unostaining for C T G F ................................................................................................ 63
Im m unostaining for TGF- ................ .. ................................... ....... 65
E strad io l E IA K it ......... ........... ................. ........................... .....................................6 7









4 RESULTS AN D D ISCU SSION .................................................... ............................... 71

APPENDIX

A PRO TO COL #480-2005 ............................................................ ........................... 100

B C A SE R E P O R T FO R M S ............................................................................ ....................105

C IN FO R M ED C O N SEN T ............................................................................. ... ............ 110

D IH C PR O TO CO L FO R CTG F ......... ................. ................................... .........................118

E T G F -B IH C PR O T O C O L ......... ...... ........... ................. ................................................. 12 1

F PA TIE N T D A T A ........... ... .... .. .. .......... .... .... ........................ ..... ......... 123

L IST O F R E F E R E N C E S ......... ...... ........... ................. ...................... ...................................13 1

B IO G R A PH IC A L SK E T C H ......... ................. ........................................... ........................... 137



































6










LIST OF TABLES


Table


3-1 P late 2 set up for E stradiol E IA ........................................ ...................... .....................70

4-1 Estradiol levels for adult males and females during each phase of the menstrual cycle
and after m menopause ....................... .. ................ ....... ........ ...........81


page









LIST OF FIGURES

Figure page

1-1 Ratio of male to female burn victims in the United States .............................................14

1-2 Ratio of burn victim s by age group .............................. .......................................15

1-3 Ratio of partial thickness versus full thickness bum injuries ..........................................16

2-1 C ross section of hum an skin. .................................................................... ..................47

2-2 C ross section of the epiderm is ........................................ ............................................48

2 -3 H em id esm o so m es ........................................ .......................................... ................. .. 4 9

2-4 Histologic view of the epidermis and dermis ............................ ...............50

2-5 Collagen formation ................................ ......... ..... .................. 51

2-6 Extracellular m atrix ......................... ......... .............. .. ............52

2-7 V ariou s skin appendages ......................................................................... ....................53

2 -8 F in g er n ails ................................................................54

2 -9 H a ir fo llic le .................................................................................................................. 5 5

2-10 H air follicle bulge stem cells ..................................................................... ..................56

2-11 C oagulation cascade ........................... .......................... .. .... ........ ........ 57

2-12 Conversion of arachidonic acid to ecosanoids....................................... ............... 58

2-13 Steps of the inflam m atory response.............................................................................. ...59

2-14 Diapedesis and extravastion of neutrophils ........................... ....................... 60

2 -15 A n g io g en esis ........................................................................... 6 1

4-1 Effects of hormone replacement, hormone inhibitor, and percent burn on estrdiol
lev els ................................................................. ................ 8 2

4-2 Average estradiol levels at debridem ent 1 ........................................ ...... ............... 83

4-3 Average estradiol levels at debridem ent 2................................ ................................. 84

4-4 Average estradiol levies at debridem ent 3 ........................................ ...... ............... 85




8









4-5 Estradiol levels versus percent burn for males and females following thermal burn
inju ry ........ ........ ............................................................................ 8 6

4-6 Estradiol levels versus age following thermal bum injury. .............................................87

4-7 Quantitative image analysis versus visual analog scale for TGF- ..................................88

4-8 Quantitative image analysis versus visual analog scale for CTGF..............................89

4-9 Immunohistology for TGF-P in human bum injured and donor site tissue (males)..........90

4-10 Immunohistology for TGF-P in human bum injured and donor site tissue (pre-
menopausal females) ...................... ........................ .......... 91

4-11 Immunohistology for TGF-P in human bum injured and donor site tissue (post-
m en op au sal fem ales).......... ......................................................................... ....... ......... .. 92

4-12 Immunohistology for TGF-P in human "normal" tissue (males) ....................................93

4-13 Immunohistology for TGF-P in human "normal" tissue (pre and post-menoapusal
fe m a le s) ................... .......................................................... ................ 9 4

4-14 Immunohistology for CTGF in human burn injured and donor site tissue (males) ..........95

4-15 Immunohistology for CTGF in human burn injured and donor site tissue (pre-
menopausal females) ........................................ ..... .......... 96

4-16 Immunohistology for CTGF in human burn injured and donor site tissue (post-
m en op au sal fem ales).......... ......................................................................... ....... ......... .. 97

4-17 Immunohistology for CTGF in human "normal" tissue (males) ....................................98

4-18 Immunohistology for CTGF in human "normal" tissue (pre and post-menoapusal
fe m a le s) ................... .......................................................... ................ 9 9









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ANALYSIS FOR CTGF AND TGF-P LEVLES IN DEEP PARTIAL THICKNESS THERMAL
BURN INJURY TISSUE VERSUS NORMAL TISSUE

By

Donald E. McCurry II

August 2007

Chair: Donna Duckworth
Major: Medical Sciences

We know from previous studies that TGF-P is found in several different cell types and that

these cells all possess TGF-P receptors. We also know that CTGF is not expressed unless

induced as in the case of wound healing. The presence of estrogen receptors on all of the major

cells involved in wound healing has been shown to stimulate these cells to produce TGF-P in the

presence of estrogen. Pre menopausal women having a higher level of estrogen versus post

menopausal women and men, show a faster rate of wound healing compared with the other two

groups: however, an increase in hypertrophic scar formation is also noted.

We hypothesize that the release of TGF-P and the subsequent production of CTGF during

the initial phases of wound healing following a thermal bum injury will decline over time as the

wound heals. We further hypothesize that immunohistochemical staining of tissue samples will

show higher levels of these growth factors in pre menopausal women, due to higher estrogen

levels, versus men and post menopausal women with lower levels of estrogen.

Based on our research we were able to conclude the following: estradiol levels increase in

response to the trauma of thermal burn injury; all of the cells involved in wound healing have

estrogen receptors and that there are two isoforms of these receptors, ERa and ERP; and that









males and pre-menopausal females have increased intensity of staining of TGF-P and CTGF over

post menopausal females.









CHAPTER 1
INTRODUCTION

Burns

A bum is a very serious injury, last year alone there were approximately 500,000 burn

injuries that required medical treatment, resulting in 40,000 hospital admissions. Fire and burn

deaths per year are approximately 4,000 this number includes an estimated 3,500 deaths from

residential fires and 500 from motor vehicle and aircraft accidents. Most bum victims are male

(70%) between 5 and 19 years of age (Figures 1-1, 1-2). The largest percentage of burn injuries

occur in the home and are the result of fire or flame. Although most burn injuries are 10% or less

of total body surface area (TBSA), some people sustain bum injuries to a significant portion of

there bodies. Advances in medical care have allowed people to survive a significant burn injury

that wound not have survived just 20 years ago; people are now able to survive after sustaining

almost 100% TBSA burn injury (Figure 1-3). The mortality rate has dropped from 6.16 % to

4.67 % over the past 10 years, and the average hospital length of stay has dropped from 13.07

days to 8.22 days over the same time period. For those who do survive a significant burn injury

they are left with multiple scars and disfigurements resulting in decrease cosmetic appearance,

loss of function and restricted growth.1

Hypothesis

We know from previous studies that TGF-P is found in several different cell types to

include platelets, macrophages, granulocytes, fibroblast, and keratinocytes. TGF-P receptors are

found on essentially all cell types.2 We also know that CTGF is not expressed unless induced as

in the case of wound healing.3'4

The presence of estrogen receptors on all of the major cells involved in wound healing

have been shown to stimulate these cells to produce TGF-P in the presence of estrogen. Pre-









menopausal women having a higher level of estrogen versus post-menopausal women and men,

show a faster rate of wound healing compare with the other two groups; however an increase in

hypertrophic scar formation is also noticed.

We hypothesize that the release of TGF-P and subsequent production of CTGF during the

initial phases of wound healing following a thermal burn injury will decline over time as the

wound heals, and that immunohistochemical staining of tissue samples will show a correlation

with previous studies and show higher levels of these growth factors in pre-menopausal women,

due to higher estrogen levels, versus men and post-menopausal women with lower levels of

estrogen.

It is our hope that this study may one day lead to a better wound healing protocol in the

treatment of burned injured patients, allowing faster healing rates, while decreasing hypertrophic

scar formation.










Male
69.9%


Female
30.1%


Figure 1-1. Ratio of male to female burn victims in the United
States(http:www.ameribum.org, last accessed January 20th, 2005)











18,000

16,000

14,000

12,000

10,000

8,00-

6,000

4,000

2,000

0


0- 1.9 2- 4.9 5 19.920 29.930 39.940 49.950 59.960- 69.9 ? 70

Age Group

Ratio of bum victims by age group(http:www.ameriburn.org, last accessed
January 20th, 2005) 1


Figure 1-2.










50,000


0.1-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89


% TBSA


Ratio of partial thickness versus full thickness bum injuries, and combined
total(http:www.ameriburn.org, last accessed January 20th, 2005) 1


Figure 1-3.









CHAPTER 2
BACKGROUND

Skin

Epidermis

Adult human skin is a very large organ, covering approximately 2 m 2 in area, with

approximately 2.5 mm thickness and has an average density of 1.lkg/m3. Together these provide

for a 5-6 kg mass value for skin, in other words, skin constitutes an impressive 6% of our total

body weight.5 The skin provides a physical barrier with the external environment and is

designed to protect us against a wide range of insults including: temperature, electrolyte/fluid

balance, mechanical, chemical and microbial. Further protection is provided by the ultraviolet

radiation (UVR) absorbing pigmentation system and the complex immuno-regulatory sentinel

networks, which sense tissue micro-environments for foreign or abnormally expressed

components. The skin has two layers, (Figure 2-1) an epidermis and dermis, separated by a

basement membrane which is supported by a hypodermis or superficial fascia.6

The normal epidermis is the outermost layer of the skin and is derived from the embryonic

ectoderm, the outermost germ layer. The major cell, making up 95% of the total, is the

keratinocyte, which moves progressively from attachment to the epidermal basement membrane

towards the skin surface, forming several well defined layers in transit.7 Other cells that reside

within the epidermis include: melanocytes which are derived from the neural crest and are

responsible for the production of melanin, langerhans cells (ie. dendritic like cells) are derived

from a bone marrow precursor that act as antigen presenting cells interacting with T-cells,

merkel cells are also derived from the neural crest and are involved in tactile sensation.8 The

stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale









are the distinct layers of keratinocytes that make up the epidermis. (Figure 2-2) The epidermis is

an avascular layer and depends on the capillary beds in the dermis for oxygen and nourishment.9

The stratum corneum is the outermost layer and is composed of several layers, usually 15-

30, of dead, but biochemically active cells called corneocytes. These cells contain keratin a

protein that helps keep the skin hydrated by preventing water evaporation. This layer is

constantly being shed into the environment through bathing, hand washing, and mechanical

disruption when changing clothes. This layer is replaced by the layers below, stratum lucidum or

the stratum spinosum.6

The stratum lucidum is a thin, clear layer of dead cells in the epidermis. It is found beneath

the stratum corneum in areas of thick skin, such as the palms of the hand and soles of the feet. It

is recognized by some histologist as an intermediate layer above the stratum granulosum and

beneath the stratum corneum. However, no distinctive cytologic features are significantly

apparent. Cells present in the stratum lucidum release a small amount of lipid material into the

extra cellular space after fusion with the plasma membrane to form a neutral lipid rich coating

that protects these cells from disruption.6'8

The stratum granulosum, sometimes referred to as the granular layer, is generally one to

three cells thick and is seen as a flat layer whose cells still have active nuclei. These non dividing

keratinocytes produce granules of a protein called keratinohyalin. These cells flatten as dividing

cells below progressively push them toward the skin surface. At the same time their organelles

and nuclei breakdown and the granules of keratinohyalin release their lipid components into the

intercellular space which causes the cell membranes to become increasingly impermeable

playing an important role in barrier function and intercellular cohesion.76









The keratinocytes of the stratum spinosum have a flattened polygonal shape with a distinct

ovoid nucleus. This layer is generally two to six cells thick and is distinguished by numerous

desmosomal (Figure 2-3) connection plaques, which interact with adjacent keratinocytes forming

a stabilizing network of surface interconnections. Desmosomes are symmetrical, laminated

structures formed in apposed plasma membranes with linear intercellular and intracellular

components. Cytoskeletal tonofilaments seem to attach close to desmosomes, providing stability

across the cell layers.6'7

The stratum basale is the inner most epidermal layer and is the layer that has actively

dividing cells undergoing mitosis. Along with the stratum spinosum, the stratum basale are

collectively called the stratum of Malpighi. The stratum of Malpighi is named for the Italian

scientist Marcello Malpighi who is known as the first microanatomist and the father of histology.

The cells of the stratum basale are attached to the noncellular basement membrane by

hemidesmosomes, a type of cell junction that forms between cells and matrix. The basement

membrane separates the epidermis from the dermis. Once the cells of this layer divide, they push

upward, and as they complete their journey to the stratum corneum in approximately two to three

weeks they undergo complete differentiation to become keratinized. It is the fate of every

keratinocyte of the stratum basale to mature and differentiate into a keratinized cell to contribute

to a tough external skin surface layer.6'5'8

Basement Membrane

The basement membrane is a thin acellular layer that separates the epidermis from the

dermis. The basement membrane anchors the epidermis to the dermis and is made up of three

layers, the lamina lucida, the lamina densa, and the lamina fibrorecticularis. The lamina lucida is

named because its an electron translucent zone compared with the electron dense zone of the

lamina densa. The lamina densa lies parallel to and below the lamina lucida. The main









component of the lamina fibroreticularis is anchoring fibrils. These are short, often curved

structures, with an irregularly spaced cross banding, that inserts into the lamina densa and extend

into the upper part of the dermis. Another component of the lamina fibroreticularis is the elastic

microfibril. This may occur singly, but is best recognized in a bundle consisting of many

microfibrils that extend into the dermis and may enmesh with the microfibrillar system of dermal

elastic fibers. The major proteins found in the basement membrane are fibronectin, an adhesive

glycoprotein; laminin, also a glycoprotein; type IV collagen, a nonfiber forming collagen; and

heparin sulfate proteoglycan, a glycosaminoglycan that probably acts as a type of ground

substance. A lesser amount of type VII collagen is present, but it plays a very important role in

attachment of cells to the lamina densa.6'7'10

Dermis

The dermis is derived from the embryonic mesoderm, the middle germ layer. The dermis is

formed by two layers without distinct boundaries the upper "papillary" and a lower "reticular"

layer, reflecting there respective connective tissue components, cell number, and supply of blood

vessels and nerves. The papillary region is composed of loose areolar connective tissue. It is

named for its fingerlike projections called papillae, which extend toward the epidermis. The

papillary dermis interdigitates with the epidermis, this strengthens the connection between the

two layers.(Figure 2-4) The reticular dermis lies deep to the papillary dermis and is usually much

thicker and blends with the subcutaneous fat. It is composed of dense irregular connective tissue,

and receives its name from the dense concentration of collagenous, elastic and reticular fibers

that weave throughout it. These protein fibers give the dermis its properties of strength,

extensibility, and elasticity.5'6'7

Despite its greater volume, the dermis contains far fewer cells than the epidermis and

instead much of its bulk consists of fibrous and amorphous extracellular matrix (ECM). The









main cell type of the dermis is the fibroblast, a heterogenous migratory cell that makes and

degrades ECM components. The ECM is a highly complex mixture of bioactive macromolecules

produced by dermal cells that is either secreted intact or assembled later outside the cells. ECM

is the defining feature of connective tissue. Collagens are the principal ECM component and

make up about 90% of total dermal protein. While at least 25 collagens exist, half are present in

skin; consisting predominantly of collagen type I and III. Collagen is an exceedingly tough

fibrous protein that is secreted as tropocollagen by the fibroblast into the extracellular

environment, (Figure 2-5) where it undergoes further processing by enzymes that cleave the

propeptide portion of the polypeptide to form mature collagen fiber bundles. Collagen is the

protein that gives the skin its tensile strength. The primary amino acids that constitute collagen

are proline, glycine, hydroxyproline, and hydroxylysine.6'10'5'7

The other major protein found in the ECM is elastin. Elastin is the protein that returns skin

continuity when it is pinched or stretched, and is secreted by fibroblast as tropoelastin into the

dermis. In the extracellular space of the dermis the proelsatin which interacts with fibrillin, a

microfibrillar protein whose anatomical distribution closely parallels that of elastin, to organize

immature elastic fibers this aggregates to form mature elastic fibers. These mature elastic fibers

are composed of amorphous and fibrous components; the amorphous portion is composed of the

protein elastin while the fibrous portion is composed of the protein fibrillin. Oxytalan and

elaunin are two other elastic fibers also found in dermal connective tissue.6'10'5'7

The other category of proteins found occupying the space between collagen and elastin

fibers is referred to as the ground substance. (Figure 2-6) This amorphous and hydroscopic

material is biologically active, highly diverse and organized. It consists of proteoglycans a

modified core protein to which are attached polymers of unbranched disaccharides, and









glycosaminoglycans that act as linkers between certain cell surface receptors and ECM

scaffolding components. Proteoglycans and glycosaminoglycans are capable of binding up to

1000 times their volume and thus play a role in regulating the water binding capacity of the

dermis that determines the dermal volume and compressibility.6'5'7

The hypodermis, or superficial fascia, (Figure 2-1) forms a subcutaneous layer below the

dermis. This is an adipose layer containing a subdermal plexus of blood vessels giving rise to the

cutaneous plexus in the dermis, which in turn gives rise to the papillary plexus and loops of the

papillary dermis. This layer is not considered part of the skin but is the underlying supporting

and connecting layer that attaches the dermis to underlying structures. The hypodermis provides

padding and insulation and accounts for differences in body shape.6'10

Appendages

In addition to the two major structural layers, the epidermis and dermis, the skin is also

populated by different appendages. These appendages include glands, such as the sweat glands

and sebaceous glands. Along with the different glands are the hair follicle, hair fiber, and nail.

(Figure 2-7)

The sweat glands can be divided into two separate groups the eccrine and apocrine glands.

As warm blooded animals humans have approximately 3-4 million eccrine glands in their skin,

each producing a watery perspiration that serves principally to cool and maintain the core body

temperature at 37.5 C. Eccrine glands have long thin ducts opening directly onto the skin

surface and proximal secretary coiled section consisting of secretary cells and contractile

myoepithelial cells. The coiled portion of the eccrine gland lies in the deep dermis or

hypodermis. The apocrine glands are larger than the eccrine glands, but like the eccrine glands

they are located in the dermis and hypodermis. Instead of opening onto the skin surface the

apocrine glands open into the hair follicle. The apocrine glands are found after puberty are









present in the groin, anal region, axilla, areola of the breast, and beard of adult males. Apocrine

gland "sweat" is thicker, more viscous and with a milky consistency due to its higher content of

fatty acids and other compounds. Some of these are odoriferous, especially after there

decomposition on the skin surface by bacteria.5'

The sebaceous gland is a holocrine simple saccular gland extending over the entire surface

of the skin except for the palms and soles of the hands and feet, respectively. The secretary

portion of the sebaceous gland lies in the dermis, and the excretory duct opens into the neck of

the hair follicle. Sebaceous glands can be independent of the hairs and open directly onto the

skin of the lips, corner of the mouth, glans penis, labia minora, and the mammary nipple. The

oily secretion of the gland sebumm) is released on the surface of the hair and the epidermis.8 The

nature of sebum's function in humans, the "naked ape" with no fur to protect, remains perplexing

especially as significant evolutionary pressure must have driven the remarkable variation in the

composition of sebum. While sebum production is the most obvious function of sebaceous

glands, recent evidence suggest other important roles for these glands in the regulation of

steroidogenesis, local androgen synthesis, skin barrier function, interaction with neuropeptides,

potential production of both anti- and pro-inflammatory compounds and synthesis of anti-

-5
microbial lipids.5

The nails are the least well studied of the skin appendages and are hard keratin plates on

the dorsal surface of the terminal phalanges of the fingers and toes. (Figure 2-8) The nail plate

covers the nail bed, the surface of the skin which consists of the stratum basale and stratum

spinosum only. The cells that go to make up the nail plate are similar to those generating the

stratum corneum of the epidermis and the trichocytes that make up the hair shaft. Up to 90% of









the nail keratins are of the hard "hair" keratin type; the remainder are soft "epidermal" keratins.

Also present in the nail are water, lipids, and trace elements including iron, zinc, and calcium.5'8

The hair follicle is a tubular invagination of the epidermis and is responsible for the growth

of hair. (Figure 2-9) The hair follicle is our body's only permanently regenerating organ, as it

transits through life-long periods of growth (anagen), regression (catagen), and relative

quiescence (telogen). Five million hair follicles reside in the skin, though only 2% are on the

head. People with little body hair or with myriad amounts have the same amount of hair follicles,

with hair visibility depending on hair size and distribution rather than number of follicles. The

hair bulb is the end portion of the invaginated hair follicle. A vascularized connective tissue core,

dermal papilla, projects into the hair bulb. The hair follicle consists of an external root sheath and

an internal root sheath. The external root sheath is an invagination of the epidermis and the

internal root sheath is made up of three layers of soft keratin.8'511

The epidermis has classically been viewed as a stratified squamous epithelium maintained

by cell division within the basal layer, which is attached to the basement membrane.

Differentiating cells are gradually displaced outwards through the stratum spinosum to the

stratum corneum. The anucleate corneal cells, corneocytes, or cornified cells, which protect the

viable cell layers, are continually shed from the skin surface, and the rate of production of cells

in the basal layer must match the rate of loss from the surface to produce normal skin thickness.7

Stem Cells

The simple definition of a stem cell is the cell of origin for terminally differentiated cells in

adult tissues. These cells have two distinct characteristics: self renewal, the ability to go through

numerous cycles of cell division while maintaining the undifferentiated state and multipotency,

the ability to generate progeny of several distinct cell types. The epidermis is a dynamic

epithelium that is constantly renewed throughout life. Its turnover is estimated at about 60 days









for humans. This rapid replacement demands, as with other epithelial tissues, that an adult have

stem cells capable of supplying differentiated cells throughout life. Stem cell niches are

composed of microenvironmental cells that nurture stem cells and enable them to maintain tissue

homeostasis. Skin epidermis and its associated structures arise from two stem cell populations

within the hair follicle and interfollicular regions. One, in the basal layer of skin, normally gives

rise to the stratified layers of skin. A second, the hair follicle stem cell, (Figure 2-10) resides in a

region of the outer root sheath called the bulge, and is responsible for the regeneration of hair

and sebaceous glands. It has been suggested that bulge stem cells are also responsible for the

long term replenishment of the interfollicular epidermis. However, it is now clear that bulge stem

cells are not required for normal epidermal homeostasis, although they can contribute transiently

during wound healing.12,13,14

Skin Function

Skin, as the outer most organ in the human body, continuously confronts the external

environment and serves as a primary defense system. Thus, knowledge of skin function is as

important as knowledge of skin anatomy. Protection from the environment is one of the most

fundamental functions of skin. Skin functions as a barrier to protect internal organs from

exposure to the outside environment and maintains an internal homeostatic environment.15'6

Keratin, the insoluble protein found in the squames of the stratum corneum and sebum,

protects the skin against aqueous and chemical assaults. The fibroelastic connective tissue of the

dermis provides the skin with mobility and skin pigmentation provides protection against

ultraviolet radiation.6

Circulation and sweating are two of the primary thermoregulatory mechanisms. In warm

environments there is an increased blood flow and heat is dissipated by dilated vessels through

conduction, convection, radiation, and evaporation. Vasoconstriction in a cold environment









shunts blood flow to other vital organs to maintain function and warmth. Sweating occurs when

there is increased activity by the sweat glands. Cooling occurs when the secreted fluid is

evaporated from the skin surface. Skin or body odor associated with sweating, is largely a result

of bacterial flora.6

The skin has an elegant system of nerve receptors to sense pain, touch, pressure, vibration,

tickle, and temperature. Nerve receptors in the epidermis and dermis transmit impulses to the

cerebral cortex for interpretation. Burning, itching, and tickling are considered to be a

combination of the four basic sensations. Unmyelinated free nerve endings, Merkel cells,

Meissner corpuscles, Krause end bulbs, Ruffini terminals, and pacinian corpuscles propagate all

these sensations. Attempts to identify particular responses with these specific nerve structures

have not been entirely successful because many of these nerve receptors respond to more than a

single stimulus. However, free nerve endings respond to touch, pain, and temperature; pacinian

corpuscles respond to pressure, coarse touch, vibration, and tension; and Meissner corpuscles are

involved in touch reception.6

Vitamin D is important in the mineralization of bone and in calcium and phosphate

metabolism. Vitamin D is formed in the skin by the conversion of 7-dehydrocholesterol to

cholecalciferol following exposure to ultraviolet light. The main function of vitamin D is to

stimulate calcium reabsorption by the intestinal mucosa. Vitamin D, like all steroids, is

transported to the nucleus of the intestinal cell to induce the synthesis of a calcium binding

protein required for the transport of calcium across the intestinal epithelium.8

As a protective interface between internal organs and the environment, the skin encounters

a host of toxins, pathogenic organisms, and physical stresses. To combat these attacks on the

cutaneous microenvironment, the skin functions as more than a physical barrier: it is an active









immune organ. Immune responses in the skin involve an armamentarium of immune competent

cells and soluble biologic response modifiers including cytokines. Langerhans cells are

recognized as the major antigen presenting cells of the skin and play a key role as the peripheral

component of the immune system. Langerhans cells are one of a large family of class II major

histocompatibilty complex (MHC) bearing dendritic cells critical to antigen processing and

presentation to specific T lymphocytes. Accounting for more than 90% of epidermal cells,

keratinocytes not only create a complex physical barrier to environmental agents but also serve

as accessory cells in cutaneous immune responses. Keratinocytes may play a role in initiating

cell mediated immune responses in the skin by releasing cytokines and expressing cellular

adhesion molecules to facilitate the movement of immune competent cells. Although

melanocytes have been traditionally considered to have no immunologic role, recent findings

suggest that these cells may contribute to the immune function of the epidermis by secreting

biologic response modifiers. Melanocytes represent 2-5% of epidermal cells; the number of

melanocytes varies from person to person and in different anatomic sites of the same person.

Like Langerhans cells, they possess numerous dendritic like processes and are strategically

placed in the lower epidermis, close to the dermis, where they may play important roles in

immune responses.16

Phases of Wound Healing

Hemostasis

Wounds heal through a series of dynamic and overlapping processes that begin in response

to tissue injury. The first major event following injury is hemostasis. Wounding destroys the

various layers of skin and underlying soft tissue just as a hurricane or bombing destroy a house.

Arteries, veins, and nerves become exposed just as plumbing and electric conduits are exposed.

The initial phase of construction is to cap off these conduits in order to prevent further









destruction and loss. Hemostasis serves as the initiating step in the wound healing process. The

formation of a clot serves a dual purpose of preserving vascular integrity and providing a

temporary scaffold for the wound healing process to begin.1

Platelets, the smallest of the human blood cells, are actually a part of a much larger

progenitor cell, the megakarocyte and are central players in the process of hemostasis. When a

vessel wall is damaged, platelets are recruited from the circulation to the unveiled subendothelial

matrix forming a hemostatic plug to close the leak in the vessel wall. Platelets become activated

when exposed to extravascular collagen and initiate the intrinsic part of the coagulation cascade.

Platelet adhesion to collagen activates them to release soluble mediators (growth factors and

cyclic AMP) and adhesive glycoproteins from their a granules, which are in turn, signals for

subsequent platelets to change their morphology, become sticky and aggregate. The key

glycoproteins released from the platelet granules include fibrinogen, fibronectin,

thrombospondin, and von Willebrand factor. Soluble mediators released from a granules include

PDGF, TGF-P, TGF-a, bFGF, and VEGF. The clot that is formed by the aggregation of platelets

is more than dried blood. On the contrary, it represents a viable, dynamic matrix of proteins and

cells that not only contribute to hemostasis but also serve as a provisional lattice for incoming

inflammatory cells, fibroblast, and growth factors. The main constituents of the visible clot are

fibrin and platelets. Fibrin is produced by the proteolytic cleavage of fibrinogen by thrombin.

Insoluble fibrin mononmers are cross linked by factor XIII (fibrin stabilizing factor), and fibrin

directly binds to platelets to produce a clot.18'10,19'20

The clotting cascade first introduced in the 1960s described an eight step process of

proenzyme-enzyme transformations beginning with the activation of factor XII (Hageman factor)

and ending with the activation of fibrinogen (factor I) by thrombin (factor IIa) to form fibrin









(factor Ia) 21. Over the next 40 years there have been changes in our understanding of how this

cascade works.

Factor X (Stuart factor) is the common point of the intrinsic and extrinsic coagulation

pathways. The intrinsic and extrinsic pathways were so named because of the location of their

protein components, within the blood or outside the blood, respectively. The intrinsic pathway is

initiated when prekallikrein is converted to kallikrein and factor XII is converted to factor XIIa.

Factor XIIa converts factor XI to factor XIa. Factor XIa activates factor IX, which with its co-

factor, factor Villa, form the tenase complex which activates factor X to factor Xa. The extrinsic

pathway is initiated by the proteolysis of Factor VII. Factor VIIa then binds with tissue factor.

The factor VIIa/tissue factor complex activates factors IX and X. From the activation of factor X

both pathways converge into a common pathway down to the formation of fibrin from

fibrinogen.

The intrinsic and extrinsic pathways are now called the contact and tissue factor pathways,

respectively. (Figure 2-11) These changes were made in response to how each pathway is

initiated. The contact (intrinsic) pathway is initiated when the blood comes into contact with a

negatively charged biological surface in vivo, such as collagen and platelet membranes. The

tissue factor (extrinsic) pathway requires the presence of tissue factor constitutively expressed on

cells separated from flowing blood; that upon vascular injury, tissue factor comes in contact with

flowing blood, initiating reactions that lead to the generation of fibrin and a fibrin clot.22,23,24

A cofactor is any substance that is required in addition to an enzyme that needs to be

present for a reaction to take place, or to make the reaction more efficient. In the case of the

blood clotting cascade vitamin K and Ca+ act as cofactors. Vitamin K is essential for the

synthesis of prothrombin, by converting glutamate to y-carboxyglutamate, which makes up the









N-terminal portion of the prothrombin molecule. The y-carboxyglutamte is a strong chelator of

Ca" which brings the prothrombin molecule into close proximity to the platelet by binding to the

lipid membrane.25

The reactions of blood coagulation are carefully controlled by several anticoagulant

mechanisms, which under normal conditions prevail over the procoagulant forces. Vitamin K

dependent protein C is the key component of an important natural anticoagulant pathway. The

procoagulant properties of thrombin are lost on binding to thrombomodulin because

thrombomodulin occupies the functionally important exosite I in thrombin and thereby blocks

interactions with other thrombin binding proteins. On the endothelial surface thrombin and

thrombomodulin form a complex with the endothelial protein C receptor. Protein C binds to its

receptor on the endothelial surface and becomes activated. The activated protein C exerts its

anticoagulant effect by regulating the activities of factor Villa and factor Va, the cofactors in the

tenase and prothrombinase complexes, respectively. The anticoagulant activity of activated

protein C is enhanced by protein S, which is sufficient for the inactivation of factor Va.26

Eicosanoids are among the mediators and regulators of inflammation and are generated

from 20-carbon polyunsaturated fatty acids found in the lipid membranes of cells. Arachidonic

acid is usually the major substrate for eicosanoid synthesis. (Figure 2-12) Eicosanoids,

prostaglandins, thromboxanes, and leukotrienes, are generated from arachidonic acid by

metabolic processes involving the enzymes cyclooxygenase and lipoxygenase. Eicosanoids are

involved in the intensity and duration of inflammatory responses, have cell and stimulus specific

sources, and frequently have opposing effects. Thus, the overall

physiological/pathophysiological outcome will depend on the cells present, the nature of the









stimulus, the timing of the eicosanoid generation, the concentration of different eicosanoids

generated and the sensitivity of the target cells and tissues to the eicosanoids generated.27

Prostaglandins were first discovered in the 1930s and named because they were thought to

be produced by the prostate. It is now known that prostaglandins are produced by the metabolism

of arachidonic acid. Phospholipase A2 mediates the release of arachidonic acid from membrane

phospholipids, a rate limiting step in the formation of eicosanoids in nonpathologic states. There

are two secretary forms of phospholipase A2, designated as PLA2I and PLA2II. PLA2I is

produced by the pancreas and plays a role in digestion of phospholipids, where as, PLA2II is

produced by inflammatory cells and is increased in conditions such as sepsis and acute

respiratory distress syndrome. One member of the prostaglandin family is PGE2. PGE2 is a

vasodialator, a natriuretic factor, an inhibitor of gastric acid secretion, and an agent that contracts

uterine tissue. Another member of the prostaglandin family is PGF2a, and is synthesized in many

tissues in varying amounts and is increased in sepsis. It is a bronchoconstrictor and

venoconstrictor, and contracts uterine smooth muscle. There are other prostaglandins with

varying and often opposite actions.28

Prostcyclin, PGI2, is synthesized by endothelial cells, macrophages, the lungs and the

kidneys. It is a potent vasodialator and an inhibitor of platelet aggregation that spontaneously

hydrolyzes to form the stable but inactive metabolite 6-keto-PGF1a.28

Thromboxane, TX, was so named because it induces platelet aggregation, and is also a

potent vasoconstrictor and bronchoconstrictor. TXA2 is synthesized in large quantities by

platelets, macrophages, monocytes, and the lungs. It is unstable and spontaneously hydrolyzes to

form the stable but inactive product TXB2.28









Leukotrienes were so named because they are produced by leukocytes. Leukotiene B4,

LTB4, is synthesized by white blood cells, macrophages, and synoviocytes. It is a chemotactic

substance for white blood cells. The cysteine containing leukotrienes include LTC4 which is

synthesized by white blood cells, lung parenchymal tissue, and macrophages. It is converted to

LTD4 an active metabolite of LTC4. The cysteinyl leukotrienes are vasoconstrictors and

bronchoconstrictors, and they increase capillary permeability and bronchial mucous secretion

through activation of cysteinyl leukotriene receptors 1 and 2.28

Inflammatory Phase

Inflammation begins within 24 hours after injury and can last for up to 2 weeks in normal

wounds. (Figure 2-13) An increase in vasodilation and vascular permeability can begin in as

little as 1 hour after injury. The major cell types involved in the inflammatory phase of wound

healing are the neutrophil, macrophage, and mast cell.

The first nucleated cell to infiltrate the wound bed after the integrity of the skin has been

disrupted is the neutrophil. This innate immune cell mediates the first line of defense and marks

the start of the inflammatory phase of wound healing.29 The neutrophils leave the vascular

system by a process known as extravasation. (Figure 1-14)

The first step in this process is the appearance ofP selection on the endothelial surface,

after exposure to leukotriene B4, the compliment fragment C5a, or histamine. E selection appears

a few hours later after exposure to TNF-a. The interaction of the P and E selections with

glycoproteins on the surface of the neutrophils allows reversible binding, so that the neutrophil

appears to "roll" along the endothelium. Next, the neutophil attaches firmly to the endothelium

by the binding of their leukocyte functional antigen-1 (LFA-1) and compliment receptor-3 (CR-

3) to the intercellular adhesion molecules-1 (ICAM-1) on the surface of the endothelium. In the

third step, the neutrophil is able to squeeze between the endothelial cells and is able to penetrate









the basement membrane with the aid of enzymes that breakdown the extracellular matrix

proteins. The movement through the basement membrane is known as diapedesis. Finally, the

neutrophils follow a chemokine gradient to the site of the wound.30 The main function of

neutrophils at the wound site is to decontaminate the open wound by the destruction of invading

microbes. In the circumstance of injury to barrier surfaces, such as the skin, a very rapid and

effective response by neutrophils minimizes the chance for infectious complications.29

Macrophages themselves are differentiated monocytes, and result from monocytes

responding to certain chemical stimuli. There are known to be three types of macrophage

important to the wound environment; cytocidal, inflammatory, and repair macrophages.

Monocytes become inflammatory macrophages in the presence of 1,3-P-glucan, and differentiate

into repair macrophages in the presence of hyaluronan. Cytocidal macrophages are formed in

response to polyinosinate-polycytidylate and are "killer" cells that remove bacteria and other

debris from the wound site. Each type of macrophage produces different growth factors and

cytokines. Inflammatory macrophages produce PDGF, TGF-P, and bFGF. Repair macrophages,

which help remodel the extracellular matrix of the wound, produce IL-GF-1 and also PDGF. It is

the balance between the inflammatory and repair macrophage populations that appears to be

crucial for successful wound healing.31

Once activated by direct injury, the mast cell located at the wound edges releases, by

means of degranulation, the mediators essential to trigger the inflammatory reaction of the

injured tissue, which mainly influence the local endothelial cells. Mast cells, through the release

of vasoactive mediators, such as histamine, proteases, tumor necrosis factor, and metabolites of

archidonic acid induce vasodilation and increase vascular permeability.32









Histamine enhances the permeability of arterioles, capillaries, and venules to albumin, then

globulin and finally fibrinogen. Histamine increases the permeability of these vascular structures

by inducing contractions of the endothelial cells and partially removing fenestral diaphragms that

cover the gaps or fenestrae of the endothelium. Histamine is released from preformed and stored

sources as well. All known varieties of tissue injury cause the de novo synthesis of histamine.33

The kinins are biologically active and nearly indistinguishable peptides in areas of tissue

destruction. The most familiar kinin, bradykinin, is a potent inflammatory substance that is

released in injured tissue from plasma proteins by the plasma enzyme, kallikrein. The action of

the kinins on the microvasculature is similar to that of histamine. Kinins are rapidly destroyed by

tissue proteases, suggesting that their importance is limited to the early inflammatory stage of

wound healing.34

Proteases form a large group of enzymes that are capable of hydrolyzing proteins.

Proteases are grossly divided into two major groups, depending on their site of action.

Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate,

whereas endopeptidases cleave peptide bonds distant from the termini of the substrate. Based on

the functional groups present at the active site, proteases are further classified into four

prominent groups; serine proteases, aspartic proteases, cysteine proteases, and

metalloproteases.35

Serine proteases are characterized by the presence of a serine group in their active site.

They are numerous and wide spread among viruses, bacteria, and eukaryotes, suggesting that

they are vital to the organisms. Aspartic proteases, commonly known as acidic proteases, are the

endopeptidases that depend on aspartic acid residues for their catalytic activity. Cysteine

proteases occur in both prokayotes and eukaryotes. About 20 families of cysteine proteases have









been recognized. The activity of all cysteine proteases depends on a catalytic dyad consisting of

cysteine and histadine. Metalloproteases are the most diverse of the catalytic types of proteases.

They are characterized by the requirement for a divalent metal ion for their activity. About 30

families of metalloproteases have been recognized.35

The two main proteases involved with wound healing are the serine and metalloproteases.

Almost one third of all proteases can be classified as serine proteases. The serine proteases are

endopeptidases that have a catalytic site made up by three amino acids, serine, aspartic acid, and

histadine, the "catalytic triad." They represent a vast class of proteases with members with a

broad variety of functions. Nearly all members are synthesized as zymogens, which are activated

by proteolytic removal of an N-terminal part of the molecule that covers and hides the active site.

Serine protease can be further classified into four different clans, chymotrypsin, subtilisin,

carboxypeptidase Y, and Clp protease. Chymotrypsin-like proteases are the most abundant in

nature. Chymotrypsin-like proteases are involved in many critical physiological processes.

Cascades of sequential activation of serine proteases drive blood coagulation, complement

fixation, and fibrinolysis. Similar protease cascades appear to be involved in development,

matrix remodeling, differentiation, and wound healing.36

Matrix metalloproteases (MMPs) are zinc endopeptidases involved in numerous

physiological and pathological processes. There have been 24 MMPs identified in humans, with

a wide variety of substrates, such as, components of the extracellular matrix, cellular receptors,

growth factors, and cytokines. Functions of MMPs embrace tissue growth and remodeling during

development and disease, including endometrial cycling, angiogenesis, cellular migration,

skeletal formation, inflammation, wound healing, coagulation; periodontal, rheumatic,

cardiovascular, and pulmonary diseases, neuroinflammtion, cancer, and metastasis. Expression









of MMPs is tightly regulated at the transcriptional level by hormones, growth factors, cytokines,

and cell-to-cell and cell-to-matrix interactions.37

The MMPs alter the extracellular matrix during the process of wound healing. In wound

healing, MMPs control platelet aggregation, macrophage and neutrophil function, cell migration

and proliferation, neoangiogenesis, and collagen secretion and deposition. MMPs have a

significant role in all phases of wound healing. They promote cell migration, break down

damaged extracellular matrix, and assist with remodeling. However, a disparity between the

levels of MMPs and tissue inhibitors of metalloproteases (TIMPs) can be harmful because a

disproportionate MMP activity can result in an increased degradation of fibronectin, collagen,

and diverse growth factors. A normal fluctuation of MMPs is seen in healing wounds, whereas

chronic wounds tend to have prolonged elevations.38

Triggers such as microbial invaders, tissue injury, and surgical trauma activate the release

and formation of arachidonate derived prostaglandins, which regulate the early events in the

inflammatory response. At sites of inflammation, prostaglandins initiate many responses relevant

in inflammatory events, but most notably they signal the end of inflammation by activating the

transcriptional regulation of 15-lipoxygenase (15-LO) in neutrophils, which in turn leads to the

temporal dissociation and production of lipoxins from arachidonic acid, which have "pro-

resolving" and anti-inflammatory functions. This is referred to as class switching of the

arachidonic acid derived eicosanoids from prostaglandins and leukotrienes to lipoxins that

initiate the termination of inflammation. The omega-3 fatty acids eicosapentaenoic acid and

docosahexaenoic acid, EPA and DHA respectively, are converted to new families of lipid

mediators that are pivotal in promoting the resolution of inflammation. These new families of

lipid mediators are called resolvins and protecting.39









Proliferative Phase

The proliferative phase includes the most prominent events in wound healing. It begins

about 4-5 days after wounding and last for a few weeks. The main processes that take place

during this phase are angiogenesis, fibroplasia, re-epithelialization, and extracellular matrix

formation.40

Angiogenesis refers to new vessel growth or neovascularization and occurs in a wound in

at least three ways: generation of a de novo vascular network in the wound space, anastomosis to

preexisting vessels, and coupling or recoupling of vessels throughout the wound space. (Figure

2-15) Arising from new blood vessels adjacent to the wound, new capillary buds extend into the

wound itself34 The endothelial cell is the most important cell involved in angiogenesis, and

through intercellular adhesion molecules plays an active role in leukocyte migration into the

wound environment. Endothelial cells have other critical roles during the wound healing process,

including formation of new vessels providing transport of oxygen and nutrients into the wound

and secretion of biologically active substances. Soluble factors released by cells such as

macrophages are believed to stimulate angiogenesis during the wound healing process. In

addition, factors such as low oxygen tension, lactic acid, and biogenic amines can potentiate

angiogenesis.41

One of the most common and expected signs that wound healing is progressing in a

normal, orderly fashion is the development of granulation tissue. Granulation tissue that forms

during healing consists of new vessels that migrate into the wound and proliferate, along with the

accumulation of fibroblast and ground substances. The most important cell in the production of

the dermal matrix is the fibroblast. Fibroblasts migrate into the wound within 48 to 72 hours.34'41

Connective tissues are composed of three elements: cells, fibers, and amorphous ground

substance. The fibers and ground substance are collectively referred to as extracellular matrix.









Ground substance is an amorphous viscous gel secreted by fibroblast occupying the spaces

between the cells and fibers of the connective tissue. It helps determine compliance, flexibility,

and integrity of the dermis; provides strength, support, and density to tissue; reduces friction

between connective tissue fibers during tissue stress or strain; and protects tissue from invasion

by microorganisms.34

Fibroblasts migrate into the wound site from the surrounding tissue, become activated and

begin synthesizing collagen, and proliferate. PDGF and EGF are the main signals to fibroblasts

and are derived from platelets and macrophages. Fibroblasts already located in the wound site

begin synthesizing collagen and transform into myofibroblasts for wound contraction.42 The

phenotypic changes that occur to the fibroblast correlate with the cells' various roles in wound

healing. The first role of the fibroblast is migration. The fibroblast then begins producing large

amounts of matrix materials, including collagen, proteoglycans, and elastin. Type I collagen is

the major collagen in the normal adult dermis. Type III collagen, present in large quantities in

fetal dermis, is a minority component of normal adult collagen but during wound healing is the

predominant type of collagen synthesized. The new connective tissue also contains

glycosaminoglycans and proteoglycans. The synthesis of these matrix proteins occurs

concomitantly with the production of the new collagen. During this time, fibroblasts alter their

phenotypic character to become myofibroblast. As a myofibroblast, the cell can also participate
3441
in wound contracture.3441

The contractile forces produced by granulation tissue in wounds are derived from

myofibroblasts that contain contractile proteins. Wound contraction is thought to be a result of

these actin rich myofibroblast, which are the most prominent cells in granulation tissue.

Myofibroblast within the wound align along the lines of contraction and differ from other









cellular components, including leukocytes and endothelial cells, which do not exhibit such

organized orientation. This muscle like contraction of myofibroblast is mediated by PGF, 5-

hydroxytryptamine, angiotensin, vasopressin, bradykinins, epinephrine, and norepinephrine.

Contraction is unified and requires cell-cell and cell-matrix communication.34'41

Re-epithelialization is critical to optimal wound healing, not only because of the

reformation of the cutaneous barrier, but also because of its role in wound contraction.43 The

features observed near the wound margin and in the migrating epidermal tongue suggest that two

independent and complementary mechanisms are involved in wound reepithelialization.44 The

classic mechanism of epithelial cell migration over the wound surface is the "leap frog" model.34

In this model, epidermal cells migrate no more than two or three cell lengths from their initial

position, sliding or rolling over epidermal cells previously implanted. The migrating cells then

become fixed at this site. Other epidermal cells successively migrate over these cells. The

epidermal layer will progressively advance and close the epithelial defect.34 A second model of

reepithelialization is the "train" model. In this model each cell maintains its original position in

the chain. This continues until migration is halted by contact with other epidermal cells or by a

complete reepithelialization of the wound surface. In either model a migrating "tongue" of

epithelium develops.7'34

Growth factors play a role in signaling cell migration. Among the two that are thought to

be very important are TGF-3 and EGF. TGF-P, an important mediator in dermal growth

regulation, stimulates keratinocyte production of fibronectin and its deposition in the

extracellular matrix. TGF-3 also stimulates epidermal migration, but it is not known whether this

is solely an effect of increased fibronectin production. It is known that TGF-3 is a potent

inhibitor of keratinocyte proliferation in vitro, and while these cells are migrating they do not









proliferate. This may allow the cells at the forefront to remain in migrating mode. Until the

wound is closed, a zone of proliferating cells remains between the migrating tongue of

epithelium and the normal cells at the wound edge. At that point all of the keratinocytes enter the

proliferative mode. The stimulus for cells undergoing rapid proliferation is not known, however,

several growth factors maybe involved, including EGF and its homologue TGF-a. In culture,

EGF induces hypertrophy and hyperplasia of epithelial cells; this and its presence in human

epidermis suggest a role as a stimulus for epidermal cell proliferation in vivo.41

Remodeling Phase

The maturation or remodeling phase is the final stage of wound healing. Collagen fibers

reorganize in this phase. Fibroblast, MMPs, and the inhibitors of MMPs play a role in this

process, as do some of the growth factors.45 However, the main feature of this phase is the

deposition of collagen in an organized and well mannered network.42

The clinical manifestations of remodeling include contraction, decreased redness,

decreased thickness, decreased induration, and increased strength. Although these changes

continue to develop over a period of weeks, months, and even years their initiation overlaps the

production of granulation tissue during the proliferative phase of wound healing. Wound

contraction is a cell directed process: cell division is required, but collagen synthesis is not. In an

unsutured wound, wound edges move toward one another at a rate of approximately 0.6 to 0.75

mm/day.18

Net collagen synthesis will continue for at least 4 to 5 weeks after wounding. The collagen

that is initially laid down is thinner than collagen in uninjured skin and is oriented parallel to the

skin. Over time the initial collagen threads are reabsorbed and deposited thicker and organized

along the stress lines of the wound. These changes are also accompanied by a wound with an

increase in tensile strength, indicating a positive correlation between collagen fiber thickness /









orientation and tensile strength. The collagen found in granulation tissue is biochemically

different from collagen found in uninjured skin. Granulation tissue collagen has a greater

hydroxylation and glycosylation of lysine residues, and this increase of glycosylation correlates

with the thinner fiber size. The collagen in the scar will never become as organized as the

collagen found in uninjured skin. Wound strength also never returns to 100%. At 1 week, the

wound has only 3% of its final strength; at 3 weeks, 30%; and at 3 months and beyond,

approximately 80%.42

As scars mature, they become less red. This change mirrors a change in the density of

capillaries within the wound over time. Whereas young wounds are characterized by granulation

tissue containing a relatively high level of capillary density, mature wounds are less vascular.

Fibroblasts also decrease over time. Together, these changes result in a relatively acellular

mature scar. The absence of appendages is another characteristic associated with mature scars.

The most clinically obvious appendageal loss involves missing hair follicles. The inability of

scar tissue to reproduce a suitable appendage specific niche may contribute to the absence of

these structures in mature scars.18

Growth Factors

Growth factors and cytokines are both members of a larger group of polypeptide regulatory

molecules released by many cell lines in the body. In fact, although they have often been

described separately from cytokines, growth factors are cytokines whose primary role is

directing the maturation of cells during normal turnover and in the post injury tissue repair

response.46 It has been shown that the fibrocytes from bur injured patients produce higher levels

of the growth factors TGF-P and CTGF than non burned patients.47









Transforming Growth Factor-Beta

The TGF-P super family consists of a diverse range of proteins that regulate many different

physiological processes, including embryonic development, homeostasis, and wound healing,

chemotaxis, and cell cycle control. The TGF-P super family includes nearly 30 proteins in

mammals. 3 Initially when discovered, TGF-P was termed sarcoma growth factor. Five different

mature forms of TGF-P have been isolated and designated 1 through 5 based on their

chronological identification.48

TGF-P is a major modulator of wound healing. It is constitutionally present in platelets, but

is also produced by several cell types present in wounds, including activated macrophages,

granulocytes, fibroblast, and keratinocytes. TGF-P receptors are widely distributed and found on

essentially all cell types. TGF-P is released as an inactive peptide bound to its propeptide, and

requires activation either by proteolysis or as a result of the acid environment found within a

wound. 2 Over expression can be unfavorable to wound repair, and TGF-P can induce its own

expression through a positive feed back loop. This can lead to elevated scaring and tissue

damage.49

TGF-P directs the functions of monocytes, endothelial cells, fibroblast, and keratinocytes

during all phases of wound healing, and is one of the most powerful chemoattractants.48

Local release of TGF-P at the site of tissue damage comes primarily from a-granules of

platelets. Once released, one of the initial actions of TGF-P is recruitment of inflammatory cells,

primarily neutrophils and macrophages. These cells are stimulated in a positive feedback

mechanism to produce more TGF-P to perpetuate its activity.48

There is conflicting evidence as to whether TGF-P promotes or inhibits angiogenesis. A

report by Goumans et al, suggests that the direction TGF-P takes endothelial cells, stimulatory or

inhibitory, may depend on the particular R-Smad that becomes activated intracellularly.48









The relationship of fibroblast and TGF-P is fundamental to the proliferative and

maturational phases of wound healing. TGF-P stimulates fibroblast to proliferate and migrate

into a wound while producing extracellular matrix. This extracellular matrix allows other healing

processes to occur. Fibroblast transform into myofibroblast under the stimulation of TGF-P,

which causes wound contraction and maturation.48

Epithelialization is the process in which keratinocytes both proliferate and migrate from

wound edges to create a barrier over the wound. The overall effect of TGF-P in this process is

not as fully understood as its other functions because studies have suggested that it both inhibits

and stimulates keratinocytes. TGF-P does influence keratinocytes and epithelialization of

wounds, but whether it plays an overall stimulatory or inhibitory role is yet to be fully

determined.48

A summary of some of the functions of TGF-P

Three isoforms; TGF-1, TGF-02, TGF-33
Is up regulated in human cancers
Plays crucial role in tissue regeneration, differentiation, embryonic development
synthesis of matrix proteins
metalloproteinase inhibitors
[ levels of proteolytic enzymes
Excess TGF-P can lead to hypertrophic scar formation
TGF-P inhibits production of IFN-a and IL-2
TGF-P promotes IL-10 production, suppressing macrophage function
TGF-P appears to shift immune reactions to a Th-2 response


Connective Tissue Growth Factor

In adult skin, CTGF normally is not expressed unless induced, for example, during the

normal wound repair process.3 CTGF is a 38 kD protein that contains 38 conserved cysteine

residues and a heparin binding domain and is chemotactic and mitogenic for connective tissue

cells. CTGF functions as a downstream mediator of TGF-P action on connective tissue cells,









stimulating cell proliferation and extracellular matrix synthesis. The induction of CTGF by TGF-

p is cell type specific, as it occurs in connective tissue cells but not epithelial cells or

lymphocytes. In addition to fibroblast; endothelial cells, vascular smooth muscle cells, epithelial

cells, and chondrocytes produce CTGF mRNA.50

CTGF, a member of the CCN family of matricellular proteins, promotes fibroblast

proliferation, matrix production, and granulation tissue formation. CTGF promotes cell adhesion

and migration of a wide variety of cell types as well as collagen matrix contraction in fibroblast.

Although, a specific CTGF receptor has yet to be identified, CTGF appears to perform many of

its functions through integrins, heparin sulfate containing proteoglycans, and the low density

lipoprotein receptor related protein.3 Studies have shown that rhCTGF has been successful in

treating burn injuries in non human primates.5

A summary of some of the functions of CTGF

CTGF is secreted by fibroblast after activation by TGF-P
CTGF is a downstream mediator of TGF-P
CTGF stimulates cell proliferation and ECM synthesis
CTGF is a more specific target for selective intervention


Hormones

The term "hormone" comes from the Greek "hormaein". The original use of hormone

implied an agent that can excite or arouse; in the modern era a hormone is perceived to be a

defined chemical entity that is secreted or produced by specific glands or cells and that acts as a

chemical messenger or signal molecule.52

Androgens are steroid hormones that induce the differentiation and maturation of the male

reproductive organs, the development of male secondary sex characteristics, and the behavioral

manifestations consistent with the male role in reproduction. The two most important steroid









hormones of the adult male are testosterone and 5a-dihydrotestosterone. Testosterone is the

principal male androgen produced by the testes. The testes also produce approximately 10-20%

of the estrogens found in males; the remainder is produced in a variety of nonendocrine tissues

including brain, liver, fat, and skin, all of which have low levels of the cytochrome P450

aromatase necessary to transform androgens into estrogens.5

The two most important steroid hormones of the adult female are estradiol and

progesterone. There are three naturally occurring estrogens in women, estradiol, estriol, and

estrone. 17p-estradiol is the most abundant of the estrogens from puberty until menopause.

Estrone is weaker than estradiol, and in post menopausal women more estrone is present than

estradiol.

Menopause is defined as the cessation of ovulation by the ovaries. This occurs in women

between the ages of 40 and 50 years due to the utilization of the fixed number of follicles that

were established in the fetal ovaries. The endocrinological consequences of cessation of

ovulation are varied, and a variety of medical problems may develop, depending upon the exact

nature of the reproductive hormonal balance achieved. While the postmenopausal ovary is not

totally devoid of the ability to secrete steroids, the production level is markedly reduced in

comparison to a younger ovulating woman.52

Estrogen

There is striking evidence from animal studies dating back to 1962 that estrogens play a

crucial role in cutaneous wound healing, and repair is significantly delayed in its absence.53 The

presence of estrogen receptors (ER) in human skin fibroblast and inflammatory cells suggest that

local estrogen levels may influence cutaneous physiology, including the wound healing

process.53 One way that estrogen may influence wound healing rates is by the regulation of the

growth factor TGF-P. TGF-1 is the cytokine that has received the greatest attention in the









context of wound repair. TGF-3 is involved in cell proliferation, differentiation and matrix

production, and also scar formation. In experiments by Ashcroft, et al wound healing rates

among post menopausal women and men were determined to be relatively equal while pre

menopausal women had faster healing rates. This was thought to be secondary to the increased

levels of estrogen in the pre menopausal women. Wound healing rates among the post

menopausal women and men were shown to improve with the addition of a topical estrogen

patch. Although, estrogen has been shown to improve wound healing rates a negative sequale

was noted. The pre menopausal women had the quicker healing rates, but also the more

hypertrophic scarring. The scars of the post menopausal women and men were consistently pale

and flat, compared with the pigmented, everted lesions of the pre menopausal women.54

A summary of how estrogen assists in wound healing

Estrogen accelerates wound healing by dampening local inflammation
Estrogen suppresses the chemotaxis of PMNs
Estrogen alters the expression of endothelial adhesion molecules
With a decrease in the numbers of PMNs found in the wound, there is also a decrease in
Elastase activity
Elastase is a serine protease derived from PMNs
Elastase degrades structural and functional proteins, such as, proteoglycans, collagen,
and fibronectin
Fibronectin directs the migration of keratinocytes, increases the amount of fibroblast and
collagen deposition, and stimulates wound contraction




























Cross section of human skin showing the two layers of the epidermis and dermis
with the underlying hypodermis that connects the skin to underlying structures
(http://www.realage.com/realbeauty/lm.aspx. last accessed Febuary 13th, 2007).5


Epidermis --


Dermis -



Hypodermis -


Figure 2-1.















Stramlw ornewum


Strattn luciduin
Stratun graniulosin



Stratiam spinosum




Sirattui basale


0E anne_rats


Figure 2-2. Cross section of the five layers that make up the
epidermis(http://www.ratbehavior.org/images/epidermis, last accessed Febuary 13th
2007)56
































Basement membrane (1) Tonofilaments provide stability between cells, (2)
desmosomes that stabilize cells with each other, (3) hemidesomsomes that keep
cells attached to the basement membrane, (4) basement
membrane(http://www.unifr. ch/hi stologie/elearningfree/francais/epitel/epithel05.h
tml Last accessed Febuary 13th, 2007) 57


Figure 2-3.








































Dermal papillae interdigitates with the epidermis to strengthen the connection
between the two layers(www.cytochemistry.net/microanatomy/skin/skin_and
mammary_glands.html, last accessed.Julyl3, 2007).58


Figure 2-4.


1--7-7,71




































I- SPt, f E(J"I COrne I


2 F1- t CPtr10t f rp4(
6 C pit "awl b Cni Ldp IL


Aoflt C~T4 91c1.i (Aj)t4Z W
0 old, 4Wnl f~f

8Yusa1 (jmwint
EjaLH~A" n f a~~Lk bc


Diagram of fibroblast laying down collagen fiber bundles from the precursor
tropocollagen(www.udel.edu/biology/wags/histopage/illuspace/icta/icta6.gif. last
accessed Febuary 13th, 2007)59


Figure 2-5.


i sh'.)






















Macrophage.


Elastic fibres

Mast cell-


Figure 2-6.


-Basal lamina

Fibroblasts



Capillary




GAGs


Cells and other proteins that make up the extracellular matrix
(www.steve.gb.com/science/extracelluarmatrix.htm, last accessed Febuary 13th,
2007).60
















Sweet glnd


J Epidermia


Dermls

) Subcutlrmam

-- Fat ca


-Doeml pIapa
6Subeou4 gand


Cross section of skin showing various appendages including hair follicle, and
sweat glands(www.tuffrhino.com/artcles.asp?id= 113, last accessed Febuary 13,
2007)61


Figure 2-7.












Finger6Nad


nail bed finger bone


nail root iermris


The nails are another appendage of skin, 90% of the nail made up of hard "hair"
keratin, the remainder is made of soft "epidermal"
keratin(www.becomehealthynow.com/popups/nails.htm, last accessed Febuary
13, 2007).62


I


Figure 2-8.














Hair fibre
Medulla
Sebaceous gland



"Bulge" region
Inner root sheath

Outer root sheath

Dermal papilla


Diagram showing structure of the hair follicle and relation to epidermis and
dermis(www.thegentletouch.com/hairbiol/h-morph3.html. Last accessed
February 13, 2007).63


Epidermis


Dermis







Subcutaneous
tissue


Figure 2-9.









padeirn mIs


ha"ir
to I 1i CI'


M abix


Figure 2-10.


msl over


Hair follicle: Hair follicle stem cell is responsible for the regeneration of hair and
sebaceous glands, They are not responsible for long term replenishment of
epidermis, although they can contribute during wound healing
(www.virtualaboratory.net/biofundamentals/lecturenotes/topic5-3_stemcells.htm.,
Last accessed Febauary 13, 2007)64


basal kayer









Contact Factor Pathway


Kallikrein
,HK
XII


Figure 2-11.


4 Prekallikrein


Tissue Damage


tHK +
HK Tissue Factor
- XIIa I
VIIa xi-C Xa

XI XIa VIIa ^ VII


IXa
SL, CaC
VIII ILA Vi llat

Xa
L, CHa
v Ila a

lHa

Fibrinogen Fibrin Monomer


Soft Fibrin Clot

XIII IIa XIla I
Ca
Hard Fihrin Clat


Pro enzyme-enzyme pathway leading to hard fibrin clot during the process of
hemostasis (www.facstaff.bloomu.edu/tbel2/research-blood.htm., Febuary 13,
2007)65


Tissue Factor Pathway









AadW-e h-* km
bt4 ph.Fw pktwdyicclikr.W


PMtp44lp A,.


Pqa"i / \, C


faW ITE 12 -PETE










p 4 1|


LTa


Figure 2-12.


Generalized pathway for the conversion of arachidonic acid to ecosanoids. COX,
cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; HPETE,
hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; PG,
prostaglandin; TX, thromboxane (Reprinted with permission from Am. J.
Clin.Nutr.2006; 83:1505 S-1519S).27


mtT
/I\






LM.


\tE










Steps of the
Irnliamm.-itur Respornsc-
The infmmmawo~y respiume is a ba y*s
B.ofld liroo of dolenrs against inrwaion
by padwyns. Why Is It IMPlratant th'at
cditting facst-um the -iroitorv -yk
tami han accet to tba Inavied uara




.- I -~-- -
-


Figure 2-13.


The inflammatory phase of wound healing encompasses many processes going
on at the same time(www.biologymad.com/immunology/inflammation.jpg., Last
accessed July 30th, 2007)66










































Figure 2-14. Diagram showing the process of diapedesis and extravastion of neutrophils
during the early inflammatory process(Reprinted from Immunobiology: the
immune system in health and disease. 2005; 6th edition: 37-100).30


Btood flwY --1













V' I P. Lup .psit I





pnkiwWipn


K






' LO' frn wn


V cuLwlIr l b ubL&


Figure 2-15.


Angiogenesis is one of the major processes that take place during the
proliferative phase of wound healing
(www.angio.org/providers/woundcar/woundcare.html., Last accessed July28th,
2007)67









CHAPTER 3
MATERIALS AND METHODS

Tissue Collection

This project was approved by the University of Florida Institutional Review Board (IRB)

on October 5, 2005. All tissue samples were collected in the operating room. All burn tissue

samples were collected in the burn unit operating room except for patient number 38 which was

collected in the main operating room at Shands at the University of Florida. Tissue samples

collected from patients undergoing elective plastic surgery procedure were collected at Alachua

General Hospital, Ayers Surgical Center, Florida Surgical Center, or Shands at the University of

Florida. One di-potassium ethylenediaminetetraacetic acid (K2 EDTA) Vactutainer (BD

Vacutainer, North America) of blood was collected either from an existing intravenous line or by

venipuncture. Tissue samples collected from burned injured patients and from non burned plastic

surgery patients were collected using a 6 mm punch biopsy tool.

Biopsies were taken from excess skin debrided during surgery. Biopsies were taken from

the burned injured area, from the donor site of the same patient and from excess skin debrided

during normal plastic surgery procedures. Tissue from one 6 mm punch biopsy was placed in a

10% buffered formalin solution; tissue from another 6 mm punch biopsy was placed in RNAlater

solution. These samples were placed in a 40 C cooler for 24 48 hours after which the tissue

placed in 10% buffered formalin solution was taken to be imbedded by an independent

contractor in the department of Anatomy and Cell Biology at the University of Florida. The

samples were placed in paraffin block and fixed to slides. The tissue samples that were placed in

RNAlater solution were transferred to separate microcentifuge tubes labeled and stored in -80 C

freezer to be archived for future studies









After tissue samples were collected the blood was spun down in order to collect the

plasma. The blood was spun for 10 minutes at 40 C at 3000 RPM's in an Eppendorf 5810R with

the acceleration and brake set at 9. Approximately 1.5 ml of plasma was obtained and aliquoted

into 3 separate microcentifuge tubes of approximately 0.5 ml each. These tubes were labeled and

stored in a -80 C freezer. Only one of these tubes was needed for this study, the extra tubes of

plasma were archived for future studies. .

Immunostaining for CTGF

Immunohistochemistry staining for CTGF was done first by deparafinizing slides as

follows:

* Xylene-2 minutes
* Xylene-2 minutes
* 100% ethyl alcohol-1 minute
* 95% ethyl alcohol-1 minute
* 75% ethyl alcohol-1 minute
* 50% ethyl alcohol-1 minute
* Distilled H20-1 minute


The slides were then laid out on a tray and wax circles were immediately made around

samples using ImmEdge pen (Vector Laboratories, Burlingame, Ca., USA) Blocking buffer, 1%

fetal bovine serum (Gibco/Invitrogen Corp, Carlsbad, Ca.) in PBS, was applied to each sample.

Non-specific receptor blocking was allowed to take place for 3.5 hours. After blocking, the

blocking buffer was removed from the right (test) side only, while the left (control) side

remained covered in blocking buffer. The 1 antibody (1 lg/ml CTGF obtained from Dr. Gary

Grotendorst, Lovelace, New Mexico) was then applied to the test side only and was allowed to

incubate in a humidity chamber over night. The next morning 1 antibody was collected and

saved to be used in other experiments. The slides were then washed in PBS for 2 minutes and 1

minute, respectively. One drop of 20 antibody (biotinylated rabbit anti-goat, Vector Laboratories,









Burlingame, Ca.) was added to 10 ml of blocking buffer and applied to both control and test

sides of slides. The 20 antibody was allowed to incubate for 30 minutes. The slides were then

washed 3 times in PBS for 2 minutes, 2 minutes, and 1 minute. The ABC-AP working solution

was made by adding 2 drops of reagent A and 2 drops of reagent B to 10 ml of blocking buffer,

this solution was allowed to stand for 30 minutes prior to use. The slides were next allowed to

incubate for 30 minutes in VECTASTAIN ABC-AP Reagent (Alkaline Phosphatase, Vector

Laboratories, Burlingame, Ca.). Slides were washed 3 times in PBS for 2 minutes, 2 minutes,

and 1 minute. After the final wash, slides were placed in tub of substrate (Vector Red, Vector

Laboratories, Burlingame, Ca.). The substrate was made as follows:

Substrate

* To 200 mL of 100 mM Tris-HCL, (pH 8.2-8.5), approximately 80 drops of reagent 1 was
added
* 80 drops of reagent 2
* 80 drops of reagent 3
* 40 drops of levamisol was added to inhibit the endogenous alkaline phosphatase activity


The slides were allowed to incubate in this substrate for 20 minutes, followed by

dehydration as follows:

* Distilled H20-1 minute
* 50% ethyl alcohol-1 minute
* 75% ethyl alcohol-1 minute
* 95% ethyl alcohol -1 minute
* 100% ethyl alcohol-1 minute
* Xylene-2 minutes
* Xylene-2 minutes

After dehydration the samples were mounted with Permount (Fisher Scientific, USA) and

cover slips were applied.









Immunostaining for TGF-p

Immunohistochemistry staining for TGF-3 was initiated by first making all solutions

necessary for assay, solutions were made as follows:

* 10 antibody, 500kg of mouse monoclonal anti-human TGF-3 antibody (R&D Systems,
Minneapolis, Mn., USA) was reconstituted with ImL of sterile PBS.
* 1% H202 solution was made to quench endogenous peroxidase acitity
* Blocking serum was made by combining 75 Cl of normal blocking serum (goat) with 5ml
of stock PBS
* Biotinylated 20 antibody was made by combining 75Cl normal blocking serum (goat), 5ml
stock PBS, and 251l biotinylated 20 antibody stock
* AB enzyme reagent was made by combining 50Cl reagent A (avidin), 50pl reagent B
(biotinylated HRP), and 2.5 ml PBS. This was allowed to stand for 30 minutes prior to use
* Peroxidase substrate was made by combining 1.6 ml dH20, 10 drops of 10x substrate
buffer, 1 drop 50x DAB (diaminobenzidine), and 1 drop peroxidase substrate

Immunohistochemistry staining for TGF-P was done using pre made kit (Santa Cruz

Biotechnolgy, Santa Cruz, Ca, USA), procedure started as follows:

Deparafinize

* Xylene-2 minutes
* Xylene-2 minutes
* 100% ethyl alcohol-1 minute
* 95% ethyl alcohol -1 minute
* 75% ethyl alcohol-1 minute
* 50% ethyl alcohol-1 minute
* Distilled H20-1 minute

After the slides were deparafinized, the slides were then laid out on a tray and wax circles

were immediately made around samples using ImmEdge pen (Vector Laboratories, Burlingame,

Ca., USA). They were then incubated in a 1% hydrogen peroxide solution for 10 minutes to

quench the endogenous peroxidase activity. This was followed by washing with PBS two washes

of 5 minutes each. The slides were then blocked with 1.5% blocking solution made earlier. Non

specific blocking was allowed to take place for one hour. After one hour the blocking buffer was

removed from the test (right) side only while blocking buffer was allowed to stay on control









(left) side. The 10 antibody was then applied to test side and allowed to incubate for 30 minutes.

The 10 antibody was titrated to 5 g/ml concentration for this assay. Following incubation with

the 10 antibody the slides were washed with PBS three washes of 5 minutes each. The slides

were next incubated with the biotinylated 20 antibody for 30 minutes, followed by three washes

of PBS for 5 minutes each. The slides were then allowed to incubate with the AB enzyme

reagent for 30 minutes, again followed by three washes of PBS for 5 minutes each. The substrate

was now applied and allowed to incubate for 10 minutes. Following incubation with the substrate

the slides were dehydrated as follows:

Dehydration

* dH20-1 minute
* 50% ETOH-1 minute
* 75% ETOH-1 minute
* 95% ETOH -1 minute
* 100% ETOH-1 minute
* Xylene-2 minutes
* Xylene-2 minutes

After dehydration the samples were mounted with Permount (Fisher Scientific, USA) and

cover slip applied. After all immunostaining was complete Zeiss Axiovision microscope was

used to take photographs of sample at varying magnifications. After all photographs were taken

they were analyzed using Image J68 to quantify the level of growth factor staining.










Estradiol EIA Kit


The estrogen concentration of each sample was assayed using Estradiol EIA Kit (Cayman

Chemicals, Ann Arbor, Michigan, USA). Prior to beginning the assay all solutions and buffers

were made as follows:

EIA Buffer

* Dilute the contents of one vial of EIA buffer concentrate (Vial #4) with 90 ml of Ultra Pure
water

Wash Buffer

* Dilute the contents of the vial (5 ml) of wash buffer concentrate (vial #5) to a volume of 2
liters with Ultra Pure water, then add 1 ml of Tween 20 (vial 5a)


Estradiol Standard

* A pipette tip was equilibrated with ethanol, using the equilibrated pipette tip 1001l of
Estradiol Standard (vial #3) was placed into a clean test tube, then diluted with 900.l Ultra
Pure water. Bulk standard concentration will be lOng/ml
* Eight clean test tubes numbered 1 through 8 were labled
* 900p1l EIA Buffer was aliquoted into tube #1
* 5001 EIA buffer was aliquoted into tubes #2 through #8
* 1001 of bulk standard (10ng/ml) was transferred into tube #1 and mixed thoroughly
* Serial dilutions were made by removing 5001 from tube #1 and placing it in tube #2,
mixed thoroughly
* 5001 was removed from tube #2 and placed into tube #3, mixed thoroughly
* The process was repeated for tubes #4 through #8

Estradiol Acetylcholinesterase Tracer

* 100 dtn estradiol tracer (vial #2) was reconstituted with 6ml of EIA buffer

Estradiol Antiserum

* 100 dtn estradiol antiserum (vial #1) was reconstituted with 6 ml EIA buffer


Plate Set Up

* See table 3-1











Performing the Assay


EIA Buffer

* 100l EIA buffer was added to Non Specific Binding (NSB) wells and 501l EIA buffer to
Maximum Binding (Bo) wells

Estradiol Standard

* 50!.l from tube #8 was added to both of the lowest standard wells (S8)
* 50l from tube #7 was placed in the next lowest standard wells (S7)
* This process was continued until all the standard wells were aliquoted
* The same pipette tip was used to aliquot all of the samples, the tip was equilibrated in each
standard prior to pipetting that standard

Samples

* 501l of sample was added to corresponding wells

Estradiol Acetylcholinesterase Tracer

* 50l of estridiol tracer was added to each well except the Total Activity (TA) and Blank
(Blk) wells

Estradiol Antiserum

* 50tl of estradiol antiserum was added to each well except the Total Activity (TA), the Non
Specific Binding (NSB), and the Blank (Blk) wells


After all wells were filled the plate was covered with plastic film and allowed to incubate

for one hour at room temperature. During the one hour incubation 100dtn Ellman's Reagent (vial

#8) was reconstituted with 20ml of Ultra Pure water. Since Ellman's Reagent is unstable once it

is prepared it was protected from the light. When the one hour incubation was finished the wells

were emptied and washed five times with wash buffer, followed by the addition of 2001l of

Ellman's to each well and 5 l of tracer to the Total Activity (TA) wells. The plate was then









covered with plastic film and allowed to incubate in the dark for one hour. After this final

incubation the plate was read using Wallac 1420 multilabel counter.












Table 3-1. Plate 1 set up for Estradiol EIA


1 2 3 4 5 6 7 8 9 10 11 12

A Blank Blank Blank S5 S5 S5 04-00-01 04-00-01 04-00-01 09-00-03 09-00-03 09-00-03

B NSB NSB NSB S6 S6 S6 05-00-01 05-00-01 05-00-01 09-00-04 09-00-04 09-00-04

C Bo Bo Bo S7 S7 S7 06-00-01 06-00-01 06-00-01 10-00-01 10-00-01 10-00-01

D TA TA TA S8 S8 S8 06-00-02 06-00-02 06-00-02 11-00-01 11-00-01 11-00-01

E S1 S1 S1 01-00-01 01-00-01 01-00-01 07-00-01 07-00-01 07-00-01 11-00-02 11-00-02 11-00-02

F S2 S2 S2 02-00-01 02-00-01 02-00-01 08-00-01 08-00-01 08-00-01 11-00-03 11-00-03 11-00-03

G S3 S3 S3 02-00-02 02-00-02 02-00-02 09-00-01 09-00-01 09-00-01 12-00-01 12-00-01 12-00-01

H S4 S4 S4 03-00-01 03-00-01 03-00-01 09-00-02 09-00-02 09-00-02 12-00-02 12-00-02 12-00-02




Table 3-la. Plate 2 set up for Estradiol EIA
1 2 3 4 5 6 7 8 9 10 11 12


A 13-00-01 13-00-01 13-00-01 19-00-02 19-00-02 19-00-02 26-00-01 26-00-01 26-00-01 34-00-01 34-00-01 34-00-01


B 14-00-01 14-00-01 14-00-01 20-00-01 20-00-01 20-00-01 27-00-01 27-00-01 27-00-01 35-00-01 35-00-01 35-00-01


C 15-00-01 15-00-01 15-00-01 21-00-01 21-00-01 21-00-01 28-00-01 28-00-01 28-00-01 36-00-01 36-00-01 36-00-01


D 16-00-01 16-00-01 16-00-01 22-00-01 22-00-01 22-00-01 29-00-01 29-00-01 29-00-01 37-00-01 37-00-01 37-00-01


E 17-00-01 17-00-01 17-00-01 22-00-02 22-00-02 22-00-02 30-00-01 30-00-01 30-00-01 38-00-01 38-00-01 38-00-01


F 17-00-02 17-00-02 17-00-02 23-00-01 23-00-01 23-00-01 31-00-01 31-00-01 31-00-01 39-00-01 39-00-01 39-00-01


G 18-00-01 18-00-01 18-00-01 24-00-01 24-00-01 24-00-01 32-00-01 32-00-01 32-00-01 40-00-01 40-00-01 40-00-01


H 19-00-01 19-00-01 19-00-01 25-00-01 25-00-01 25-00-01 33-00-01 33-00-01 33-00-01 12-00-03 12-00-03 12-00-03










CHAPTER 4
RESULTS AND DISCUSSION

The normal estrogen levels of pre-menopausal women vary greatly during the menstrual

period (Table 4-1). Although, estrogen levels decline as women get older, post menopausal

women still produce estrogen albeit at a much lower amount. Men are also producers of small

amounts of estrogen. Estrogens and androgens are synthesized from the neutral lipid cholesterol.

Dehydroepiandrosterone (DHEA), the precursor of both androgens and estrogens, is synthesized

from cholesterol as a result of a series of transformations. It is subsequently converted to

testosterone via either androstenedione (ADione) or androstenediol (ADiol). The estrogens

estrone and 17p-estradiol are respectively synthesized from ADione and testosterone by the

action of the enzyme aromatase.69

As far as was known there was only one patient on hormone replacement therapy (HRT)

and one patient on an estrogen inhibitor. Both of these patients were from the non burned injured

group so that their estradiol levels were not affected by burn injury. The patient on HRT was

taking Estratest (Solvay Pharmaceuticals, Inc.) which is a mixture of the sodium salts of the

sulfate esters of the estrogenic substances. The primary source of estrogen in normally cycling

adult women is the ovarian follicle and estradiol (E2) is the principal intracellular human

estrogen and is substantially more potent than its metabolites, estrone and estriol, however after

menopause most endogenous estrogen is produced by conversion of androstenedione, secreted

by the adrenal cortex, to estrone by peripheral tissues. Therefore, estrone and the sulfate

conjugated for estrone sulfate are the most abundant circulating estrogens in post menopausal

women.70 The patient on estrogen inhibitor was taking Femara (Novartis). In post menopausal

women estrogens are mainly derived from the action of the aromatase enzyme, which converts

adrenal androgens to estrone and estridiol. Femara (Letrozole) is a nonsteroidal competitive









inhibitor of the aromatase enzyme system; blocking conversion of androgens to estrogens.71 The

difference in estradiol levels of each of these individuals can be seen in Figure 4-1. Each of these

patients were considered to be post menopausal, the patient on estrogen inhibitor is a 57 year old

female and was taking the estrogen inhibitor because of a diagnosis of breast cancer and the

patient taking the estrogen replacement although only 39 years old was status post total

hysterectomy. Although showing a large difference in range both of these values lie within the

normal range for a pre menopausal female in mid cycle (Table 4-1). Although, on estrogen

inhibitor, this 57 year old patient, in this study exhibited an estradiol level above what is

considered normal for post menopausal females. This could be due to continued estrogen

synthesis by the liver, adrenals, or other peripheral organs including fat and muscle tissue.

Something of interest that was noted was the estradiol levels of the female with the highest

percentage of total body surface area burned (TBSA) and the male with the highest TBSA. The

male had a TBSA that was almost twice as much as the female that coincided with an estradiol

level that was almost twice that of the female. This led to the thought that maybe TBSA had

something to do with the amount of estrogen produced following injury (Figure 4-1), however no

literature could be found to support this idea and the rest of the data did not support this trend.

The estradiol levels of the male and post menopausal patients showed a significant increase

above normal values (Figure 4-2). This is consistent with studies that show increased estradiol

levels in men and post menopausal women following trauma such as burns.72 Other situations

that could raise estradiol levels among these groups include other forms of trauma, sepsis, how

over weight an individual is as fat cells produce more estrogen, and how much alcohol some one

consumes.72,73 However, the pre menopausal group had estradiol levels that were with in the

normal range. This could be because the male and post menopausal groups had a significantly









lower estradiol level prior to injury and due to the trauma of bum injury and sepsis following

injury they produced more estradiol than the pre menopausal group who already had a significant

amount of estradiol on board at time of injury and therefore did not need to produce as much. An

ANOVA statistical analysis was performed (p=0.9) which indicates that there is not a statistical

difference in these three groups.

These initial estradiol levels were taken at time of the first surgical debridement following

burn injury the average days for each group are listed in Figure 4-2. There were 8 patients that

received a second surgical debridement (Figure 4-3) of these 8 patients, 5 were male and 3 were

female. A two-tailed T-test was performed (p=0.8) which indicated that there was not a

significant difference in male and female estradiol levels at the time of the second surgical

debridement. The females were not separated into pre or post menopausal because the total

number of females receiving a second debridement was so small. There were only 2 females and

no males that received a third debridement. Figure 4-4 shows the decline in estradiol levels of

females at the first, second, and third debridements. An ANOVA statistical analysis was

performed on this data (p=0.6) which indicates no significant difference among these groups.

There was not a decrease in estradiol levels from the second to third debridements. The sample

size of females receiving a second and third debridement being so small, n=3 and n=2

respectively, could skew the results and give a false correlation between estradiol levels

following injury and time since injury.

Previously mentioned was the fact that the male patient with the highest percentage of

TBSA also had an estradiol level twice that of the female with the highest TBSA. Figure 4-5

shows a break down of estradiol levels based on percentage TBSA for males, females, and

"normal" non burn injured males and females. An ANOVA statistical analysis was performed









(p=0.9) which indicates no significant difference among these groups. The largest proportion of

patients fall into a category of TBSA <19 percent, which is consistent with data from the

American Burn Association1. The estradiol levels except for one or two outliers all fall between

100 and 600 pg/ml which is noted to be the normal range for pre menopausal females.74

Something unusual that was noted was the estradiol levels of the "normal" non burned males,

both of which were well above the normal expected values for adult males. There could be a

number of reasons for this, a closer look at body mass index (BMI), medications (prescribed and

OTC), alcohol intake, illegal drug use as well as other parameters could give a better explanation

for these two values being so much higher than expected.73'72

The estradiol levels are also listed according to age (Figure 4-6). Again, all values range

between 100 and 600 pg/ml which is consistent with the estradiol levels from Figure 4-5, and

again an ANOVA statistical analysis was performed (p=0.4) which again indicates no significant

difference among groups. What is interesting is that there is no clear cut difference among age

groups. What would be interesting to see would be estradiol levels from these same patients prior

to injury to compare estradiol levels prior to injury and after injury and see which group actually

had the most increase? Another piece of information that would have been interesting to know

would have been asking the pre menopausal females where they were in their menstrual cycle as

the estradiol levels peak as they get closer to mid cycle and then start to decline again. Another

good question would be, following thermal bum injury, do estradiol levels go up more when

women are in the early, late, or mid cycle? It is probably not probable that all pre menopausal

women were near mid cycle as the reason that their estradiol levels did not show as a dramatic an

increase as the post menopausal and male groups.









Estrogens increase TGF-P secretion by dermal fibroblast in vitro, regardless of age.7 In

fact there are estrogen receptors on all the major cell types involved with wound healing

suggesting that estrogen may directly modulate the function of these cells.54 In adult human skin

CTGF is normally not expressed unless induced, TGF-3 induces CTGF expression.3 Therefore,

we did immunohistochemistry staining for TGF-P and for CTGF to look for localization of these

growth factors in relation to estradiol levels.

The first task after immunostaining was complete was to grade the level of staining based

on a visual analog scale (VAS) of 0-5. This was accomplished by a two tester grading system.

Each grader was blinded to whether the slide being graded was from a male or female or pre or

post menopausal female. After each grader completed all slides, the numbers were averaged

together to come up with a final grade for each slide. These grades were then used to select slides

from each category; male, pre menopausal, and post menopausal female. Photographs of these

slides were made using the Zeiss Axiovision microscope and staining intensity quantified using

computerized digital image analysis by Image J.68

After comparisons were made from the VAS, image analysis using Image J68 was used to

verify the accuracy of the VAS for the TGF-P stained slides. This was done by normalizing the

tissue sample by dividing the percentage of staining by the total area of the sample. What was

noted from this was that the post menopausal females had a lower normalized amount of staining

as compared to the pre menopausal and males groups, this was consistent with data from the

VAS (Figure 4-7). The post-menopausal staining intensity was very similar to the staining

intensity of the normal donor site tissue. A Pearson's correlation test was performed (r=0.5)

which indicated a weak positive correlation between the VAS and the computerized digital

image analysis by Image J.68 This was further verified by a two tailed T-test (p=0.003) which









indicated that r is significantly greater than 0. The in laid bar graph (Figure 4-7) shows the

averages of the individual scores versus the normalized values, this gives a more dramatic

presentation of how much more staining intensity there is among the burn injured tissue over the

normal non injured donor site tissue. A Mann-Whitney statistical test was performed and the p

value comparing male bum tissue to male donor tissue was 0.02 which is a significant difference,

the p value comparing pre-menopausal female burn tissue to pre-menopausal donor tissue was

0.08 which is considered not significant, and the p value comparing post-menopausal burn tissue

to post-menopausal donor tissue was 0.01 which is considered very significant. Even though the

difference between the pre-menopausal burn and donor tissue was not significant the trend

remains the same with the bum injured tissue showing a greater staining intensity for TGF-0

among all three groups, and among the three groups the post-menopausal females showing the

lowest intensity for staining for burn injured tissue.

After the data was collected from the TGF-0 slides the process was repeated for the CTGF

stained slides (Figure 4-8). A Pearson's correlation test was again used to verify the relationship

between the VAS and the computerized digital image analysis. However, this time the

correlation showed an increase in the r value indicating a good positive correlation (r=0.6). A

two tailed T-test was performed (p<0.0001) which confirmed the results of the Pearson's

correlation test indicating that r is significantly greater than 0. The trend for the CTGF staining

intensity is very similar to the TGF-0 staining intensity. However, a Mann-Whitney test

performed on male bum tissue versus male donor tissue, pre-menopausal burn tissue versus pre-

menopausal donor tissue, and post-menopausal burn tissue versus post-menopausal donor tissue

determined that there was not a significant difference among these three groups with p values of

0.15, 0.08, and 0.12 respectively. Although, there was not a significant difference among these









groups the trend continues to be that the bum injured tissue has a higher staining intensity for the

growth factors TGF-3 and CTGF with the post-menopausal group having the lowest intensity of

staining among all the bum injured tissue.

There are two types of estrogen receptors, ERa and ERP. These receptors are found on all

the major cell types involved in wound healing. These receptors are co-expressed with ERP

playing a dominant role over ERa.76 Ankrom et al77 showed that the ERa increased with

increasing age and Haczynski et al76 showed in an experiment using only post menopausal

women that the ERa out numbered the ERP. In a review by Pelletier78 the ERa did not

immunolocalize to any specific structure of human skin. However, ERP was found to be highly

expressed in the epidermis. These ER localization studies have shown that ERP is widely

expressed in human skin and its appendages, whereas ERa is only found in very few subsets of

structures.8 It is the therefore concluded that if in the presence of estrogen certain cells are

stimulated to produce TGF-3 in response to wound healing, that this is accomplished through the

ERP and that with increasing numbers of ERa, such as in post menopausal females, there is a

decrease in the amount of TGF-P produced by these cells. This would explain why the amount of

staining of TGF-P, as seen by the blinded two grader VAS and by the computerized digital image

analysis, was lowest among the post menopausal women (Figure 4-7). The fact that TGF-P

stimulates the production of CTGF in response to injury would explain why the post menopausal

women also had the lowest amount of staining for CTGF. An interesting addition to this would

have been to separate young males from aged males to determine if there was a staining intensity

difference such as between the pre and post menopausal women.

It has already been established that estrogen levels are increased as a response to injury,

and this was confirmed by the estradiol levels obtained after thermal burn injury which showed









elevated estradiol levels among male and post-menopausal female thermal bum injured victims.

It is difficult to say whether the pre-menopausal females had elevated estradiol levels because all

values fell with in a normal range. Immunohistological photographs confirm that TGF-3 staining

intensity after thermal burn injury increases as seen in Figure 4-9 comparing the thermal burned

injured tissue and donor site tissue of a male burn victim. Note the difference in intensity

between the thermal injured tissue compared to the donor site tissue. Even though the donor site

tissue is from a burned injured patient its staining pattern is similar to that of a "normal" non

burn injured patient (Figure 4-12). This same staining pattern is seen among the pre and post

menopausal women where the burn injured tissue shows increased staining intensity while the

donor site tissue shows a staining pattern similar to that of "normal" non bum injured females

(Figures 4-10, 4-11, 4-13). This would indicate that the response to injury is localized to the area

of injury and not a systemic response.

CTGF is expressed in many different tissues and organs and stimulates proliferation and

chemotaxis of fibroblast directly. Most interestingly, it is a potent inducer of extracellular matrix

proteins, such as collagen type I and fibronectin and their integrin receptors, and it acts as a

mediator of TGF-P in these processes.79 However, CTGF is not normally expressed unless it is

induced by TGF-3.3 Experiments by Igarashi et also show the presence of TGF-3 and CTGF in

wound tissue. TGF-3 was present at the earliest times following injury with a peak at day 3

followed by a rapid decrease, however, CTGF exhibited a much different expression pattern with

a peak in levels at day 9. Levels of CTGF continue to accumulate for up to 24 hours after

exposure to TGF-1.80

Immunohistological photographs of CTGF stained tissue shows staining to both the bum

injured and donor site tissue (Figure 4-14) of a bum injured male victim with dermal and









epidermal involvement. This same pattern is also seen in a pre menopausal female bum injured

victim (Figure 4-15), however, the post menopausal pattern of staining (Figure 4-16) is

noticeably decreased to both the dermis and epidermis. This remains consistent with the previous

mentioned blinded two grader VAS and computerized digital image analysis (Figure 4-8) which

indicated a much lower intensity of CTGF staining among the post menopausal females

compared to the pre menopausal females and male groups. In "normal" non bum injured tissue

CTGF is not normally expressed 80, this is demonstrated in the immunohistological photographs

of "normal" non bum injured tissue from male (Figure 4-17) and pre and post menopausal

females (Figure 4-18) in which there is no or very little evidence of CTGF staining noted.

In summary, we know that estradiol levels increase in response to the trauma of thermal

burn injury, We know that all of the cells involved in wound healing have estrogen receptors and

that there are two isoforms of these receptors, ERa and ERP. It is known that these receptors are

co expressed and that ERP is dominate over ERa, however ERa levels seem to increase with age.

It is our belief that it is the ERP that is responsible for stimulating wound healing cells to

produce TGF-3 following thermal bum injury which in turn stimulates CTGF allowing wound

healing to occur. We know that males and pre menopausal females had increased intensity of

staining of TGF-3 and CTGF over post menopausal females. What is still left unanswered is why

the pre menopausal females did not increase estradiol levels as drastically as the males and post

menopausal females in response to thermal burn injury? Why did both of the "normal" non bum

injured males have such high estradiol levels? We believe that most unanswered questions could

be answered by taking a closer more in depth medical history and medication list. Some things

that are left for future studies would be performing extraction assays to compare quantitative

levels of TGF-3 and CTGF to estradiol levels and histological stainings. Also, it would be good









to do some immunostaining for TGF-3 receptors. A final thought would be to set up a knock out

assay to verify that it is truly the ERB that is responsible for signaling the cell to produce TGF-3.












Table 4-1. Estradiol levels for adult males and females during each phase of the menstrual cycle
and after menopause74
Total Estradiol (pg/ml)
Adult Male 10-50 Adult Female
Pre Menopausal
Early Follicular 20-150
Late Follicular 40-350
Mid-Cycle 150-750
Luteal 30-450
Post Menopausal <20
















600


500


400 -Females
SMales
300-

S200


100



Estrogen Replacement Estrogen hhibitor 79 yt Female 35% Bum 35 ylo Male 66% Bum


Figure 4-1. The Effects of Hormone Replacement, Hormone Inhibitor, and Percent Burn on
Estadiol Levels (n=l). (A)Estradiol levels of a 39 y/o female non burn injured
patient on estrogen replacement compared to a 57 y/o female non bum injured
patient on estrogen inhibitor. (B) The estradiol levels of the female with the
highest percentage burn injury compared with the male with the highest
percentage burn injury.











600

500 *


MYIn r 3. L21y l 0-IqI aP; L S P-air)
aI D ayS POST-i 1 r 3c OI;FyS 06T-01ri 5 L12V P QSJ-a 1 r


p > 0.05


Estradiol Levels of Males vs. Pre- and Post-Menopausal Females at First
Debridement, (i +/- o)The average estradiol levels of all males compared to all
pre menopausal and post menopausal females at the time of the first debridement
following burn injury. Average days from time of injury until first debridement
are listed for each group. An ANOVA test was performed (p=0.9) which indicated
that there was no significant difference among these groups.


Figure 4-2.






















I -





I-
0
:5
- II
ni
Di
I. I


III I


, I.*I I


Figure 4-3.


I L i EI-i ir


Estradiol Levies of Males vs. Females at Second Debridement, (i+/- o). The

average estradiol levels for all males and females undergoing a second

debridement are listed with the average days since injury until second

debridement listed. A two-tailed T-test was performed (p=0.8) which indicated no

significant difference among these two groups.


11-



















.300

"o

200



IDD


L LI aa' roi-l-ilrl


Sge rle m El :
i=F l'iiyi PoIT-8TIn


I ol- IEmEm I.i


p > 0.05


Figure 4-4.


Female Estradiol Levels at Debridements 1-3, (i +/- o). The average estradiol
levels for all females at the first debridement, all females undergoing a second and
third debridement along with the average days since time of injury for each group.
An ANOVA was performed (p=0.6) which indicated that there is no significant
difference among these groups.

























4.


p

*i


r !


9
*
i


Mull PirrunO Is M1*i Frmrlhk Mfm*k Pwimll Malmi- Fi4* MMa'u "HmMnr'N" nmr"
) t-f 1 [- -] 4341 Miau Fnri l
%TlSA Eurned


p > 0.05


Estradiol Levels vs. Percent Burn for males and Females Following Burn injury.
The estradiol levels of male and female burned injured patients broken down by
percent total body surface area (TBSA) burned compared to the estradiol levels of
non burned "normal" tissue. An ANOVA test was performed; single values could
not be used therefore were left out of the statistical analysis. Results among the
remaining groups returned a p value of 0.9 which indicates that there is no
significant difference among these groups. Horizontal lines indicate the mean for
each group.


.3
no


Figure 4-5.

























I







p > .O5


. -
.


A pi4o-MM"p r**44pi*4l4uI, M" W p
~rlr ~ M~~p ~mTW~ CI~U t ~A TGIU fy-JM~I~n


Estradiol Levels vs. Age for Males and Females Following Burn injury. The
average estradiol levels of male, pre menopausal, and post menopausal broken
down by age. An ANOVA was performed; single values could not be used
therefore were left out of the statistical analysis. Results among the remaining
groups returned a p value of 0.4 which indicates that there is no significant
difference among these groups. Horizontal bars indicate the mean for each group.


Figure 4-6.


U9




















. is knzai""
i i,-nrc xi. HailndTpjl
* mahdar BjnTmVE
*PNn~iqa M RmT


1
A


0 0.5 1 1.5 2 25 3 3.5
Visual Andog Scale Score


4 4.5 5


Quantitative Image Analysis vs. Visual Analog Scale for TgF-P. The individual
scores from two masked graders using a visual analog scale versus computerized
digital image analysis to determine staining intensity of TGF-P in human skin
tissue samples following thermal burn injury. The in laid bar graph shows the
averages of these scores. A Pearson's correlation test was performed and a weak
positive correlation was determined (r=0.5). The coefficient of determination (r2)
was determined to be 0.3, and a two tailed T-test (p=0.003) determined that r is
significantly greater than 0. Also, a Mann-Whitney test was performed to
determine significance of staining intensity of TGF-3 between burned injured
tissue and normal donor site tissue. Comparing male burn tissue to male donor
site tissue the p value was 0.02 which is determined to a significant difference.
Comparing pre-menopausal female bum tissue to pre-menopausal female donor
site tissue the p value was 0.08 which is not a significant difference. Comparing
post-menopausal female bum tissue to post-menopausal donor site tissue the p
value is 0.01 which is a very significant difference.


Figure 4-7.

















S20 [

I-
~15


.1

S5


0
0


Figure 4-8.


i~icbiF


4 + )'' "
0


Visual Analog Sale Score


Quantitative Image Analysis vs. Visual Analog Scale for CTGF. The individual
scores from two masked graders using a visual analog scale versus computerized
digital image analysis to determine staining intensity of CTGF in human skin
tissue samples following thermal burn injury. The in laid bar graph shows the
averages of these scores. A Pearson's correlation test was performed and a
positive correlation was identified (r=0.6). The coefficient of determination (r2)
was determined to be 0.3, and a two tailed T-test (p<0.0001) determined that r
was significantly greater than 0. Also, a Mann-Whitney test was performed to
determine significance of staining intensity of CTGF between burned injured
tissue and normal donor site tissue. Comparing male burn tissue to male donor
tissue the p value was 0.2 which is not significant. Comparing pre-menopausal
burn tissue to pre-menopausal donor tissue the p value was 0.08 which is not
significant. Comparing post-menopausal burn tissue to post-menopausal donor
tissue the p value was 0.12 which is not significant.


L
















A C

hair follicle



epidermis
destroyed by
thermal injury dermis

dermis



burned injured test donor site test sample
sample
B ,
1 hair follicle


l,,,,-,, n ls ^derm is
,Ji,_.-:, ed by
I 1.il injury

epidermis







Figure 4-9. Immunohistology for Tgf-P in human burned injured and donor site tissue (males).
(A) Burn injured control sample, omission of the 1 Ab was used as the control. (B)
Burned injured test sample immunohistochemistry staining for TGF-P, sample taken
from burned injured area. Significant structures are labeled. (C) Donor site control
sample, omission of the 10 Ab was used the control. (D) Donor site test sample
immunohistochemistry staining for TGF-P, sample taken from donor site prior to
donor tissue harvest. Significant structures are labeled. Note differences in dermal
staining among bum injured tissue and donor site tissue. (100x magnification)


burn injured control sample


donor site control sample















burned injured control sample


donor site control sample


A C
epidermis
destroyed
by thermal
injury

Sepidermi
S dermis s

dermis





donor site test sample
burned injured test
samle b f
B
epidermis
'i epidermis
destroyed by
thermal



4 -.mi






Figure 4-10. Immunohistology for Tgf-P in human burned injured and donor site tissue (pre-
menopausal females). (A) Burn injured control sample, omission of 1 Ab was used
as the control. (B) Bum injured test sample immunohistochemistry staining for TGF-
P, sample taken from burn injured area. Significant structures are labeled. Although
the dermal tissue appears to have some staining in the control sample, it is important
to note the difference in the staining intensity of the epidermis among the control and
test sample. (C) Donor site control sample, omission of the 1 Ab was used as the
control. (D) Donor site test sample immunohistochemistry staining for TGF-3,
sample taken from donor site prior to donor tissue harvest. Significant structures are
labeled. Note the differences in dermal staining intensity among the bum injured and
donor site tissues. (100x magnification)














burned injured control sample


4 collagen fibers in
dermis


4 blood vessel


burn injured test sample
B


4 collagen fibers in
dermis


blood vessel


donor site test sample
DEll

epidermis


dermis





A*l


Figure 4-11.


Immunohistology for Tgf-3 in human burned injured and donor site (post-
menopausal females). (A) Burn injured control sample, omission of the 1 Ab
was used as the control. (B) Burn injured test sample immunohistochemistry
staining for TGF-3 sample taken from burn injured area. Significant structures are
labeled. (C) Donor site control sample, omission of the 1 Ab was used as the
control. (D) Donor site test sample immunohistochemistry staining for TGF-3,
sample taken from donor site prior to donor tissue harvest. Significant structures
are labeled. Note differences in dermal staining intensity among bur injured and
donor site tissue. (100x magnification)


epidermis.



dermis


donor site control sample
















A C


d ermis
dermis ..... dermal papilla (papillary dermis)
Sepidermal ndge

dermis (reticular dermis)


"Normal" tissue test sample (pt. 31)
"Normal" tissue test sample (pt. 31)
"Normal" tissue test sam pie (pt. 33)
B
D
Sepidermis
epidermis
4._ dermal papilla (papillary
dermis)

dermis (reticular
dermis)
At* epidermal ndge




Figure 4-12. Immunohistology for Tgf-P in human "normal" tissue (males). (A) "Normal"
tissue control sample from patient #31, omission of 1 Ab was used as the control.
(B) "Normal" tissue test sample immunohistochemistry staining for TGF-3 from
patient #31, significant structures are labeled. This sample was taken from elective
plastic surgery site. (C) "Normal" tissue control sample from patient #33, omission of
1 Ab was used as the control. (D) "Normal" tissue test sample
immunohistochemistry staining for TGF-3 from patient #33, significant structures are
labeled. This sample was taken from elective plastic surgery site. Patient #31 showing
slight increase in dermal staining compared to patient #33 many variables could be
responsible including pre morbid conditions. A closer look at pre morbid conditions
and medications would be helpful in future studies. (100x magnification)


"Normal" tissue control sample (pt. 31)


"Normal" tissue control sample (pt. 33)











"Normal" pre-menopausal control
sample
A


"Normal" post-menopausal
control sample
C


. epidermis


Sepidermis


"I ormral pre-enEnopau s.al test


4'



pidermls
Sdermis


"Normal" post-menopausal test
sln-iplei

7_ epider
mis

___sweai gland
(eccnne)


Figure 4-13.


Immunohistology for Tgf-3 in human "normal" tissue (pre- and post-menopausal
females). (A) "Normal" tissue control sample from a pre menopausal female,
omission of 10 Ab was used as the control. (B) "Normal" tissue test sample
immunohistochemistry staining for TGF-3 from pre menopausal female, significant
structures are labeled. This sample was taken from elective plastic surgery site. (C)
"Normal" tissue control sample from a post menopausal female, omission of 1 Ab
was used as the control. (D) "Normal" tissue test sample immunohistochemistry
staining for TGF-P from post menopausal female, significant structures are labeled.
This sample was taken from elective plastic surgery site. (100x magnification)












burned injured control
samAle


donor site control sample
C 'V
epidermi
s


4 ? dermis


burned injured test sample


epidermis
T destroyed by
thermal injury


donor site test sample


Figure 4-14.


Immunohistology for CTGF in human burned injured and donor site tissue
(male). (A) Bur tissue control sample, omission of the 1 Ab was used as the
control. (B) Burn tissue test sample immunohistochemistry staining for CTGF, the
sample was taken from the burn injured area. Significant structures are labeled.
(C) Donor site control sample, omission of the 1 Ab was used as the control. (D)
Donor site test sample immunohistochemistry staining for CTGF, the sample was
taken from the donor site prior to donor tissue harvest. Significant structures are
labeled. (100x manafication)


Sepidermis
destroyed by
thermal injury
Sderm
is


papilla

















burned injured control sample
A


4 dermis


burned injured test sample




S' collagen fibers in
dermis


donor site test sample
I. Mr~


d. w IIIIe


Figure 4-15.


Immunohistology for CTG in human bum injured and donor site tissue (pre-
menopausal females). (A) Burn injured control sample, omission of the 1 Ab
was used as the control. (B) Burn injured test sample immunohistochemistry
staining for CTGF; the sample was taken from the bum injured area. Significant
structures are labeled. (C) Donor site control sample, omission of 1 Ab was used
as the control. (D) Donor site test sample immunohistochemistry staining for
CTGF; the sample was taken from the donor site prior to donor tissue harvest.
Significant structures are labeled. (100x magnification)


donor site control
sarmnple I


L-I ,J,-~ ;












burned injured control sample


donor site control

epidermis


Sdermis


4 dermis


burned injured test sample
B .,'' 4-* :
B r,, < .


Sderml
s


donor site test sample
|_ .. e p id e rm is note
., cornefied outer layer
S and dermal papilla with
dermal ridges

i '.. is


Figure 4-16.


Immunohistology for CTGF in human burn injured and donor site tissue (post-
menopausal females). (A) Bum tissue control sample, omission of the 1 Ab was
used as the control. (B) Burn tissue test sample, the sample was taken from the
burn injured area. Significant structures are labeled. (C) Donor site control
sample, omission of the 10 Ab was used as the control. (D) Donor site test sample
immunohistochemistry staining for CTGF, the sample was taken from the donor
site prior to donor tissue harvest. Significant structures are labeled. (100x
magnification)












"Normal" tissue control sample


Sepidermi
Blood vessel


"Normal" tissue control sample
C.e
IA4"


epidermal ride


dermal papilla


, dermis


derml


"Normal" tissue test sample
B
.o- epidermi


blood vessels


blood vessels
-/'


"Normal" tissue test sample (pt.


epidermrus
epidermal rldae


dermal paplla

dermis


Figure 4-17.


Immunohistology for CTGF in human "normal" tissue (males). (A) "Normal"
tissue control sample from patient #31, omission of the 1 Ab was used as the
control. (B) "Normal" tissue test sample immunohistochemistry staining for
CTGF in patient #31, the sample was taken from elective plastic surgery site area.
Significant structures are labeled. (C) "Normal" tissue control sample from
patient #33, omission of the 1 Ab was used as the control. (D) "Normal" tissue
test sample immunohistochemistry staining for CTGF in patient #33, the sample
was taken from elective plastic surgery site. Significant structures are labeled.
Note the lack of staining in patient #33 which is what would be expected since
CTGF is not normally expressed in "normal" tissue. However, patient #31 does
show some slight increase staining intensity which could be indicative of on going
pre morbid condition since patient #31 also had increase in TGF-3 staining. (100x
magnification)














"Normal" pre-menopausal control
sample
A
epider
mis


d dermis


4 dermis


"Normal" pre-menopausal test
sample
B
.- epidermis
4 dermal papilla


blood vessel
(capillaries)


__ reticular
dermis


Figure 4-18.


Immunohistology for CTGF in human "normal tissue (pre-and post-mneopausal
females). (A) "Normal" tissue control sample in pre menopausal female,
omission of the 1 Ab was used as the control. (B) "Normal" tissue test sample
immunohistochemistry staining for CTGF in pre menopausal female. Tissue
sample was taken from elective plastic surgery site. Note the very little staining
intensity which is consistent with "normal" tissue not expressing CTGF unless
induced to. Significant structures are labeled. (C) "Normal" tissue control sample
in post menopausal female, omission of the 1 Ab was used as the control. (D)
"Normal" tissue test sample immunohistochemistry staining for CTGF in post
menopausal female, tissue sample was taken from elective plastic surgery site.
Significant structures have been labeled. Note the almost non existent staining for
CTGF in the post menopausal female, this is consistent with "normal" tissue not
expressing CTGF unless induced to, also consistent with post menopausal females
having less staining intensity than pre menopausal females or men per visual
analog scale and computerized digital imaging analysis. (100x magnification)


Sepidermi
s


epidermi
s


_ dermis









APPENDIX A
PROTOCOL #480-2005


1. Project Title:

Analysis for CTGF and TGF-3 levels in deep partial thickness thermal bum injury tissue
versus normal tissue.

2. Investigatorss:

David W. Mozingo, M.D.
Winston Richards, MD
Gregory Schultz, Ph.D.
Lyle L. Moldawer, Ph.D.
Brent Seagle, M.D.
Wayne Lee, MD
Donald McCurry, P.T.
Karen Perrin, MSN, ARNP
Tera Thigpin

3. Abstract:

Hormones are chemical signaling molecules produced in one site of the body that then
travel to another site to have an effect. In this way one cell can communicate with
distantly located cells.

The ovaries produce the steroid hormones, estrogen and progesterone, that are
responsible for the development of secondary sexual characteristics and develop and
maintain the reproductive function in the female. The normal development and
maturation of the female is dependent on estrogens. Besides stimulating development of
the reproductive structures and secondary sexual characteristics, estrogens are also
necessary for the health of the skin and vascular system, and bone homeostasis.(7)

Estrogen has been shown to play a crucial role in the wound healing process in both men
and women.(1,2) Premenopausal women are shown to have a faster rate of healing
following injury, while postmenopausal women and men heal slower but have decrease
scar tissue formation.(2) Estrogen has been shown to prevent a decrease in collagen levels
and to increase levels of mucopolysaccharids and hyaluronic acid keeping the skin thick
and moist, respectively. The lack of estrogen has been shown to improve the quality of
scar formation.(4)

Transforming growth factor P(TGF-P) and connective tissue growth factor (CTGF) are
involved in the wound healing process. TGF-3 induces synthesis and inhibits degradation
of extracellular matrix (ECM) and stimulates granulation tissue formation and collagen
deposition(2). CTGF is a major autocrine growth stimulator for connective tissue cells,









and its production is regulated by TGF-P.(3) Since estrogen is known to play a crucial role
in wound healing and the growth factors TGF-P and CTGF are also involved in the
wound healing process; this study will focus on the relationship between TGF-P, CTGF
levels and the hormone estrogen.


4. Specific Aims:

The purpose of this study is to investigate the relationship between estrogen and CTGF
and TGF-P levels in pre/postmenopausal women and men of varying ages following
thermal burn injury versus the CTGF and TGF-3 levels in pre/postmenopausal women
and men of varying ages with normal tissue. To better understand these relationships,
these questions will be addressed: How do estrogen levels correlate with healing rates
and quality of scar tissue following a thermal burn injury? How do TGF-P and CTGF
levels vary with estrogen levels?

5. Background and Significance:

Each year in the United States, trauma is the leading cause of death for people between
the ages of 1 and 24.(6) One type of trauma injury is a burn. A bum is defined as tissue
damage caused by a variety of agents, such as heat, chemicals, electricity, sunlight, or
nuclear radiation. The most common bums are caused by scalds, building fires, and
flammable liquids and gases.(6) Burns often lead to infection, due to damage to the skin's
protective layer. The larger the burn, the more increased chance of infection. In the
United States, 10,000 people die every year of burn related infections. Therefore, it is the
goal of health care providers to reduce the size of a burn wound as quickly as possible in
order to decrease the chance of such infections.

One of the main problems involved with wounds caused from a bum injury is the
formation of scar tissue. Disfigurement from scars and burned tissue can affect self
concept, body image, comfort in interpersonal situations, and acceptance from peers in
the work place. Severe burns are more likely to result in long term quality of life
problems with both physical and psychosocial aspects.(s) Muscle contractures can
develop from protective posturing to decrease pain. Contractures lead to loss of range of
motion and require physical therapy for stretching program and serial casting. Severe
contractures may require surgical release to restore range of motion.(9) Increased levels
of estrogen have been shown to increase the rate of wound healing(1'2), however,
increased estrogen levels increase scar tissue formation.(2) Twenty years ago, a burn that
covered 50% of the body was routinely fatal; today, thanks to advances in wound
cleaning, people with bums covering 90% of their body can survive (but often with
permanent impairments). While the goal is to reduce the size of the bur as quickly as
possible in order to decrease the chance for infection, it is hoped that in the future this can
be done without increasing scar tissue formation.









6. Research Plan:


It is projected that we will enroll 80 patients into this research that will meet the criteria
below:

Inclusion Criteria:
1. Patient meets one of the following criteria:
a). Any patient admitted to the Acute Care Surgery Service following thermal
burn injury that requires one or more surgical debridements.
b). Any patient which will be undergoing an elective surgical procedure by
the Plastics Reconstructive Surgery Service that will involve excision of
normal skin that would normally be discarded.
2. Male or Female 18 years of age or older.
3. Patient must be willing to comply with protocol and protocol procedures.

Exclusion Criteria:
1. Pregnant and nursing mothers.
2. Patients suffering from terminal disease
3. Patients with chemical or electrical burns.
4. Patients who have received systemic corticosteroids within 30 days prior to
surgical procedure, unless they are receiving them for acquired adrenal
insufficiency during their thermal burn care.


The Principal Investigator or his designated research staff will perform informed consent.
After informed consent has been obtained the patient will be separated into two
categories, patients with thermal bum injury and patients without thermal burn injury.

Thermal Burn Injury Tissue Patients

Samples will be collected during surgical procedures up to four times for a possible total
of eight tissue samples. After the patient has been anesthetized and the procedure has
begun, a 6mm punch biopsy will be taken from the injured area prior to debridement as
well as a 6 mm punch biopsy from normal skin prior to donor site harvesting.

Normal Tissue Patients

Patients undergoing elective plastic/reconstructive surgeries will have a one time 6 mm
biopsy performed from the normal tissue that has been excised and will be discarded.
Patients in both groups will have blood taken during their surgical procedure from
existing intravenous lines when available. One teaspoon of blood will be collected in a
lavender/EOTA tube prior to the biopsy procedure. These blood samples will be used to
test for plasma hormone and growth factor levels.









Tissue samples collected from thermal injured and non-thermally injured patients will be
divided with 50% being placed in formalin and 50% being placed in RNAlater. These
samples will be used for molecular analysis.

Also, a database will be set up to look at specific sub groups. The subgroups will include
but are not limited to males greater than 50 years of age, males less than 50 years of age,
premenopausal women, and postmenopausal women.

7. Potential Health Risks:

The risks or discomforts associated with participation in this study are the same as those
associated with the medical and surgical care subjects receive as part of the standard of
care for treatment of their medical condition at this facility.

The risks of drawing blood from a vein include discomfort at the site of puncture; and,
uncommonly, faintness from the procedure.

8. Potential Health Benefits:

While there are no immediate direct individual patient benefits from participation in this
study, patients with thermal burn injuries may in time directly benefit from the new
knowledge generated from the tissue collected.

9. Potential Financial Risks:

None

10. Potential Financial Benefits:

None


11. Conflict of Interest:

There is no conflict of interest involved with this study beyond the professional benefit
from academic publication or presentation of the results.


12. References

1. Ashcroft, Gillian. Stuart Mills, Kejian Lei, Linda Gibbons, Moon Jin Jeong,
Marisu Taniguchi, Matthew Burow, Michael Horan, Sharon Wahl, Toshinori
Nakayama. "Estrogen modulates cutaneous wound healing by down regulating
macrophage migration inhibitory factor" The Journal of Clinical Investigation.
2003. 111:1309-1318.









2. Ashcroft, Gillian. Jason Ashworth. "Potential Role of Estrogens in Wound
Healing" American Journal of Clinical Dermatology. 2003; 4(11): 737-743.


3. Atsuyuki, Igarashi, Okochi Hitoshi, Douglas Bradham, Gary Grotendorst.
"Regulation of Connective Tissue Growth Factor Gene Expression in Human
Skin Fibroblasts and During Wound Repair" Molecular Biology of the Cell. 1993.
Vol.4,637-645.

4. Kovacs, EJ, TP Plackett, PL Witte. "Estrogen Replacement, Aging, and Cell
Mediated Immunity After Injury" Journal ofLeukocyte Biology. 2004. 76(1):36-
41.

5. Shah, MG, HI Maibach. "Estogen and Skin, an Overveiw" American Journal of
Clinical Dermatology. 2001. 2(3):143-150.

6. Burn facts figures www.nigms.nih.gov/news/facts/traumaburnfactsfigures.html., July 31,
2007.

7. Hormones, www.e-Hormones.com, July 31, 2007.

8. Weed, Roger Debra Berans. "Basics of Burn Injury: Implications for Case
Management and Life Care Planning" Lippincotts Case Management. 2005; 10
(1): 22-29

9. Brown, Melissa Phala Helm. "Life Care Planning for the Burn Patient" Life Care
Planning and Case Management Handbook (2nd Ed, p 247-262) Boca Raton, Fl:
CRC Press, LLC.

10. Burn Surgery, /www.burnsurgery.org July 31, 2007









APPENDIX B
CASE REPORT FORMS


Case Report Forms


Oct 21, 2005


Patient Initials:


Patient ID:


Screening Date:


Inclusion Criteria:


Y N
[I] [ ]


Any patient admitted to the Acute Care Surgery Service
following thermal burn injury that requires one or more surgical
debridements.


Any patient which will be undergoing an elective surgical
procedure by the Plastics Reconstructive Surgery Service that will
involve excision of rmal skin that would normally be discarded.


[ ] [ ]


Male or Female 18 years of age or older.


II] II


II] II


Patient willing to comply with protocol and protocol
Procedures.


Exclusion Criteria:

Y N N/A
[ ] [] [ ]


II] II


II] II

II] II


Pregnant and nursing mothers.


Patients suffering from terminal disease.


Patients with chemical or electrical burns.


Patients who have received systemic corticosteroids within
30 days prior to surgical procedure, unless they are receiving them
for acquired adrenal insufficiency during their normal burn care.













CFm2


Patient Initials:

General Patient Information:
Male or Female:
Age:
Menopausal Status:
Hormone Replacement Therapy, Yes / No
Explain:
Medical History:





Burn Assessment:

Date of Burn: -

Location of Bum(s):

Degree of Burn:

Percentage of TBSA of Bum:



Debridement Site #1:

Date of Biopsy #1:

Biopsy Site of Thermal Bum Tissue and Sample Number:


Biopsy Site of Normal Donor Tissue and Sample Number:


Patient ID:


Blood Sample Collection, Yes / No
Blood Sample Number:

Is Patient on Antibiotics, Yes / No
Explain:


Case ReDort Forms


Oct 21. 2005











Debridement Site #2:

Date of Biopsy #2:

Biopsy Site of Thermal Bum Tissue and Sample Number:


Biopsy Site of Normal Donor Tissue and Sample Number:


Blood Sample Collection, Yes / No
Blood Sample Number:

Is Patient on Antibiotics, Yes / No
Explain:





Debridement Site #3:

Date of Biopsy #3:

Biopsy Site of Thermal Bum Tissue and Sample Number:


Biopsy Site of Normal Donor Tissue and Sample Number:


Blood Sample Collection, Yes / No
Blood Sample Number:

Is Patient on Antibiotics, Yes / No
Explain:











Debridement Site #4:

Date of Biopsy #4:

Biopsy Site of Thermal Bum Tissue and Sample Number:


Biopsy Site of Normal Donor Tissue and Sample Number:


Blood Sample Collection, Yes / No
Blood Sample Number:

Is Patient on Antibiotics, Yes / No
Explain:





Normal Tissue Collection:

Date of Surgery/Biopsy:

Type of Surgery:

Biopsy Site of Normal Tissue and Sample Number:


Blood Sample Collection, Yes / No
Blood Sample Number:

Is Patient on Antibiotics, Yes / No
Explain:









Case Report Forms


Patient Initials:

Study Completion Date: -




Study Completion:


Did the subject complete all study related activities?
If no, explain why:


Patient ID:


Yes No
[ ] [ ]


Were there any protocol deviations or violations reported? [] []
If yes, please comment:


Were there any Serious Adverse Events reported? [ ] [ ]
If yes, please describe:


Did the subject decease during the study period? [] []
If yes, when? -

Is the patient valuable? [ ] [ ]



Additional Comments:


Principal Investigator Signature Date


Oct 21, 2005









APPENDIX C
INFORMED CONSENT

IRB# 480-2005



Informed Consent to Participate in Research
and Authorization for Collection, Use, and
Disclosure of Protected Health Information


You are being asked to take part in a research study. This form provides you with information
about the study and seeks your authorization for the collection, use and disclosure of your
protected health information necessary for the study. The Principal Investigator (the person in
charge of this research) or a representative of the Principal Investigator will also describe this study
to you and answer all of your questions. Your participation is entirely voluntary. Before you
decide whether or not to take part, read the information below and ask questions about anything
you do not understand. If you choose not to participate in this study you will not be penalized or
lose any benefits to which you would otherwise be entitled.


1. Name of Participant ("Study Subject")




2. Title of Research Study

Analysis for CTGF and TGF-3 levels in deep partial thickness thermal bum injury tissue
versus normal tissue.

3. Principal Investigator and Telephone Number(s)

David W. Mozingo, M.D. (352) 273-5667









4. Source of Funding or Other Material Support


University of Florida


5. What is the purpose of this research study?

The purpose of this study is to investigate the relationship between estrogen and healing in
pre/postmenopausal women and men of different ages following burn injury versus the
healing in pre/postmenopausal women and men of different ages with normal skin.

6. What will be done if you take part in this research study?

The Principal Investigator or his research staff will perform informed consent. After
informed consent has been obtained, you will be separated into two groups, subjects with
burn injury and subjects without bum injury.

Subjects With Burn Injury

Tissue samples will be collected during your normal standard of care surgical procedure.
After you have been sedated for your surgical procedure, the surgeon will perform a 6mm
punch biopsy (about the size of a pencil eraser) from the injured area and a sample of normal
skin will be taken from your designated donor site. This procedure could be performed up to
four times depending on your need for more surgeries as part of your standard of care.

Subjects Without Bum Injury

A tissue sample will be collected once during your elective surgical procedure. You have
been selected to participate in this research study because your surgery requires the removal
of excess skin that would normally be discarded. From this discarded skin a 6mm punch
biopsy (about the size of a pencil eraser) will be taken.

Both groups will have blood drawn while they are in surgery through an existing intravenous
line if possible. This blood will be tested for hormones and growth factors.

The tissue sample collected from both groups will be split in half and saved. These tissues
will have tests performed on them at a later date. A database will be set up to track tissue,
tissue test results and your basic demographic information such as age, sex, premenopausal
and postmenopausal status. Your confidential information, such as name and birth date, will
not be included in this database.

If you have any questions now or at any time during the study, you may contact the Principal
Investigator listed in #3 of this form.

7. If you choose to participate in this study, how long will you be expected to participate in
the research?










For those patients that do have thermal burn injuries participation will last up to the time of
your fourth or final surgical procedure, whichever comes first.

For those patients that do not have a thermal bum injury participation in this study will end
after the one time tissue sample is collected.

8. How many people are expected to participate in this research?

Approximately 80 individuals are anticipated to participate in this study.

9. What are the possible discomforts and risks?

The risks or discomforts associated with participation in this study are the same as those
associated with the medical and surgical care subjects receive as part of the standard of care
for treatment of their medical condition at this facility.

The risks of drawing blood from a vein include discomfort at the site of puncture; possible
bruising and swelling around the puncture site; rarely, an infection; and, uncommonly,
faintness.

This study may include risks that are unknown at this time.

Participation in more than one research study or project may further increase the risks to you.
Please inform the Principal Investigator (listed in #3 of this consent form) or the person
reviewing this consent with you before enrolling in this or any other research study or
project.

Throughout the study, the researchers will notify you of new information that may become
available and might affect your decision to remain in the study.

If you wish to discuss the information above or any discomforts you may experience, you may
ask questions now or call the Principal Investigator or contact person listed on the front page of
this form.


10a. What are the possible benefits to you?

There are no benefits to you if you agree to participate in this study.


10b. What are the possible benefits to others?

The information gained from this study may someday improve healing standards and practice
in burn care.









11. If you choose to take part in this research study, will it cost you anything?

Taking part in this study will not cost you anything. The sponsor of this study will perform
the blood and tissue tests and these tests will not be billed to you.

Costs for routine medical care procedures will be charged to you or your insurance company.

12. Will you receive compensation for taking part in this research study?

You will receive no money or other compensation for participation in this study.


13. What if you are injured because of the study?

If you experience an injury that is directly caused by this study, only professional medical care
that you receive at the University of Florida Health Science Center will be provided without
charge. However, hospital expenses will have to be paid by you or your insurance provider.
No other compensation is offered. Please contact the Principal Investigator listed in Item 3 of
this form if you experience an injury or have any questions about any discomforts that you
experience while participating in this study.


14. What other options or treatments are available if you do not want to be in this study?

The alternative to being in this study is to do nothing and not sign this Consent Form.
Participation in this study is entirely voluntary. You are free to refuse to be in the study, and
your refusal will not influence current or future health care you receive at this institution.


15a. Can you withdraw from this research study?

You are free to withdraw your consent and to stop participating in this research study at any
time. If you do withdraw your consent, there will be no penalty, and you will not lose any
benefits you are entitled to.

If you decide to withdraw your consent to participate in this research study for any reason, you
should contact David W. Mozingo, MD at (352) 273-5667.

If you have any questions regarding your rights as a research subject, you may phone the
Institutional Review Board (IRB) office at (352) 846-1494.

15b. If you withdraw, can information about you still be used and/or collected?

If you decide to stop your participation in this study, we will not continue to collect any more
information from you. We would ask that you allow us to maintain the information we had
already collected.











15c. Can the Principal Investigator withdraw you from this research study?

You may be withdrawn from the study without your consent for the following reasons:

the study doctor thinks it is necessary for your health or safety;
if any significant side effect occurs;
you participate in another investigational study that skews data being collected for
this research study;
the Principal Investigator stops the study;
or administration reasons

16. If you agree to participate in this research study, the Principal Investigator will create,
collect, and use private information about you and your health. Once this information is
collected, how will it be kept secret (confidential) in order to protect your privacy?

Information collected about you and your health (called protected health information), will be
stored in locked filing cabinets or in computers with security passwords. Only certain people
have the legal right to review these research records, and they will protect the secrecy
(confidentiality) of these records as much as the law allows. These people include the
researchers for this study, certain University of Florida officials, the hospital or clinic (if any)
involved in this research, and the Institutional Review Board (IRB; an IRB is a group of
people who are responsible for looking after the rights and welfare of people taking part in
research). Otherwise your research records will not be released without your permission
unless required by law or a court order.

If you participate in this research study, the researchers will collect, use, and share your
protected health information with others. Items 17 to 26 below describe how this information
will be collected, used, and shared.


17. If you agree to participate in this research study, what protected health information
about you may be collected, used and shared with others?

Your protected health information may be collected, used, and shared with others to determine
if you can participate in the study, and then as part of your participation in the study. This
information can be gathered from you or your past, current or future health records, from
procedures such as physical examinations, x-rays, blood or urine tests or from other procedures
or tests. This information will be created by receiving study treatments or participating in
study procedures, or from your study visits and telephone calls.

If you agree to be in this research study, it is possible that some of the information collected
might be copied into a "limited data set" to be used for other research purposes. If so, the
limited data set may only include information that does not directly identify you. For
example, the limited data set cannot include your name, address, telephone number, social









security number, or any other photographs, numbers, codes, or so forth that link you to the
information in the limited data set. If used, limited data sets have legal agreements to protect
your identity and confidentiality and privacy.


18. For what study-related purposes will your protected health information be collected,
used, and shared with others?

Your protected health information may be collected, used, and shared with others to make sure
you can participate in the research, through your participation in the research, and to evaluate
the results of the research study.

19. Who will be allowed to collect, use, and share your protected health information?

Your protected health information may be collected, used, and shared with others by:

-the study Principal Investigator, David Mozingo, M.D. and his/her staff
-other professionals at the University of Florida or Shands Hospital that provide study-
related treatment or procedures
the University of Florida Institutional Review Board

20. Once collected or used, who may your protected health information be shared with?

Your protected health information may be shared with:

-the study sponsor, the University of Florida
-United States and foreign governmental agencies who are responsible for overseeing
research, such as the Food and Drug Administration, the Department of Health and
Human Services, and the Office of Human Research Protections
-Government agencies who are responsible for overseeing public health concerns such
as the Centers for Disease Control and Federal, State and local health departments)

21. If you agree to participate in this research, how long will your protected health
information be used and shared with others?

Your protected health information will be collected until the end of the study. This information
will be used and disclosed forever since it will be stored for an indefinite period of time in a
secure database. If you withdraw your permission fro the use and sharing of your protected
health information, then your information will be removed form the database.

22. Why are you being asked to allow the collection, use and sharing of your protected health
information?

Under a new Federal Law, researchers cannot collect, use, or share with others any of your
protected health information for research unless you allow them to by signing this consent and
authorization.











23. Are you required to sign this consent and authorization and allow the researchers to
collect, use and share with others your protected health information?

No, and your refusal to sign will not affect your treatment, payment, enrollment, or eligibility
for any benefits outside this research study. However, you cannot participate in this research
unless you allow the collection, use and sharing ofyour protected health information by
signing this c /ii'iu .n 1 uhel i:,tiinl


24. Can you review or copy your protected health information that has been collected, used
or shared with others under this authorization?

You have the right to review and copy your protected health information. However, you will
not be allowed to do so until after the study is finished.


25. Is there a risk that your protected health information could be given to others beyond
your authorization?

Yes. There is a risk that information received by authorized persons could be given to others
beyond your authorization and not covered by the law.


26. Can you revoke (cancel) your authorization for collection, use and sharing with others of
your protected health information?

Yes. You can revoke your authorization at any time before, during, or after your participation
in the research. If you revoke, no new information will be collected about you. However,
information that was already collected may still be used and shared with others if the
researchers have relied on it to complete and protect the validity of the research. You can
revoke your authorization by giving a written request with your signature on it to the Principal
Investigator.


27. How will the researchers) benefit from your being in this study?

In general, presenting research results helps the career of a scientist. Therefore, the Principal
Investigator may benefit if the results of this study are presented at scientific meetings or in
scientific journals.









28. Signatures


As a representative of this study, I have explained to the participant the purpose, the procedures,
the possible benefits, and the risks of this research study; the alternatives to being in the study; and
how the participant's protected health information will be collected, used, and shared with others:



Signature of Person Obtaining Consent & Authorization Date

Consenting Adults. You have been informed about this study's purpose, procedures, possible
benefits, and risks; the alternatives to being in the study; and how your protected health
information will be collected, used and shared with others. You have received a copy of this Form.
You have been given the opportunity to ask questions before you sign, and you have been told that
you can ask other questions at any time.

Adult Consenting for Self. By signing this form, you voluntarily agree to participate in this
study. You hereby authorize the collection, use and sharing of your protected health information as
described in sections 17-26 above. By signing this form, you are not waiving any of your legal
rights.



Signature of Adult Consenting & Authorizing for Self Date

Parent/Adult Legally Representing the Subject. By signing this form, you voluntarily give your
permission for the person named below to participate in this study. You hereby authorize the
collection, use and sharing of protected health information for the person named below as
described in sections 17-26 above. You are not waiving any legal rights for yourself or the person
you are legally representing. After your signature, please print your name and your relationship to
the subject.



Consent & Authorization Signature Date
of Parent/Legal Representative


Print:
Name of Legal Representative of and Relationship to Participant:









APPENDIX D
IHC PROTOCOL FOR CTGF

Tissue Should NEVER Dry Out
Deparafinizing Xylene gets cloudy when using > 15 slides, change this out after
every 25 slides and wash container

Prepare blocking solution (1% Human serum) before starting, make a liter and keep @ 40C

Prepare 10 Ab (keep on ice)(prepare in 1% Human serum)(10pg/ml CTGF)

Deparafinize
Xylene 5 Min Change after every 25 slides and wash container
Xylene 5 Min Change after every 75 slides and wash container
100% ETOH 5 Min
95% ETOH 5 Min
75% ETOH 5 Min
25% ETOH 5 Min
dH20 5 Min


Record diagram of slide in notebook (control on left)(test on right)

Make wax circles around samples, try to make circles the same size, use paper circle sticky as a
guide
Add blocking buffer, Record time of first sample
Block 3 1/2 hours (this reduces variability between samples)
No need to wash at this step

Only remove Blocking buffer from test (right side of slide)

Add same volume of 10 Ab

Incubate in humidity chamber overnight (Record Time!)

Keep In Mind, Next Time You Complete Experiment, It Has To Be The EXACT Same Time For
Everything!

Collect the 10 Ab on the test side only, cover with PBS (record starting and stopping time)

Once all slides have 10 Ab removed then place into a tub of PBS
PBS 5 Min
PBS 5 Min

Then place in tub of 20 Ab (1% Human Serum) for 1 hour









Wash again in tub of PBS
PBS 5 Min
PBS 5 Min
PBS 5 Min

Tub Ready of 1 2 3 system (same lot #, same time)









Substrate (vector red)(same lot #)(levamisol)(make lots of substrate buffer)









APPENDIX E
TGF-B IHC PROTOCOL


Tissue should never dry out
Deparafinizing- Xylene gets cloudy when using >15 slides, change this out after
every 25 slides and wash container.
Prepare blocking solutions (1% FBS in PBS) before starting, make a Liter, filter
sterilize and keep at 40 C
Prepare 10 Ab (keep on ice)(prepare in blocking solution)(1:150 TGF-P) use 10 Ab
sparingly.


Deparafinize
Xylene
Xylene
100% ETOH
95% ETOH
75% ETOH
25% ETOH
dH20


2 min Change after every 25 slides and wash container
2 min Change after every 25 slides and wash container
1 min
1 min
1 min
1 min
1 min


Record diagram of each slide in notebook (control on left)(test on right) along with a
designation for each slide

Quickly make wax circle around samples, try to make circles the same size, beware not to
press immune pen too deeply-this can cause wax to run
KEEP SAMPLES HYDRATED!


Incubate samples 10 min in 1% H202 diluted in PBS
Wash in PBS twice for 5 min each

Incubate for 1 hour in blocking serum

Remove blocking serum from test side only

Keeping control side hydrated with blocking serum, incubate test side 30 min with 10 Ab
Wash with three changes of PBS for 5 min each

Incubate 30 min with biotinylated 20 Ab
Wash with three changes of PBS for 5 min each

Incubate 30 min with AB enzyme reagent
Wash with three changes of PBS for 5 min each









Incubate in 1-3 drops of peroxidase substrate

dH20 5 min
25% ETOH 1 min
75% ETOH 1 min
95% ETOH 1 min
100% ETOH 1 min
Xylene 2 min
Xylene 2 min

Mount with permount and add cover slip











APPENDIX F
PATIENT DATA


Patient Sample Date of Menopausal % Site of .Receiving Hormone
Sex Age Diabetic
Reference # # Biopsy Status Burn Biopsy Antibiotics Replacement
01 01-01-R M 62 11/21/2005 N/A 15 Buttock Yes Yes No
01-01-F
02-01-R Back
02-01-F
00-01-B
02 01-01-R M 27 11/28/2005 N/A 20 Forearm No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
01-02-R 12/9/2005 Back
01-02-F
02-02-R Back
02-02-R
00-02-B
03 01-01-R F 40 12/2/2005 Pre 3 Hand No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
04 01-01-R M 19 1/3/2006 N/A 5 Leg No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
05 01-01-R M 22 1/11/2006 N/A 7 Leg No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
06 01-01-R M 18 1/10/2006 N/A 32 Thigh No Yes No
01-01-F











Patient Sample Date of Menopausal % Site of Receiving Hormone
Sex Age Diabetic
Reference # # Biopsy Status Burn Biopsy Antibiotics Replacement
02-01-R Thigh
02-01-F
00-01-B
01-02-R 1/27/2006 Thigh No
01-02-F
02-02-R Abd
02-02-F
00-02-B
07 01-01-R M 52 1/15/2006 N/A 7 Leg No No No
01-01-F
02-01-R Leg
02-01-F
00-01-B
08 01-01-R F 70 1/30/2006 Post 35 Thigh No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
09 01-01-R F 79 2/10/2006 Post 35 Chest No Yes No
01-01-F
02-01-R Chest
02-01-F
00-01-B
01-02-R 2/15/2006 Chest Yes
01-02-F
02-02-R Thigh
02-02-F
00-02-B
01-03-R 2/27/2006 Chest Yes
01-03-F
02-03-R Thigh
02-03-F
00-03-B
01-04-R 3/9/2006 Arm Yes
01-04-F
00-04-B











Patient Sample Date of Menopausal % Site of Receiving Hormone
Sex Age Diabetic
Reference # # Biopsy Status Burn Biopsy Antibiotics Replacement
10 01-01-R M 25 2/13/2006 N/A 25 Abd No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
11 01-01-R F 72 2/13/2006 Post 25 Chest No No No
01-01-F
02-01-R Abd
02-01-F
00-01-B
01-02-R 2/27/2006 Chest Yes
01-02-F
02-02-R Thigh
02-02-F
00-02-B
12 01-01-R F 19 3/1/2006 Pre 30 Chest No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
01-02-R 3/6/2006 Arm Yes
01-02-F
02-02-R Thigh
02-02-F
00-02-B
01-03-R 3/13/2006 Abd Yes
01-03-F
02-03-R Thigh
02-03-F
00-03-B
13 01-01-R F 32 5/5/2006 Pre 13 Leg Yes Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
14 01-01-R M 57 5/5/2006 N/A 3 Hand No Yes No











Patient Sample Date of Menopausal % Site of Receiving Hormone
Sex Age Diabetic
Reference # # Biopsy Status Burn Biopsy Antibiotics Replacement
01-01-F
02-01-R thigh
02-01-F
00-01-B
15 01-01-R F 42 5/24/2006 Pre 11 Arm No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
16 01-01-R M 21 5/26/2006 N/A 27 Leg No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
17 01-01-R M 64 6/1/2006 N/A 15 Chest No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
01-02-R 6/19/2006 Hand No
01-02-F
02-02-R Thigh
02-02-F
00-02-B
18 01-01-R M 79 6/21/2006 N/A 18 Leg No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
19 01-01-R M 35 6/28/2006 N/A 66 Chest No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
01-02-R 7/14/2006 Shoulder Yes
01-02-F











Patient Sample Date of Menopausal % Site of Receiving Hormone
Sex Age Diabetic
Reference # # Biopsy Status Burn Biopsy Antibiotics Replacement
02-02-R Abd
02-02-F
00-02-B
20 01-01-R F 65 7/14/2006 Post 13 Chest No No No
01-01-F
02-01-R Abd
02-01-F
00-01-B
21 01-01-R M 21 7/21/2006 N/A 2 Foot No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
22 01-01-R M 50 9/6/2006 N/A 15 Arm No No No
01-01-F
02-01-R Hip
02-01-F
00-01-B
01-02-R 9/13/2006 Leg No
01-02-F
02-02-R Hip
02-02-F
00-02-B
23 01-01-R F 41 9/15/2006 Pre 6 Leg No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
24 03-01-R F 43 9/20/2006 Pre N/A Breast No No No
03-01-F
00-01-B
25 03-01-R F 55 9/29/2006 Post N/A Breast No No No
03-01-F
00-01-B
26 01-01-R M 43 10/18/2006 N/A 13 Leg No No No
01-01-F











Patient Sample Date of Menopausal % Site of Receiving Hormone
Sex Age Diabetic
Reference # # Biopsy Status Burn Biopsy Antibiotics Replacement
02-01-R Thigh
02-01-F
00-01-B
27 03-01-R F 56 10/26/2006 Post N/A Arm No No No
03-01-F
00-01-B
28 01-01-R F 69 12/1/2006 Post 10 Shoulder No Yes No
01-01-F
02-01-R Groin
02-01-F
00-01-B
*29 03-01-R F 57 12/4/2006 Post N/A Abd No No No
03-01-F
00-01-B
30 01-01-R F 74 1/17/2007 Post 9 Arm Yes No No
01-01-F
02-01-R Abd
02-01-F
00-01-B
31 03-01-R M 55 1/19/2007 N/A N/A Eyebrow Yes No No
03-01-F
00-01-B
32 03-01-R F 34 1/19/2007 Pre N/A Eyelid No No No
03-01-F
00-01-B
33 03-01-R M 23 1/22/2007 N/A N/A Sacrum No Yes No
03-01-F
00-01-B
**34 03-01-R F 39 1/25/2007 Post N/A Abd No No Yes
03-01-F
00-01-B
35 03-01-R F 60 1/29/2007 Post N/A Thigh Yes No No
03-01-F
00-01-B
36 03-01-R F 22 1/31/2007 Pre N/A Neck No No No
03-01-F











Patient Sample Date of Menopausal % Site of Receiving Hormone
Sex Age Diabetic
Reference # # Biopsy Status Burn Biopsy Antibiotics Replacement
00-01-B
37 01-01-R M 19 2/3/2007 N/A 15 Leg No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
38 01-01-R F 30 2/14/2007 Pre 3.5 Arm No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
39 01-01-R M 48 2/14/2007 N/A 8 Leg No Yes No
01-01-F
02-01-R Thigh
02-01-F
00-01-B
40 01-01-R M 53 2/21/2007 N/A 3 Hand No No No
01-01-F
02-01-R Thigh
02-01-F
00-01-B










The first set of two numbers = patient

The second set of two numbers = site of biopsy





The third set of two numbers = number of biopsy


01-40

01 = Sample taken from deep partial thickness thermal bum injury tissue

02 = Sample taken from non thermal bum injured donor site

03 = Sample taken of normal tissue taken from non bum injured patient

01 = First biopsy

02 = Second biopsy

03 = Third biopsy


F = Formalin

R = RNAlater

B = Blood

Example of patient and sample number: 01-01-01-R, refers to patient #1, sample taken from bum injured tissue, from the first debridement, placed in

RNAlater solution.

* Patient is on estrogen inhibitor, Femara

** Patient is on estrogen replacement, Estratest










LIST OF REFERENCES


1. Burn Incidence and Treatment in the US 2007 Fact Sheet .Chicago, IL: American Burn
Association; available from http://www.ameriburn.org/resourcesfactsheet.
Internet; accessed 20 Jan. 2005.

2. Hakvoort, T., Altun, V., van Zuijlen, P. P. and et al., Transforming growth factor-beta(l),
-beta(2), -beta(3), basic fibroblast growth factor and vascular endothelial growth factor
expression in keratinocytes of burn scars. Eur. Cytokine Netw.2000; 11:233-239.

3. Leask, A. and Abraham, D. J., TGF-beta signaling and the fibrotic response. FASEB
J.2004;18:816-827.

4. Igarashi, A., Nashiro, K., Kikuchi, K. and et al., Connective tissue growth factor gene
expression in tissue sections from localized scleroderma, keloid, and other fibrotic skin
disorders.J. Invest Dermatol. 1996; 106:729-733.

5. Tobin, D. J., Biochemistry of human skin--our brain on the outside.Chem. Soc.
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BIOGRAPHICAL SKETCH

When I was in high school, college was the last thing on my mind. I did not have any

direction or path. Halfway through my senior year in high school I got a job at a local textile

plant, I started out packing boxes and eventually moved up to internal quality control inspector

(just fancy words to say that I looked at cloth all night). I stayed at this job for three after high

school, still had no direction or anything that I particularly wanted to do. However, during the

three years at the textile plant, I was laid off three times and finally on the third time the plant

closed for good. I was twenty years old with just a high school diploma and the only skill I had

was looking at cloth. I really was in no hurry to look for another job, until one day an Army

recruiter called me. Just for the heck of it I asked him about the Army college fund. Things

happened fairly quickly from then, in less than a month I was Private McCurry, U.S. Army! I

spent 3 years in the Army stationed with a field artillery unit based at Fort Hood, Texas. During

my time in Texas I never really had the desire to make the military a career, but I did start to

think about school. I even took a few classes at Central Texas College. I was released from the

Army in May of 1990, but by the fall I was recalled back to active duty because of Operation

Desert Storm.

I started college at South Carolina State University of Florida, in fall 1991, after Desert

Storm was over. I started there as a biology major because by this time I decided that I wanted to

be a physical therapist and the requirements for PT school closely paralleled the degree

requirements for a biology degree at SCSU. I continued to work as a rehab tech, nursing assistant

and EMT while continuing to pursue my degree in biology. I finally graduated in May of 1994

with a cum laude degree in biology from South Carolina State University. This occasion was

bitter sweet because my father passed away just 2 months prior and was never able to see this

accomplishment in my life.









Why did I choose physical therapy? Well, I was in pretty good shape from being in the

military and I thought what a cool job where you can get paid to show people how to work out. I

enrolled at the Medical University of South Carolina in the summer of 1995. I continued to work

weekends in the emergency room as an ER tech, and graduated in June 1997 with a degree in

physical therapy. I passed the South Carolina State Board that October and worked the next

seven years as an acute care physical therapist at The Regional Medical Center in Orangeburg,

South Carolina.

Now, you ask, how did I get involved in wound care? There are many areas of physical

therapy and by working in an acute care setting I got to see a little bit of a lot of things, wound

care being one. The "popular" areas of PT such as orthopedics to me quickly became a little

boring. Then one day I met a nurse, Marie Gehling. She wanted to start a clinic to follow up on

wound patients that were released from the hospital. Over time this clinic grew from patients

lined up in a small closet like room and Marie on the floor to now one of the largest wound

clinics in the state of South Carolina. Marie eventually recruited (either willingly or unwillingly)

Dr. John Samies to be the medical director of the wound center. It is these two people that I

credit for where I am today as far as my career goes. As a physical therapist, I worked closely

with Marie and Dr. Samies and would perform modalities and dressing changes on the wound

clinic patients. These two people have a passion for patient care that is unequal in anyone that I

have ever met. They have taught me a great deal professionally; however there was part of me

that wanted more.

One day I was placing a dressing on a patient's foot after whirlpool therapy, I don't

remember this patient's name, but his question to me I will always remember. He asked "how

come you are using that particular dressing". I honestly could not answer his question. I knew it









was a good dressing choice, I used many times before with good results, but I could not explain

how it worked. This made me feel very foolish, I was a professional, I should know this kind of

thing.

My sister and I brought my mom to Shands at the University of Florida for a second

opinion on a medical condition. My mom was in the hospital for two weeks and there is only so

much sitting around watching someone sleep that I could do, so I went for a walk. I went up this

hall, down that hall and by pure luck or divine intervention I saw the sign that would change my

life; The Wound Healing Institute. I walked in and introduced myself and met with Dr. Gregory

Schultz, who I would later find out is pretty famous in the wound healing community. He

explained to me what was required for graduate school and when I went home to South Carolina

I applied to graduate school and a few months later I was accepted.

I started graduate school at the University of Florida in the fall of 2004. I applied for the

masters program, which is a two year program. By the time I am finished it will have taken me

three years to complete this program because I have only been able to attend on a part time basis.

I work at North Florida Regional Medical Center as a wound care physical therapist, so need less

to say the last three years of my life have been pretty hectic. I am hoping to graduate this fall,

August 2007, and looking forward to being part of the Gator Nation!

My plans for the future right now are a little up in the air, but I do plan on taking my

wound care certification through the Academy for the Advancement of Wound Care (AAWC)

once things calm down a little. I feel that my time here at UF will make me a better clinician and

more confident in being able to answer any question that patients through my way.





PAGE 1

1 ANALYSIS FOR CTGF AND TGF-BETA LEVELS IN DEEP PARTIAL THICKNESS THERMAL BURN INJURY TISS UE VERSUS NORMAL TISSUE By DONALD E. McCURRY II A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Donald E. McCurry II

PAGE 3

3 To all of the people who have sustained a burn inju ry, who at a time of crisis in their own lives gave in order to help future burn victims.

PAGE 4

4 ACKNOWLEDGMENTS I would like to thank a ll the people that have guided me along my car eer path. First, the professors who have taught me along the way, wit hout the excellent education that I received at South Carolina State University and the Medical University of South Carolina, I would not have been prepared to enter the University of Florida. I would also like to recognize my professors at the University of Florida whose commitment to excellence in education and research is an inspiration. In addition to my class professors, I have to give a special thanks to my committee members, Dr. Gregory Schultz, Dr. David Mozi ngo, and Dr. Lyle Moldawer, I truly could not have had a better group of guys to guide me thr ough this process. There are also several key people, from the University of Florida that I would like to rec ognize for all of their assistance during my time here: Karen Perrin, ARNP; Robi n Masterson, RN; Angel Sampson; Dr. Jompo Moloye; Dr. John Azeke; Paulette Sanders; Dr. Be verly Vidaurreta and all the staff on the burn unit. Special thanks go out to all of the burn vi ctims who generously donated tissue for my study. There have been many people that have made an impact on my life both personally and professionally. Dr. John Samies and Marie Gehling, RN the passi on that you show for patient care and your good moral character are worth emul ating. I am proud to be their colleague and friend. To my best friends, Big Luke and Vic, whose support and love has helped me through this stressful time. MIBs bringin da heat! A nd the rest of my fam ily and friends thanks!

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 Burns.......................................................................................................................... .............12 Hypothesis..................................................................................................................... .........12 2 BACKGROUND....................................................................................................................17 Skin........................................................................................................................... ..............17 Epidermis...................................................................................................................... ...17 Basement Membrane.......................................................................................................19 Dermis......................................................................................................................... ....20 Appendages..................................................................................................................... ........22 Stem Cells..................................................................................................................... ...24 Skin Function.................................................................................................................. .25 Phases of Wound Healing.......................................................................................................27 Hemostasis..................................................................................................................... ..27 Inflammatory Phase.........................................................................................................32 Proliferative Phase...........................................................................................................37 Remodeling Phase...........................................................................................................40 Growth Factors................................................................................................................. ......41 Transforming Growth Factor-Beta..................................................................................42 Connective Tissue Growth Factor...................................................................................43 Hormones....................................................................................................................... .........44 Estrogen....................................................................................................................... ...........45 3 MATERIALS AND METHODS...........................................................................................62 Tissue Collection.............................................................................................................. ......62 Immunostaining for CTGF.....................................................................................................63 Immunostaining for TGF.....................................................................................................65 Estradiol EIA Kit.............................................................................................................. ......67

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6 4 RESULTS AND DISCUSSION.............................................................................................71 APPENDIX A PROTOCOL #480-2005.......................................................................................................100 B CASE REPORT FORMS.....................................................................................................105 C INFORMED CONSENT......................................................................................................110 D IHC PROTOCOL FOR CTGF.............................................................................................118 E TGFIHC PROTOCOL.....................................................................................................121 F PATIENT DATA..................................................................................................................123 LIST OF REFERENCES.............................................................................................................131 BIOGRAPHICAL SKETCH.......................................................................................................137

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7 LIST OF TABLES Table page 3-1 Plate 2 set up for Estradiol EIA.........................................................................................70 4-1 Estradiol levels for adult males and fema les during each phase of the menstrual cycle and after menopause..........................................................................................................81

PAGE 8

8 LIST OF FIGURES Figure page 1-1 Ratio of male to female burn victims in the United States ...............................................14 1-2 Ratio of burn victims by age group ...................................................................................15 1-3 Ratio of partial thickness ve rsus full thickness burn injuries ...........................................16 2-1 Cross section of human skin. ............................................................................................47 2-2 Cross section of the epidermis ..........................................................................................48 2-3 Hemidesmosomes ........................................................................................................... ..49 2-4 Histologic view of the epidermis and dermis....................................................................50 2-5 Collagen formation ....................................................................................................... ....51 2-6 Extracellular matrix ..................................................................................................... .....52 2-7 Various skin appendages .................................................................................................. .53 2-8 Finger nails ............................................................................................................. ...........54 2-9 Hair follicle............................................................................................................. ...........55 2-10 Hair follicle bulge stem cells........................................................................................... ..56 2-11 Coagulation cascade ..................................................................................................... .....57 2-12 Conversion of arachidon ic acid to ecosanoids...................................................................58 2-13 Steps of the inflammatory response...................................................................................59 2-14 Diapedesis and extravastion of neutrophils ......................................................................60 2-15 Angiogenesis ............................................................................................................ .........61 4-1 Effects of hormone replacement, hormone inhibitor, and percent burn on estrdiol levels......................................................................................................................... .........82 4-2 Average estradiol le vels at debridement 1.........................................................................83 4-3 Average estradiol le vels at debridement 2.........................................................................84 4-4 Average estradiol le vles at debridement 3.........................................................................85

PAGE 9

9 4-5 Estradiol levels versus percent burn for males and females following thermal burn injury......................................................................................................................... .........86 4-6 Estradiol levels versus age following thermal burn injury................................................87 4-7 Quantitative image analysis versus visual analog scale for TGF...................................88 4-8 Quantitative image analysis versus visual analog scale for CTGF....................................89 4-9 Immunohistology for TGFin human burn injured and donor site tissue (males)..........90 4-10 Immunohistology for TGFin human burn injured and donor site tissue (premenopausal females) .........................................................................................................91 4-11 Immunohistology for TGFin human burn injured and donor site tissue (postmenopausal females)..........................................................................................................92 4-12 Immunohistology for TGFin human normal tissue (males)......................................93 4-13 Immunohistology for TGFin human normal tissue (pre and post-menoapusal females)....................................................................................................................... .......94 4-14 Immunohistology for CTGF in human burn injured and donor site tissue (males) ..........95 4-15 Immunohistology for CTGF in human bur n injured and donor site tissue (premenopausal females) ........................................................................................................96 4-16 Immunohistology for CTGF in human bur n injured and donor site tissue (postmenopausal females)..........................................................................................................97 4-17 Immunohistology for CTGF in human normal tissue (males) ......................................98 4-18 Immunohistology for CTGF in human normal tissue (pre and post-menoapusal females)....................................................................................................................... .......99

PAGE 10

10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ANALYSIS FOR CTGF AND TGFLEVLES IN DEEP PARTIAL THICKNESS THERMAL BURN INJURY TISSUE VERSUS NORMAL TISSUE By Donald E. McCurry II August 2007 Chair: Donna Duckworth Major: Medical Sciences We know from previous studies that TGFis found in several different cell types and that these cells all possess TGFreceptors. We also know that CTGF is not expressed unless induced as in the case of wound healing. The pres ence of estrogen receptors on all of the major cells involved in wound healing has been show n to stimulate these cells to produce TGFin the presence of estrogen. Pre menopausal women having a higher level of estrogen versus post menopausal women and men, show a faster rate of wound healing compared with the other two groups: however, an increase in hypertroph ic scar formation is also noted. We hypothesize that the release of TGFand the subsequent pr oduction of CTGF during the initial phases of wound hea ling following a thermal burn injury will decline over time as the wound heals. We further hypothesi ze that immunohistochemical st aining of tissue samples will show higher levels of these growth factors in pre menopausal women, due to higher estrogen levels, versus men and post menopausal women with lower levels of estrogen. Based on our research we were able to conclude the following: estradiol levels increase in response to the trauma of thermal burn injury; all of the cells involve d in wound healing have estrogen receptors and that there are two isoforms of these receptors, ER and ER ; and that

PAGE 11

11 males and pre-menopausal females have in creased intensity of staining of TGFand CTGF over post menopausal females.

PAGE 12

12 CHAPTER 1 INTRODUCTION Burns A burn is a very serious in jury, last year alone ther e were approximately 500,000 burn injuries that required medical treatment, re sulting in 40,000 hospital ad missions. Fire and burn deaths per year are approximately 4,000 this nu mber includes an estimated 3,500 deaths from residential fires and 500 from motor vehicle and aircraft accidents. Most burn victims are male (70%) between 5 and 19 years of age (Figures 1-1, 1-2). The largest percen tage of burn injuries occur in the home and are the resu lt of fire or flame. Although mo st burn injuries are 10% or less of total body surface area (TBSA), some people sust ain burn injuries to a significant portion of there bodies. Advances in medical care have allowed people to su rvive a significant burn injury that wound not have survived just 20 years ago; people are now able to survive after sustaining almost 100% TBSA burn injury (Figure 1-3). Th e mortality rate has dropped from 6.16 % to 4.67 % over the past 10 years, and the average hospital length of st ay has dropped from 13.07 days to 8.22 days over the same time period. For those who do survive a si gnificant burn injury they are left with multiple scars and disfiguremen ts resulting in decrease cosmetic appearance, loss of function and restricted growth.1 Hypothesis We know from previous studies that TGFis found in several different cell types to include platelets, macrophages, granuloc ytes, fibroblast, a nd keratinocytes. TGFreceptors are found on essentially all cell types.2 We also know that CTGF is not expressed unless induced as in the case of wound healing.3,4 The presence of estrogen receptors on all of the major cells involved in wound healing have been shown to stimulate these cells to produce TGFin the presence of estrogen. Pre-

PAGE 13

13 menopausal women having a higher level of estr ogen versus post-menopausal women and men, show a faster rate of wound healing compare with the other two groups; however an increase in hypertrophic scar formation is also noticed. We hypothesize that the release of TGFand subsequent production of CTGF during the initial phases of wound healing following a th ermal burn injury will decline over time as the wound heals, and that immunohistochemical stai ning of tissue samples will show a correlation with previous studies and show higher levels of these growth factors in pre-menopausal women, due to higher estrogen levels, versus men and post-menopausal women with lower levels of estrogen. It is our hope that th is study may one day lead to a bett er wound healing protocol in the treatment of burned injure d patients, allowing faster healing ra tes, while decreasing hypertrophic scar formation.

PAGE 14

14 Figure 1-1. Ratio of male to female burn victims in the United States(http:www.ameriburn.org, last accessed January 20th, 2005) 1 Female 30.1% Male 69.9%

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15 Figure 1-2. Ratio of burn victims by age group(http:www.ameri burn.org, last accessed January 20th, 2005) 1. 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 0 1.92 4.95 19.920 29.930 39.940 49.950 59.960 69.9? 70Age Group N o. of Cases

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16 Figure 1-3. Ratio of partial thickness versus full thickness burn injuries, and combined total(http:www.ameriburn.org, last accessed January 20th, 2005) 1. 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 45,000 50,000 0.1 910 1920 29 30 39 40 49 50 5960 6970 7980 89 Total Full PartialNo. of Cases % TBSA

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17 CHAPTER 2 BACKGROUND Skin Epidermis Adult human skin is a very large organ, covering approximately 2 m 2 in area, with approximately 2.5 mm thickness and ha s an average density of 1.1kg/m3. Together these provide for a 5-6 kg mass value for skin, in other words, sk in constitutes an impressive 6% of our total body weight.5 The skin provides a physical barrier with the external environment and is designed to protect us against a wide range of insults including: temperature, electrolyte/fluid balance, mechanical, chemical and microbial. Fu rther protection is prov ided by the ultraviolet radiation (UVR) absorbing pigmentation system and the complex immuno-regulatory sentinel networks, which sense tissue micro-environm ents for foreign or abnormally expressed components. The skin has two layers, (Figure 2-1) an epidermis and dermis, separated by a basement membrane which is supported by a hypodermis or superficial fascia.6 The normal epidermis is the outermost layer of the skin and is derived from the embryonic ectoderm, the outermost germ layer. The majo r cell, making up 95% of the total, is the keratinocyte, which moves progressively from a ttachment to the epidermal basement membrane towards the skin surface, forming seve ral well defined layers in transit.7 Other cells that reside within the epidermis include: melanocytes whic h are derived from the neural crest and are responsible for the production of melanin, langerhans cells (ie. de ndritic like cells) are derived from a bone marrow precursor that act as antig en presenting cells interacting with T-cells, merkel cells are also derived from the neural crest and are involved in tactile sensation.8 The stratum corneum, stratum lucidum, stratum gra nulosum, stratum spinosum, and stratum basale

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18 are the distinct layers of keratinocytes that make up the epidermis. (Figure 2-2) The epidermis is an avascular layer and depends on the capillary beds in the de rmis for oxygen and nourishment.9 The stratum corneum is the outermost layer a nd is composed of seve ral layers, usually 1530, of dead, but biochemically active cells calle d corneocytes. These cel ls contain keratin a protein that helps keep the skin hydrated by preventing water evaporation. This layer is constantly being shed into the environment through bathi ng, hand washing, and mechanical disruption when changing clothes. This layer is replaced by the layers below, stratum lucidum or the stratum spinosum.6 The stratum lucidum is a thin, clear layer of d ead cells in the epidermi s. It is found beneath the stratum corneum in areas of th ick skin, such as the palms of th e hand and soles of the feet. It is recognized by some histologis t as an intermediate layer ab ove the stratum granulosum and beneath the stratum corneum. However, no dis tinctive cytologic features are significantly apparent. Cells present in the stratum lucidum re lease a small amount of lipid material into the extra cellular space after fu sion with the plasma membrane to form a neutral lipid rich coating that protects these cells from disruption.6,8 The stratum granulosum, sometimes referred to as the granular layer, is generally one to three cells thick and is seen as a flat layer w hose cells still have active nuclei. These non dividing keratinocytes produce granules of a protein calle d keratinohyalin. These cells flatten as dividing cells below progressively push them toward the sk in surface. At the same time their organelles and nuclei breakdown and the granules of kerati nohyalin release their lipid components into the intercellular space which causes the cell memb ranes to become increasingly impermeable playing an important role in barrie r function and intercellular cohesion.7,6

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19 The keratinocytes of the stratum spinosum have a flattened polygonal sh ape with a distinct ovoid nucleus. This layer is gene rally two to six cells thick a nd is distinguished by numerous desmosomal (Figure 2-3) connection plaques, whic h interact with adjacent keratinocytes forming a stabilizing network of surf ace interconnections. Desmosomes are symmetrical, laminated structures formed in apposed plasma membrane s with linear intercellu lar and intracellular components. Cytoskeletal tonofilaments seem to attach close to desmosomes, providing stability across the cell layers.6,7 The stratum basale is the inner most epider mal layer and is the layer that has actively dividing cells undergoing mitosis. Along with th e stratum spinosum, the stratum basale are collectively called the stratum of Malpighi. The stratum of Malpighi is named for the Italian scientist Marcello Ma lpighi who is known as the first microa natomist and the father of histology. The cells of the stratum basale are attach ed to the noncellular basement membrane by hemidesmosomes, a type of cell junction that forms between cells and matrix. The basement membrane separates the epidermis from the dermis Once the cells of this layer divide, they push upward, and as they complete thei r journey to the stratum corneum in approximately two to three weeks they undergo complete differentiation to b ecome keratinized. It is the fate of every keratinocyte of the stratum basale to mature and differentiate into a keratinized cell to contribute to a tough external skin surface layer.6,5,8 Basement Membrane The basement membrane is a thin acellular layer that separates the epidermis from the dermis. The basement membrane anchors the epid ermis to the dermis and is made up of three layers, the lamina lucida, the lamina densa, and th e lamina fibrorecticularis. The lamina lucida is named because its an electron translucent zone compared with the electron dense zone of the lamina densa. The lamina densa lies parallel to and below the lamina lucida. The main

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20 component of the lamina fibroreticularis is an choring fibrils. These are short, often curved structures, with an irregularly spaced cross bandin g, that inserts into the lamina densa and extend into the upper part of the dermis Another component of the lamina fibroreticularis is the elastic microfibril. This may occur singly, but is best recognized in a bundle consisting of many microfibrils that extend into the dermis and may en mesh with the microfibrillar system of dermal elastic fibers. The major proteins found in the ba sement membrane are fi bronectin, an adhesive glycoprotein; laminin, also a gl ycoprotein; type IV collagen, a nonfiber forming collagen; and heparin sulfate proteoglycan, a glycosaminoglycan that probably acts as a type of ground substance. A lesser amount of type VII collagen is present, but it plays a very important role in attachment of cells to the lamina densa.6,7,10 Dermis The dermis is derived from the embryonic mesoderm, the middle germ layer. The dermis is formed by two layers without distinct boundaries the upper papillary and a lower reticular layer, reflecting there respectiv e connective tissue components, cell number, and supply of blood vessels and nerves. The papillary region is composed of loose ar eolar connective tissue. It is named for its fingerlike projecti ons called papillae, which exte nd toward the epidermis. The papillary dermis interdigitates with the epidermis, this strengthens the connection between the two layers.(Figure 2-4) The reticular dermis lies d eep to the papillary dermis and is usually much thicker and blends with the subcut aneous fat. It is composed of dense irregular connective tissue, and receives its name from the dense concentrat ion of collagenous, elastic and reticular fibers that weave throughout it. These protein fibers give the dermis its properties of strength, extensibility, and elasticity.5,6,7 Despite its greater volume, the dermis contai ns far fewer cells than the epidermis and instead much of its bulk consists of fibrous and amorphous extracellular matrix (ECM). The

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21 main cell type of the dermis is the fibroblas t, a heterogenous migratory cell that makes and degrades ECM components. The ECM is a highly complex mixture of bioactive macromolecules produced by dermal cells that is either secreted intact or assembled later outside the cells. ECM is the defining feature of connective tissue. Collagens are the principal ECM component and make up about 90% of total dermal protein. While at least 25 collagens exist, half are present in skin; consisting predominantly of collagen ty pe I and III. Collagen is an exceedingly tough fibrous protein that is secreted as tropoco llagen by the fibroblast into the extracellular environment, (Figure 2-5) where it undergoes further processing by enzymes that cleave the propeptide portion of the polypeptide to form mature collagen fiber bundles. Collagen is the protein that gives the skin its tensile strength. The primary amino acids that constitute collagen are proline, glycine, hydr oxyproline, and hydroxylysine.6,10,5,7 The other major protein found in the ECM is elas tin. Elastin is the prot ein that returns skin continuity when it is pinched or stretched, and is secreted by fibroblast as tropoelastin into the dermis. In the extracellular space of the dermis the proelsatin which inte racts with fibrillin, a microfibrillar protein whose anatomical distribution closely parallels that of elastin, to organize immature elastic fibers this aggregates to form mature elastic fibers. These mature elastic fibers are composed of amorphous and fibrous compon ents; the amorphous portion is composed of the protein elastin while th e fibrous portion is composed of th e protein fibrillin. Oxytalan and elaunin are two other elastic fibers also found in dermal connective tissue.6,10,5,7 The other category of proteins found occ upying the space between collagen and elastin fibers is referred to as th e ground substance. (Figure 2-6) This amorphous and hydroscopic material is biologically active, highly diverse and organized. It consis ts of proteoglycans a modified core protein to wh ich are attached polymers of unbranched disaccharides, and

PAGE 22

22 glycosaminoglycans that act as linkers be tween certain cell surface receptors and ECM scaffolding components. Proteoglycans and glyc osaminoglycans are capable of binding up to 1000 times their volume and thus play a role in regulating the water bi nding capacity of the dermis that determines the dermal volume and compressibility.6,5,7 The hypodermis, or superficial fascia, (Figure 2-1) forms a subcutaneous layer below the dermis. This is an adipose layer containing a subd ermal plexus of blood vessels giving rise to the cutaneous plexus in the dermis, which in turn give s rise to the papillary plexus and loops of the papillary dermis. This layer is not considered pa rt of the skin but is the underlying supporting and connecting layer that attach es the dermis to underlying structures. The hypodermis provides padding and insulation and accounts for differences in body shape.6,10 Appendages In addition to the two major structural layers the epidermis and dermis, the skin is also populated by different appendages. These appendages include glands, such as the sweat glands and sebaceous glands. Along with the different glands are the hair follicle, hair fiber, and nail. (Figure 2-7) The sweat glands can be divide d into two separate groups th e eccrine and apocrine glands. As warm blooded animals humans have approximat ely 3-4 million eccrine glands in their skin, each producing a watery perspiration that serves principally to cool and maintain the core body temperature at 37.5 C. Eccrine glands have long thin ducts opening di rectly onto the skin surface and proximal secretory coiled section co nsisting of secretory cells and contractile myoepithelial cells. The coiled portion of th e eccrine gland lies in the deep dermis or hypodermis. The apocrine glands are larger than the eccrine glands, but like the eccrine glands they are located in the dermis and hypodermis. Instead of opening onto the skin surface the apocrine glands open into the hair follicle. The apocrine glands ar e found after puberty are

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23 present in the groin, anal region, ax illa, areola of the breast, and beard of adult males. Apocrine gland sweat is thicker, more viscous and with a milky consistency due to its higher content of fatty acids and other compounds. Some of th ese are odoriferous, esp ecially after there decomposition on the skin surface by bacteria.5,8 The sebaceous gland is a holocrine simple s accular gland extending over the entire surface of the skin except for the palms and soles of the hands and feet, respectively. The secretory portion of the sebaceous gland lies in the dermis and the excretory duct opens into the neck of the hair follicle. Sebaceous glands can be indepe ndent of the hairs and open directly onto the skin of the lips, corner of the mouth, glans pe nis, labia minora, and the mammary nipple. The oily secretion of the gland (sebum) is releas ed on the surface of the hair and the epidermis.8 The nature of sebums function in humans, the naked ape with no fur to protect, remains perplexing especially as significant evolutiona ry pressure must have driven the remarkable variation in the composition of sebum. While sebum production is the most obvious function of sebaceous glands, recent evidence suggest other important roles for these glands in the regulation of steroidogenesis, local androgen synthesis, skin barrier function, interaction with neuropeptides, potential production of both antiand pro-in flammatory compounds and synthesis of antimicrobial lipids.5 The nails are the least well studied of the sk in appendages and are hard keratin plates on the dorsal surface of the terminal phalanges of the fingers and toes. (Figur e 2-8) The nail plate covers the nail bed, the surface of the skin whic h consists of the stratum basale and stratum spinosum only. The cells that go to make up th e nail plate are similar to those generating the stratum corneum of the epidermis and the trichocytes that make up the hair shaft. Up to 90% of

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24 the nail keratins are of the hard hair keratin type; the remainder are soft epidermal keratins. Also present in the nail are water, lipids, and trace elements including iron, zinc, and calcium.5,8 The hair follicle is a tubular invagination of the epidermis and is responsible for the growth of hair. (Figure 2-9) The hair follicle is our bodys only perman ently regenerating organ, as it transits through life-long periods of growth (anagen), regres sion (catagen), and relative quiescence (telogen). Five million hair follicles reside in the skin, though only 2% are on the head. People with little body hair or with myriad amounts have the same amount of hair follicles, with hair visibility depending on hair size and distribution rather than number of follicles. The hair bulb is the end portion of the invaginated ha ir follicle. A vascularized connective tissue core, dermal papilla, projects into the ha ir bulb. The hair follicle consists of an external root sheath and an internal root sheath. The ex ternal root sheath is an inva gination of the epidermis and the internal root sheath is made up of three layers of soft keratin.8,5,11 The epidermis has classically been viewed as a stratified squamous epithelium maintained by cell division within the basal layer, whic h is attached to the basement membrane. Differentiating cells are gradually displaced ou twards through the stratum spinosum to the stratum corneum. The anucleate corneal cells, corn eocytes, or cornified cells, which protect the viable cell layers, are co ntinually shed from the skin surface, and the rate of production of cells in the basal layer must match th e rate of loss from the surface to produce normal skin thickness.7 Stem Cells The simple definition of a stem cell is the cell of origin for terminally differentiated cells in adult tissues. These cel ls have two distinct characteristics: self renewal, the ability to go through numerous cycles of cell division while maintain ing the undifferentiated state and multipotency, the ability to generate progeny of several di stinct cell types. The epidermis is a dynamic epithelium that is constantly re newed throughout life. Its turnover is estimated at about 60 days

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25 for humans. This rapid replacement demands, as with other epithelial tissues, that an adult have stem cells capable of supplying differentiate d cells throughout life. Stem cell niches are composed of microenvironmental cells that nurture stem cells and enable them to maintain tissue homeostasis. Skin epidermis and its associated structures arise from two stem cell populations within the hair follicle and interfollicular region s. One, in the basal layer of skin, normally gives rise to the stratified layers of skin. A second, the hair follicle stem cell, (Figure 2-10) resides in a region of the outer root sheath ca lled the bulge, and is responsible for the regeneration of hair and sebaceous glands. It has been suggested that bulge stem cells are also responsible for the long term replenishment of the inte rfollicular epidermis. However, it is now clear that bulge stem cells are not required for normal epidermal homeos tasis, although they can contribute transiently during wound healing.12,13,14 Skin Function Skin, as the outer most organ in the hu man body, continuously confronts the external environment and serves as a primary defense sy stem. Thus, knowledge of skin function is as important as knowledge of skin anatomy. Protecti on from the environment is one of the most fundamental functions of skin. Skin functions as a barrier to protec t internal organs from exposure to the outside environment and main tains an internal homeostatic environment.15,6 Keratin, the insoluble protein found in the squames of the stratum corneum and sebum, protects the skin against aqueous and chemical as saults. The fibroelastic connective tissue of the dermis provides the skin with mobility and skin pigmentation provides protection against ultraviolet radiation.6 Circulation and sweating are tw o of the primary thermoregulatory mechanisms. In warm environments there is an increased blood flow a nd heat is dissipated by dilated vessels through conduction, convection, radiation, and evaporation. Vasoconstric tion in a cold environment

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26 shunts blood flow to other vital organs to main tain function and warmth. Sweating occurs when there is increased activity by th e sweat glands. Cooling occurs when the secreted fluid is evaporated from the skin surface. Skin or body odor associated with sweating, is largely a result of bacterial flora.6 The skin has an elegant system of nerve recep tors to sense pain, touch, pressure, vibration, tickle, and temperature. Nerve receptors in the epidermis and dermis transmit impulses to the cerebral cortex for inte rpretation. Burning, itching, and tic kling are considered to be a combination of the four basic sensations. Un myelinated free nerve endings, Merkel cells, Meissner corpuscles, Krause end bulbs, Ruffini te rminals, and pacinian corpuscles propagate all these sensations. Attempts to identify particular responses with these specific nerve structures have not been entirely successful because many of these nerve receptors respond to more than a single stimulus. However, free nerve endings resp ond to touch, pain, and temperature; pacinian corpuscles respond to pressure, coarse touch, vibr ation, and tension; and Me issner corpuscles are involved in touch reception.6 Vitamin D is important in the mineralization of bone and in calcium and phosphate metabolism. Vitamin D is formed in the skin by the conversion of 7-dehydrocholesterol to cholecalciferol following exposure to ultraviole t light. The main function of vitamin D is to stimulate calcium reabsorption by the intestinal mucosa. Vitamin D, like all steroids, is transported to the nucleus of the intestinal cel l to induce the synthesi s of a calcium binding protein required for the transport of cal cium across the intestinal epithelium.8 As a protective interf ace between internal organs and the environment, the skin encounters a host of toxins, pathogenic organisms, and phy sical stresses. To combat these attacks on the cutaneous microenvironment, the skin functions as more than a physical barrier: it is an active

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27 immune organ. Immune responses in the skin involve an armamentarium of immune competent cells and soluble biologic response modifier s including cytokines. Langerhans cells are recognized as the major antigen pr esenting cells of the skin and pl ay a key role as the peripheral component of the immune system. Langerhans cells are one of a large fa mily of class II major histocompatibilty complex (MHC) bearing dendriti c cells critical to antigen processing and presentation to specific T lymphocytes. Account ing for more than 90% of epidermal cells, keratinocytes not only create a co mplex physical barrier to envir onmental agents but also serve as accessory cells in cutaneous immune responses Keratinocytes may play a role in initiating cell mediated immune responses in the skin by releasing cytokines and expressing cellular adhesion molecules to facil itate the movement of imm une competent cells. Although melanocytes have been traditionally consider ed to have no immunologic role, recent findings suggest that these cells may c ontribute to the immune function of the epidermis by secreting biologic response modifiers. Melanocytes repres ent 2-5% of epidermal cells; the number of melanocytes varies from person to person and in different anatomic sites of the same person. Like Langerhans cells, they possess numerous de ndritic like processes and are strategically placed in the lower epidermis, close to the dermis, where they may play important roles in immune responses.16 Phases of Wound Healing Hemostasis Wounds heal through a series of dynamic and overlapping processes that begin in response to tissue injury. The first major event following injury is hemostasis. Wounding destroys the various layers of skin and underl ying soft tissue just as a hurr icane or bombing destroy a house. Arteries, veins, and nerves become exposed just as plumbing and electr ic conduits are exposed. The initial phase of construction is to cap o ff these conduits in order to prevent further

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28 destruction and loss. Hemostasis serves as the initiating step in the wound healing process. The formation of a clot serves a dual purpose of preserving vascular inte grity and providing a temporary scaffold for the wound healing process to begin.17 Platelets, the smallest of the human blood cel ls, are actually a part of a much larger progenitor cell, the megakarocyte and are central players in the process of hemostasis. When a vessel wall is damaged, platelets are recruited from the circula tion to the unveiled subendothelial matrix forming a hemostatic plug to close the leak in the vessel wall. Platelets become activated when exposed to extravascular collagen and initia te the intrinsic part of the coagulation cascade. Platelet adhesion to collagen activates them to release soluble mediators (growth factors and cyclic AMP) and adhesive glycoproteins from their granules, which are in turn, signals for subsequent platelets to cha nge their morphology, become sticky and aggregate. The key glycoproteins released from the platelet granules include fi brinogen, fibronectin, thrombospondin, and von Willebrand factor. Soluble mediators released from granules include PDGF, TGF, TGF, bFGF, and VEGF. The clot that is fo rmed by the aggregation of platelets is more than dried blood. On the contrary, it re presents a viable, dynamic matrix of proteins and cells that not only contribute to hemostasis but also serve as a provision al lattice for incoming inflammatory cells, fibroblast, and growth factors. The main constituents of the visible clot are fibrin and platelets. Fibrin is produced by th e proteolytic cleavage of fibrinogen by thrombin. Insoluble fibrin mononmers are cross linked by factor XIII (fibrin stabilizi ng factor), and fibrin directly binds to plat elets to produce a clot.18,10,19,20 The clotting cascade first introduced in the 1960s described an eight step process of proenzyme-enzyme transformations beginning with the activation of factor XII (Hageman factor) and ending with the activation of fibrinogen (factor I) by thrombin (factor IIa) to form fibrin

PAGE 29

29 (factor Ia) 21. Over the next 40 years there have been changes in our understanding of how this cascade works. Factor X (Stuart factor) is the common point of the intrinsic and extrinsic coagulation pathways. The intrinsic and extrinsic pathways we re so named because of the location of their protein components, within the blood or outside the blood, respect ively. The intrinsic pathway is initiated when prekallikrein is c onverted to kallikrein and factor XII is converted to factor XIIa. Factor XIIa converts factor XI to factor XIa. Factor XIa activ ates factor IX, which with its cofactor, factor VIIIa, form the tenase complex whic h activates factor X to factor Xa. The extrinsic pathway is initiated by the proteoly sis of Factor VII. Factor VIIa then binds with tissue factor. The factor VIIa/tissue factor complex activates fact ors IX and X. From the activation of factor X both pathways converge into a common pathwa y down to the formation of fibrin from fibrinogen. The intrinsic and extrinsic pathways are now called the contact and tissue factor pathways, respectively. (Figure 2-11) These changes were made in response to how each pathway is initiated. The contact (intrinsic) pathway is initiated when the blood comes into contact with a negatively charged biological su rface in vivo, such as collagen and platelet membranes. The tissue factor (extrinsic) pathway requires the presence of tissue factor constitutively expressed on cells separated from flowing blood; that upon vascular in jury, tissue factor comes in contact with flowing blood, initiating reactions that lead to the generation of fibrin and a fibrin clot.22,23,24 A cofactor is any substance that is required in addition to an enzyme that needs to be present for a reaction to take place, or to make the reaction more efficient. In the case of the blood clotting cascade vitamin K and Ca++ act as cofactors. Vitamin K is essential for the synthesis of prothrombin, by converting glutamate to -carboxyglutamate, which makes up the

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30 N-terminal portion of the prothrombin molecule. The -carboxyglutamte is a strong chelator of Ca++ which brings the prothrombin molecule into cl ose proximity to the pl atelet by binding to the lipid membrane.25 The reactions of blood coagulation are care fully controlled by several anticoagulant mechanisms, which under normal conditions prevail over the procoagulant forces. Vitamin K dependent protein C is the key component of an important natural an ticoagulant pathway. The procoagulant properties of thrombin are lost on binding to thrombomodulin because thrombomodulin occupies the f unctionally important exosite I in thrombin and thereby blocks interactions with othe r thrombin binding proteins. On the endothelial surface thrombin and thrombomodulin form a complex with the endothelial protein C receptor. Protein C binds to its receptor on the endothelial surface and becomes ac tivated. The activated protein C exerts its anticoagulant effect by regulating th e activities of factor VIIIa and factor Va, the cofactors in the tenase and prothrombinase complexes, respec tively. The anticoagulant activity of activated protein C is enhanced by prot ein S, which is sufficient for the inactivation of factor Va.26 Eicosanoids are among the mediators and regul ators of inflammation and are generated from 20-carbon polyunsaturated fatty acids found in the lipid membranes of cells. Arachidonic acid is usually the major substrate for eicosa noid synthesis. (Fi gure 2-12) Eicosanoids, prostaglandins, thromboxanes, and leukotrien es, are generated from arachidonic acid by metabolic processes involvi ng the enzymes cyclooxygenase a nd lipoxygenase. Eicosanoids are involved in the intensity and durat ion of inflammatory responses, have cell and stimulus specific sources, and frequently have opposing effects. Thus, the overall physiological/pathophysiological outcome will depend on the cells present, the nature of the

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31 stimulus, the timing of the eicosanoid generati on, the concentration of different eicosanoids generated and the sensitivity of the target ce lls and tissues to the eicosanoids generated.27 Prostaglandins were first discovered in the 1930s and named because they were thought to be produced by the prostate. It is now known that prostaglandins are produced by the metabolism of arachidonic ac id. Phospholipase A2 mediates the release of arachidonic acid from membrane phospholipids, a rate limiting step in the formation of eicosanoids in nonpathologic states. There are two secretory forms of phospholipase A2, designated as PLA2I and PLA2II. PLA2I is produced by the pancreas and plays a role in digestion of phospholipids, where as, PLA2II is produced by inflammatory cells and is increas ed in conditions such as sepsis and acute respiratory distress syndrome. One member of the prostaglandin family is PGE2. PGE2 is a vasodialator, a natriuretic factor, an inhibitor of gastric acid secr etion, and an agent that contracts uterine tissue. Another member of the prostaglandin family is PGF2 and is synthesized in many tissues in varying amounts and is increased in sepsis. It is a bronchoconstrictor and venoconstrictor, and contracts uterine smooth mu scle. There are other prostaglandins with varying and often opposite actions.28 Prostcyclin, PGI2, is synthesized by endothelial cells macrophages, the lungs and the kidneys. It is a potent vasodialat or and an inhibitor of platelet aggregation that spontaneously hydrolyzes to form the stable but inactive metabolite 6-keto-PGF1 .28 Thromboxane, TX, was so named because it indu ces platelet aggrega tion, and is also a potent vasoconstrictor an d bronchoconstrictor. TXA2 is synthesized in large quantities by platelets, macrophages, monocytes, and the lungs. It is unstable a nd spontaneously hydrolyzes to form the stable but inactive product TXB2.28

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32 Leukotrienes were so named because they are produced by leukocytes. Leukotiene B4, LTB4, is synthesized by white blood cells, macrophage s, and synoviocytes. It is a chemotactic substance for white blood cells. The cyst eine containing leukotrienes include LTC4 which is synthesized by white blood cells, lung parenchyma l tissue, and macrophages. It is converted to LTD4 an active metabolite of LTC4. The cysteinyl leukotrienes are vasoconstrictors and bronchoconstrictors, and they increase capillary permeability and bronchial mucous secretion through activation of cysteinyl leukotrien e receptors 1 and 2.28 Inflammatory Phase Inflammation begins within 24 hours after inju ry and can last for up to 2 weeks in normal wounds. (Figure 2-13) An increase in vasodilati on and vascular permeability can begin in as little as 1 hour after injury. The major cell types involved in the inflammatory phase of wound healing are the neutrophil, macrophage, and mast cell. The first nucleated cell to inf iltrate the wound bed af ter the integrity of the skin has been disrupted is the neutrophil. This innate immune cell mediates th e first line of defense and marks the start of the inflammatory phase of wound healing.29 The neutrophils le ave the vascular system by a process known as extravasation. (Figure 1-14) The first step in this process is the appear ance of P selectin on the endothelial surface, after exposure to leukotriene B4, the compliment fr agment C5a, or histamine. E selectin appears a few hours later after exposure to TNF. The interaction of the P and E selectins with glycoproteins on the surface of the neutrophils allo ws reversible binding, so that the neutrophil appears to roll along the endot helium. Next, the neutophil attach es firmly to the endothelium by the binding of their leukocyte functional an tigen-1 (LFA-1) and compliment receptor-3 (CR3) to the intercellular adhesi on molecules-1 (ICAM-1) on the surf ace of the endothelium. In the third step, the neutrophil is able to squeeze between the endothelial cells a nd is able to penetrate

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33 the basement membrane with the aid of enzymes that breakdown the extracellular matrix proteins. The movement through the basement me mbrane is known as diapedesis. Finally, the neutrophils follow a chemokine grad ient to the site of the wound.30 The main function of neutrophils at the wound site is to decontaminate the open w ound by the destruction of invading microbes. In the circumstance of injury to barr ier surfaces, such as the skin, a very rapid and effective response by neutrophils minimizes the chance for infectious complications.29 Macrophages themselves are differentiated monocytes, and result from monocytes responding to certain chemical stimuli. Ther e are known to be three types of macrophage important to the wound environment; cytocida l, inflammatory, and repair macrophages. Monocytes become inflammatory macrophages in the presence of 1,3-glucan, and differentiate into repair macrophages in the presence of hya luronan. Cytocidal macrophages are formed in response to polyinosinate-polycytidylate and are k iller cells that remove bacteria and other debris from the wound site. Each type of m acrophage produces different growth factors and cytokines. Inflammatory macrophages produce PDGF, TGF, and bFGF. Repair macrophages, which help remodel the extracellular matrix of the wound, produce IL-GF-1 and also PDGF. It is the balance between the inflammatory and repa ir macrophage populations that appears to be crucial for successful wound healing.31 Once activated by direct inju ry, the mast cell located at the wound edges releases, by means of degranulation, the medi ators essential to tr igger the inflammatory reaction of the injured tissue, which mainly influence the local endothelial cells. Mast cells, through the release of vasoactive mediators, such as histamine, prot eases, tumor necrosis factor, and metabolites of archidonic acid induce vasodilation and increase vascular permeability.32

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34 Histamine enhances the permeability of arteriol es, capillaries, and venules to albumin, then globulin and finally fibrinogen. Hist amine increases the permeability of these vascular structures by inducing contractions of the e ndothelial cells and part ially removing fenestral diaphragms that cover the gaps or fenestrae of the endothelium. Histamine is rele ased from preformed and stored sources as well. All known varieties of tissue injury cause the de novo synthesis of histamine.33 The kinins are biologically activ e and nearly indistinguishable peptides in areas of tissue destruction. The most familiar kinin, bradykinin, is a potent inflammatory substance that is released in injured tissue from plasma proteins by the plasma enzyme, kallikrein. The action of the kinins on the microvasculature is similar to th at of histamine. Kinins are rapidly destroyed by tissue proteases, suggesting that their importance is limited to th e early inflammatory stage of wound healing.34 Proteases form a large group of enzymes th at are capable of h ydrolyzing proteins. Proteases are grossly divided into two majo r groups, depending on th eir site of action. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distan t from the termini of the substrate. Based on the functional groups present at the active site, proteases are fu rther classified into four prominent groups; serine proteases, aspart ic proteases, cysteine proteases, and metalloproteases.35 Serine proteases are characterized by the pres ence of a serine group in their active site. They are numerous and wide spread among viruse s, bacteria, and eukaryotes, suggesting that they are vital to the organisms. Aspartic prot eases, commonly known as acidic proteases, are the endopeptidases that depend on aspartic acid resi dues for their catalytic activity. Cysteine proteases occur in both prokayotes and eukaryotes About 20 families of cysteine proteases have

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35 been recognized. The activity of all cysteine pr oteases depends on a catalytic dyad consisting of cysteine and histadine. Metalloproteases are the mo st diverse of the catalytic types of proteases. They are characterized by the re quirement for a divalent metal ion for their activity. About 30 families of metalloproteases have been recognized.35 The two main proteases involved with wound h ealing are the serine and metalloproteases. Almost one third of all proteases can be classified as serine pr oteases. The serine proteases are endopeptidases that have a catalytic site made up by three amino acids, serine, aspartic acid, and histadine, the catalytic triad. They represent a vast class of proteases with members with a broad variety of functions. Nearly all members ar e synthesized as zymogens, which are activated by proteolytic removal of an N-terminal part of th e molecule that covers and hides the active site. Serine protease can be further classified into four different clans, chymotrypsin, subtilisin, carboxypeptidase Y, and Clp protease. Chymotryps in-like proteases are the most abundant in nature. Chymotrypsin-like proteases are involv ed in many critical phys iological processes. Cascades of sequential activation of serine proteases drive blood coagulation, complement fixation, and fibrinolysis. Similar protease cascad es appear to be invol ved in development, matrix remodeling, differentiation, and wound healing.36 Matrix metalloproteases (MMPs) are zinc endopeptidases involved in numerous physiological and pathological processes. There ha ve been 24 MMPs identified in humans, with a wide variety of substrates, su ch as, components of the extrace llular matrix, ce llular receptors, growth factors, and cytokines. Functions of MMPs embrace tissue growth and remodeling during development and disease, including endometria l cycling, angiogenesis, cellular migration, skeletal formation, inflammation, wound hea ling, coagulation; periodontal, rheumatic, cardiovascular, and pulmonary diseases, neuroi nflammtion, cancer, and metastasis. Expression

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36 of MMPs is tightly regulated at the transcriptiona l level by hormones, growth factors, cytokines, and cell-to-cell and cell-to-matrix interactions.37 The MMPs alter the extr acellular matrix during the process of wound healing. In wound healing, MMPs control platelet aggregation, macrophage and ne utrophil function, cell migration and proliferation, neoangiogene sis, and collagen secretion and deposition. MMPs have a significant role in all phases of wound healing. They promot e cell migration, break down damaged extracellular matrix, and assist with remodeling. However, a disparity between the levels of MMPs and tissue inhib itors of metalloproteases (TIM Ps) can be harmful because a disproportionate MMP activity can result in an increased degrad ation of fibronectin, collagen, and diverse growth factors. A normal fluctuati on of MMPs is seen in healing wounds, whereas chronic wounds tend to ha ve prolonged elevations.38 Triggers such as microbial i nvaders, tissue injury, and surgi cal trauma activate the release and formation of arachidonate derived prostagla ndins, which regulate the early events in the inflammatory response. At sites of inflammation, prostaglandins initiate many responses relevant in inflammatory events, but most notably they signal the end of infl ammation by activating the transcriptional regulation of 15-lipoxygenase (15-LO) in neutr ophils, which in turn leads to the temporal dissociation and production of lipoxi ns from arachidonic acid, which have proresolving and anti-inflammatory functions. This is referred to as class switching of the arachidonic acid derived eicosano ids from prostaglandins and leukotrienes to lipoxins that initiate the termination of inflammation. The omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid, EPA and DHA respectivel y, are converted to new families of lipid mediators that are pivotal in promoting the re solution of inflammation. These new families of lipid mediators are called resolvins and protectins.39

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37 Proliferative Phase The proliferative phase include s the most prominent events in wound healing. It begins about 4-5 days after wounding and last for a few weeks. The main processes that take place during this phase are angiogenesis, fibroplasia, re-epithelialization, and extracellular matrix formation.40 Angiogenesis refers to new vessel growth or neovascularization and occurs in a wound in at least three ways: generation of a de novo vasc ular network in the wound space, anastomosis to preexisting vessels, and coupling or recoupling of vessels throughout th e wound space. (Figure 2-15) Arising from new blood vessels adjacent to the wound, new capillary buds extend into the wound itself.34 The endothelial cell is the most impor tant cell involved in angiogenesis, and through intercellular adhesion mol ecules plays an active role in leukocyte migration into the wound environment. Endothelial cells have other critical roles during th e wound healing process, including formation of new vessels providing tr ansport of oxygen and nutrients into the wound and secretion of biologically ac tive substances. Soluble factors released by cells such as macrophages are believed to stimulate angioge nesis during the wound healing process. In addition, factors such as low oxygen tension, lactic acid, and bi ogenic amines can potentiate angiogenesis.41 One of the most common and expected si gns that wound healing is progressing in a normal, orderly fashion is the development of gr anulation tissue. Granul ation tissue that forms during healing consists of new ve ssels that migrate into the wound and proliferate, along with the accumulation of fibroblast and ground substances. Th e most important cell in the production of the dermal matrix is the fibroblast. Fibroblas ts migrate into the wound within 48 to 72 hours.34,41 Connective tissues are composed of three elements: cells, fibers, and amorphous ground substance. The fibers and ground substance are co llectively referred to as extracellular matrix.

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38 Ground substance is an amorphous viscous gel secreted by fibroblast occupying the spaces between the cells and fibers of the connective ti ssue. It helps determine compliance, flexibility, and integrity of the dermis; prov ides strength, support, and dens ity to tissue; reduces friction between connective tissue fibers during tissue stre ss or strain; and protects tissue from invasion by microorganisms.34 Fibroblasts migrate into the wound site from the surrounding tissue, become activated and begin synthesizing collagen, and pr oliferate. PDGF and EGF are the main signals to fibroblasts and are derived from platelets and macrophages. Fibroblasts already loca ted in the wound site begin synthesizing collagen and transform into myofibr oblasts for wound contraction.42 The phenotypic changes that occur to the fibroblast correlate with th e cells various roles in wound healing. The first role of the fibroblast is migr ation. The fibroblast then begins producing large amounts of matrix materials, in cluding collagen, proteoglycans, and elastin. Type I collagen is the major collagen in the normal adult dermis. T ype III collagen, present in large quantities in fetal dermis, is a minority component of norma l adult collagen but durin g wound healing is the predominant type of collagen synthesize d. The new connective tissue also contains glycosaminoglycans and proteoglycans. The s ynthesis of these matrix proteins occurs concomitantly with the production of the new collag en. During this time, fibroblasts alter their phenotypic character to become myofibroblast. As a myofibroblast, the cell can also participate in wound contracture.34,41 The contractile forces produced by granul ation tissue in wounds are derived from myofibroblasts that contain cont ractile proteins. Wound contraction is thought to be a result of these actin rich myofibroblast, which are the most prominent cells in granulation tissue. Myofibroblast within the wound align along the lines of contraction and differ from other

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39 cellular components, in cluding leukocytes and endothelial ce lls, which do not exhibit such organized orientation. This musc le like contraction of myofibroblast is mediated by PGF, 5hydroxytryptamine, angiotensin, va sopressin, bradykinins, epine phrine, and norepinephrine. Contraction is unified and requires cell-cell and cell-matrix communication.34,41 Re-epithelialization is criti cal to optimal wound healing, not only because of the reformation of the cutaneous barrier, but also because of its role in wound contraction.43 The features observed near the wound margin and in the migrating epidermal tongue suggest that two independent and complementary mechanisms are involved in w ound reepithelialization.44 The classic mechanism of epithelial cell migrati on over the wound surface is the leap frog model.34 In this model, epidermal cells migrate no more th an two or three cell lengths from their initial position, sliding or rolling over epidermal cells pr eviously implanted. The migrating cells then become fixed at this site. Other epidermal ce lls successively migrate over these cells. The epidermal layer will progressively adva nce and close the epithelial defect.34 A second model of reepithelialization is the train model. In this model each cell maintains its original position in the chain. This continues until migration is halted by contact with other epidermal cells or by a complete reepithelializa tion of the wound surface. In either model a migrating tongue of epithelium develops.7,34 Growth factors play a role in signaling cell migration. Among the two that are thought to be very important are TGFand EGF. TGF, an important mediator in dermal growth regulation, stimulates keratinocyte production of fibronectin and its deposition in the extracellular matrix. TGFalso stimulates epidermal migrati on, but it is not know n whether this is solely an effect of increased fibr onectin production. It is known that TGFis a potent inhibitor of keratinocyte prolifer ation in vitro, and while these cells are migrating they do not

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40 proliferate. This may allow the cells at the fo refront to remain in migrating mode. Until the wound is closed, a zone of proliferating cel ls remains between the migrating tongue of epithelium and the normal cells at the wound edge. At that point all of the keratinocytes enter the proliferative mode. The stimulus for cells unde rgoing rapid proliferation is not known, however, several growth factors maybe involve d, including EGF and its homologue TGF. In culture, EGF induces hypertrophy and hyperplasia of epitheli al cells; this and its presence in human epidermis suggest a role as a stimulus for epidermal cell proliferation in vivo.41 Remodeling Phase The maturation or remodeling phase is the fi nal stage of wound healing. Collagen fibers reorganize in this phase. Fibrobl ast, MMPs, and the inhibitors of MMPs play a role in this process, as do some of the growth factors.45 However, the main feature of this phase is the deposition of collagen in an or ganized and well mannered network.42 The clinical manifestations of remodeli ng include contractio n, decreased redness, decreased thickness, decreased induration, a nd increased strength. A lthough these changes continue to develop over a period of weeks, mont hs, and even years their initiation overlaps the production of granulation tissue during the proliferative phase of wound healing. Wound contraction is a cell directed proce ss: cell division is re quired, but collagen s ynthesis is not. In an unsutured wound, wound edges move toward one anot her at a rate of approximately 0.6 to 0.75 mm/day.18 Net collagen synthesis will continue for at least 4 to 5 weeks after wounding. The collagen that is initially laid down is thin ner than collagen in uninjured skin and is oriented parallel to the skin. Over time the initial collagen threads are reabsorbed and deposited thicker and organized along the stress lines of the wound. These change s are also accompanied by a wound with an increase in tensile strength, indicating a positive co rrelation between collagen fiber thickness /

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41 orientation and tensile strength. The collagen f ound in granulation tissue is biochemically different from collagen found in uninjured ski n. Granulation tissue co llagen has a greater hydroxylation and glycosylation of ly sine residues, and this increa se of glycosylation correlates with the thinner fiber size. The collagen in th e scar will never become as organized as the collagen found in uninjured skin. Wound strength also never returns to 100%. At 1 week, the wound has only 3% of its final strength; at 3 weeks, 30%; and at 3 months and beyond, approximately 80%.42 As scars mature, they become less red. This change mirrors a change in the density of capillaries within the wound over time. Whereas young wounds are characte rized by granulation tissue containing a relatively high level of capillary density, matu re wounds are less vascular. Fibroblasts also decrease over ti me. Together, these changes resu lt in a relatively acellular mature scar. The absence of appendages is anothe r characteristic associated with mature scars. The most clinically obvious appendageal loss involv es missing hair follicles. The inability of scar tissue to reproduce a suitab le appendage specific niche ma y contribute to the absence of these structures in mature scars.18 Growth Factors Growth factors and cytokines are both members of a larger group of polypeptide regulatory molecules released by many cell lines in the body. In fact, although th ey have often been described separately from cytokines, growth factors are cytokines whose primary role is directing the maturation of cells during normal turnover and in the post injury tissue repair response.46 It has been shown that th e fibrocytes from burn injured patients produce higher levels of the growth factors TGFand CTGF than non burned patients.47

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42 Transforming Growth Factor-Beta The TGFsuper family consists of a diverse rang e of proteins that regulate many different physiological processes, includ ing embryonic development, homeostasis, and wound healing, chemotaxis, and cell cycle control. The TGFsuper family includes nearly 30 proteins in mammals. 3 Initially when discovered, TGFwas termed sarcoma growth factor. Five different mature forms of TGFhave been isolated and desi gnated 1 through 5 based on their chronological identification.48 TGFis a major modulator of wou nd healing. It is constitutionall y present in platelets, but is also produced by several cell types presen t in wounds, including activated macrophages, granulocytes, fibroblast, and keratinocytes. TGFreceptors are widely distributed and found on essentially all cell types. TGFis released as an inactive pe ptide bound to its propeptide, and requires activation either by prot eolysis or as a result of the acid environment found within a wound. 2 Over expression can be unfa vorable to wound repair, and TGFcan induce its own expression through a positive feed back loop. This can lead to elevated scaring and tissue damage.49 TGFdirects the functions of monocytes, endo thelial cells, fibroblas t, and keratinocytes during all phases of wound healing, and is one of the most powerful chemoattractants.48 Local release of TGFat the site of tissue damage comes primarily from -granules of platelets. Once released, one of the initial actions of TGFis recruitment of inflammatory cells, primarily neutrophils and macrophages. These ce lls are stimulated in a positive feedback mechanism to produce more TGFto perpetuate its activity.48 There is conflicting evidence as to whether TGFpromotes or inhibi ts angiogenesis. A report by Goumans et al, suggests that the direction TGFtakes endothelial cells, stimulatory or inhibitory, may depend on the particular R-Sm ad that becomes activ ated intracellularly.48

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43 The relationship of fibroblast and TGFis fundamental to the proliferative and maturational phases of wound healing. TGFstimulates fibroblast to proliferate and migrate into a wound while producing extr acellular matrix. This extracellular matrix allows other healing processes to occur. Fibroblast transform into myofibroblast under th e stimulation of TGF, which causes wound contraction and maturation.48 Epithelialization is the process in which kera tinocytes both prolifer ate and migrate from wound edges to create a barrier over the wound. The overall effect of TGFin this process is not as fully understood as its other functions because studies have s uggested that it both inhibits and stimulates keratinocytes. TGFdoes influence keratinocytes and epithelialization of wounds, but whether it plays an overall stimulator y or inhibitory role is yet to be fully determined.48 A summary of some of the functions of TGFThree isoforms; TGF1, TGF2, TGF3 Is up regulated in human cancers Plays crucial role in tissue regenera tion, differentiation, embryonic development synthesis of matrix proteins metalloproteinase inhibitors levels of proteolytic enzymes Excess TGFcan lead to hypertrophic scar formation TGFinhibits produ ction of IFNand IL-2 TGFpromotes IL-10 production, s uppressing macrophage function TGFappears to shift immune r eactions to a Th-2 response Connective Tissue Growth Factor In adult skin, CTGF normally is not expre ssed unless induced, for example, during the normal wound repair process. 3 CTGF is a 38 kD protein that contains 38 conserved cysteine residues and a heparin binding domain and is ch emotactic and mitogenic for connective tissue cells. CTGF functions as a downstream mediator of TGFaction on connective tissue cells,

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44 stimulating cell proliferation and extracellular ma trix synthesis. The induction of CTGF by TGFis cell type specific, as it occurs in connective tissue cell s but not epithelial cells or lymphocytes. In addition to fibroblast; endothelia l cells, vascular smooth muscle cells, epithelial cells, and chondrocytes produce CTGF mRNA.50 CTGF, a member of the CCN family of ma tricellular proteins, promotes fibroblast proliferation, matrix producti on, and granulation tissue formati on. CTGF promotes cell adhesion and migration of a wide variety of cell types as we ll as collagen matrix c ontraction in fibroblast. Although, a specific CTGF receptor has yet to be identified, CTGF appears to perform many of its functions through integrins, heparin sulfate containing prote oglycans, and the low density lipoprotein receptor related protein.3 Studies have shown that rh CTGF has been successful in treating burn injuries in non human primates.51 A summary of some of the functions of CTGF CTGF is secreted by fibroblast after activation by TGFCTGF is a downstream mediator of TGFCTGF stimulates cell prolif eration and ECM synthesis CTGF is a more specific target for selective internvention Hormones The term hormone comes from the Greek hormaein. The original use of hormone implied an agent that can excite or arouse; in the modern era a hormone is perceived to be a defined chemical entity that is se creted or produced by specific gla nds or cells and that acts as a chemical messenger or signal molecule.52 Androgens are steroid hormones that induce th e differentiation and maturation of the male reproductive organs, the development of male sec ondary sex characteristics, and the behavioral manifestations consistent with the male role in reproduction. The two most important steroid

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45 hormones of the adult male are testosterone and 5 -dihydrotestosterone. Testosterone is the principal male androgen produced by the testes. The testes also produce approximately 10-20% of the estrogens found in males; the remainder is produced in a variety of nonendocrine tissues including brain, liver, fat, and skin, all of which have low levels of the cytochrome P450 aromatase necessary to transf orm androgens into estrogens.52 The two most important steroid hormones of the adult female are estradiol and progesterone. There are three naturally occurri ng estrogens in women, estradiol, estriol, and estrone. 17 -estradiol is the most abundant of th e estrogens from puberty until menopause. Estrone is weaker than estradiol, and in post menopausal women more estrone is present than estradiol. Menopause is defined as the cessation of ovul ation by the ovaries. This occurs in women between the ages of 40 and 50 years due to the utilization of the fixed number of follicles that were established in the fetal ovaries. The endocrinological conseque nces of cessation of ovulation are varied, and a vari ety of medical problems may de velop, depending upon the exact nature of the reproductive hormonal balance ach ieved. While the postmenopausal ovary is not totally devoid of the ability to secrete steroi ds, the production level is markedly reduced in comparison to a younger ovulating woman.52 Estrogen There is striking evidence from animal studies dating back to 1962 th at estrogens play a crucial role in cutaneous wound healing, and repa ir is significantly de layed in its absence.53 The presence of estrogen receptors (E R) in human skin fibroblast and inflammatory cells suggest that local estrogen levels may influence cuta neous physiology, incl uding the wound healing process.53 One way that estrogen may influence wound h ealing rates is by the regulation of the growth factor TGF. TGF1 is the cytokine that has receiv ed the greatest attention in the

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46 context of wound repair. TGFis involved in cell proliferat ion, differentiation and matrix production, and also scar fo rmation. In experiments by Ashcroft, et al wound healing rates among post menopausal women and men were dete rmined to be relatively equal while pre menopausal women had faster healing rates. This was thought to be secondary to the increased levels of estrogen in the pre menopausal women. Wound healing rates among the post menopausal women and men were shown to impr ove with the addition of a topical estrogen patch. Although, estrogen has been shown to im prove wound healing rates a negative sequale was noted. The pre menopausal women had the quicker healing rates, but also the more hypertrophic scarring. The scars of the post menopausal women and men were consistently pale and flat, compared with the pigmented, ev erted lesions of the pre menopausal women.54 A summary of how estrogen assists in wound healing Estrogen accelerates wound healing by dampening local inflammation Estrogen suppresses the chemotaxis of PMNs Estrogen alters the expression of endothelial adhesion molecules With a decrease in the numbers of PMNs found in the wound, there is also a decrease in Elastase activity Elastase is a serine prot ease derived from PMNs Elastase degrades structural and functional proteins, such as, proteoglycans, collagen, and fibronectin Fibronectin directs the migra tion of keratinocytes, increases the amount of fibroblast and collagen deposition, and stimulates wound contraction

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47 Figure 2-1. Cross section of hu man skin showing the two layers of the epidermis and dermis with the underlying hypodermis that connects the skin to underlying structures (http://www.realage.com/realbeauty/lm.aspx last accessed Febuary 13th, 2007).55

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48 Figure 2-2. Cross section of th e five layers that make up the epidermis(http://www.ratbehavior.org/images/epidermis last accessed Febuary 13th 2007)56

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49 Figure 2-3. Basement membrane (1) Tonofilaments provide stability between cells, (2) desmosomes that stabilize cells with each other, (3) hemidesomsomes that keep cells attatched to the basement membrane, (4) basement membrane(http://www.unifr.ch/histologie/elearn ingfree/francais/epitel/epithel05.h tml Last accessed Febuary 13th, 2007) 57

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50 Figure 2-4. Dermal papillae in terdigitates with th e epidermis to strengthen the connection between the two layers(www.cytochemistry.net/microanatomy/skin/skin_and mammary_glands.html last accessed.July13, 2007).58

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51 Figure 2-5. Diagram of fibr oblast laying down collagen fiber bundles from the precursor tropocollagen(www.udel.edu/biology/wags/histop age/illuspace/icta/icta6.gif last accessed Febuary 13th, 2007)59

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52 Figure 2-6. Cells and other proteins that make up the extracellular matrix (www.steve.gb.com/science/extracelluar_matrix.htm last accessed Febuary 13th, 2007).60

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53 Figure 2-7. Cross section of skin showing various appendages includi ng hair follicle, and sweat glands(www.tuffrhino.com/artcles.asp?id=113 last accessed Febuary 13, 2007)61

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54 Figure 2-8. The nails are anothe r appendage of skin, 90% of the nail made up of hard hair keratin, the remainder is made of soft epidermal keratin(www.becomehealthynow.com/popups/nails.htm last accessed Febuary 13, 2007).62

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55 Figure 2-9. Diagram showing structure of the hair follicle and relation to epidermis and dermis(www.thegentletouch.com/hairbiol/h -morph3.html. Last accessed Febuary 13 2007).63

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56 Figure 2-10. Hair follicle: Hair follicle stem ce ll is responsible for the regeneration of hair and sebaceous glands, They are not respon sible for long term replenishment of epidermis, although they can c ontribute during wound healing (www.virtualaboratory.net/biofundamentals /lecturenotes/topic5-3_stemcells.htm ., Last accessed Febauary 13, 2007)64

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57 Figure 2-11. Pro enzyme-enzyme pathway leading to hard fibrin clot during the process of hemostasis (www.facstaff.bloomu.edu/tbel2/research-blood.htm ., Febuary 13, 2007)65

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58 Figure 2-12. Generalized pathway for the convers ion of arachidonic acid to ecosanoids. COX, cyclooxygenase; HETE, hydroxyeicos atetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin; TX, thromboxane (Repri nted with permission from Am. J. Clin.Nutr.2006; 83:1505S-1519S ) .27

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59 Figure 2-13. The inflammatory phase of wound healing encompasses many processes going on at the same time(www.biologymad.com/immunology/inflammation.jpg ., Last accessed July 30th, 2007)66

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60 Figure 2-14. Diagram showing the process of diapedesis and extravastion of neutrophils during the early inflammatory proce ss(Reprinted from Immunobiology: the immune system in health and disease. 2005; 6th edition: 37-100).30

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61 Figure 2-15. Angiogenesis is one of the ma jor processes that ta ke place during the proliferative phase of wound healing (www.angio.org/providers/woundcar/woundcare.html., Last accessed July28th, 2007)67

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62 CHAPTER 3 MATERIALS AND METHODS Tissue Collection This project was approved by the University of Florida Institutiona l Review Board (IRB) on October 5, 2005. All tissue samples were coll ected in the operating room. All burn tissue samples were collected in the burn unit operating room except for patient number 38 which was collected in the main operating room at Shands at the University of Florida. Tissue samples collected from patients undergoi ng elective plastic surgery proce dure were collected at Alachua General Hospital, Ayers Surgical Center, Florida Su rgical Center, or Shands at the University of Florida. One di-potassium et hylenediaminetetraacetic acid (K2 EDTA) Vactutainer (BD Vacutainer, North America) of bl ood was collected either from an existing intravenous line or by venipuncture. Tissue samples collected from burne d injured patients and fr om non burned plastic surgery patients were collected using a 6 mm punch biopsy tool. Biopsies were taken from excess skin debrid ed during surgery. Biopsies were taken from the burned injured area, from the donor site of th e same patient and from excess skin debrided during normal plastic surgery procedures. Tissu e from one 6 mm punch biopsy was placed in a 10% buffered formalin solution; tissue from anot her 6 mm punch biopsy wa s placed in RNAlater solution. These samples were placed in a 4 C cooler for 24 48 hours after which the tissue placed in 10% buffered formalin solution was taken to be imbedded by an independent contractor in the department of Anatomy and Cell Biology at th e University of Florida. The samples were placed in paraffin block and fixed to slides. The tissue samples that were placed in RNAlater solution were transferre d to separate microcen tifuge tubes labeled and stored in -80 C freezer to be archived for future studies

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63 After tissue samples were collected the blood was spun down in order to collect the plasma. The blood was spun for 10 minutes at 4 C at 3000 RPMs in an Eppendorf 5810R with the acceleration and brake set at 9. Approximately 1.5 ml of plasma was obtained and aliquoted into 3 separate microcentifuge tubes of approxi mately 0.5 ml each. These tubes were labeled and stored in a -80 C freezer. Only one of these tube s was needed for this study, the extra tubes of plasma were archived for future studies. Immunostaining for CTGF Immunohistochemistry staining for CTGF was done first by deparafinizing slides as follows: Xylene-2 minutes Xylene-2 minutes 100% ethyl alcohol-1 minute 95% ethyl alcohol-1 minute 75% ethyl alcohol-1 minute 50% ethyl alcohol-1 minute Distilled H2O-1 minute The slides were then laid out on a tray and wax circles were im mediately made around samples using ImmEdge pen (Vector Laboratories, Burlingame, Ca ., USA) Blocking buffer, 1% fetal bovine serum (Gibco/Invitrogen Corp, Carlsba d, Ca.) in PBS, was applied to each sample. Non-specific receptor blocking was allowed to take place for 3.5 hours. After blocking, the blocking buffer was removed from the right (test) side only, while the left (control) side remained covered in blocking buffer. The 1 antibody (1g/ml CTGF obtained from Dr. Gary Grotendorst, Lovelace, New Mexico) was then applied to the test side only and was allowed to incubate in a humidity chamber over night. Th e next morning 1 antibody was collected and saved to be used in other experiments. The slid es were then washed in PBS for 2 minutes and 1 minute, respectively. One drop of 2 antibody (bioti nylated rabbit anti-goat, Vector Laboratories,

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64 Burlingame, Ca.) was added to 10 ml of blocki ng buffer and applied to both control and test sides of slides. The 2 antibody was allowed to incubate for 30 minutes. The slides were then washed 3 times in PBS for 2 minutes, 2 minutes and 1 minute. The ABC-AP working solution was made by adding 2 drops of reagent A and 2 drops of reagent B to 10 ml of blocking buffer, this solution was allowed to stand for 30 minutes prior to use. The slides were next allowed to incubate for 30 minutes in VE CTASTAIN ABC-AP Reagent (Alkaline Phosphatase, Vector Laboratories, Burlingame, Ca.). Slides were wa shed 3 times in PBS for 2 minutes, 2 minutes, and 1 minute. After the final wash, slides were placed in tub of substrat e (Vector Red, Vector Laboratories, Burlingame, Ca.). Th e substrate was made as follows: Substrate To 200 mL of 100 mM Tris-HCL, (pH 8.2-8.5), approximately 80 drops of reagent 1 was added 80 drops of reagent 2 80 drops of reagent 3 40 drops of levamisol was added to inhibit the endogenous alkaline phosphatase activity The slides were allowed to incubate in this substrate for 20 minutes, followed by dehydration as follows: Distilled H2O-1 minute 50% ethyl alcohol-1 minute 75% ethyl alcohol-1 minute 95% ethyl alcohol -1 minute 100% ethyl alcohol-1 minute Xylene-2 minutes Xylene-2 minutes After dehydration the samples were mounted wi th Permount (Fisher Scientific, USA) and cover slips were applied.

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65 Immunostaining for TGFImmunohistochemistry staining for TGFwas initiated by first making all solutions necessary for assay, solutions were made as follows: 1 antibody, 500g of mouse monoclonal anti-human TGFantibody (R&D Systems, Minneapolis, Mn., USA) was reconst ituted with 1mL of sterile PBS. 1% H2O2 solution was made to quench endogenous peroxidase acitity Blocking serum was made by combining 75l of normal blocking serum (goat) with 5ml of stock PBS Biotinylated 2 antibody was made by comb ining 75l normal blocking serum (goat), 5ml stock PBS, and 25l biotinylated 2 antibody stock AB enzyme reagent was made by combining 50l reagent A (avidin), 50l reagent B (biotinylated HRP), and 2.5 ml PBS. This was allowed to stand for 30 minutes prior to use Peroxidase substrate was made by combining 1.6 ml dH2O, 10 drops of 10x substrate buffer, 1 drop 50x DAB (diaminobenzidine) and 1 drop peroxidase substrate Immunohistochemistry staining for TGFwas done using pre made kit (Santa Cruz Biotechnolgy, Santa Cruz, Ca, USA), procedure started as follows: Deparafinize Xylene-2 minutes Xylene-2 minutes 100% ethyl alcohol-1 minute 95% ethyl alcohol -1 minute 75% ethyl alcohol-1 minute 50% ethyl alcohol-1 minute Distilled H2O-1 minute After the slides were deparafinized, the slides were then laid out on a tray and wax circles were immediately made around samples using I mmEdge pen (Vector Laboratories, Burlingame, Ca., USA). They were then incubated in a 1% hydrogen peroxide solu tion for 10 minutes to quench the endogenous peroxidase activity. This was followed by washing with PBS two washes of 5 minutes each. The slides were then blocke d with 1.5% blocking solu tion made earlier. Non specific blocking was allowed to take place for one hour. After one hour the blocking buffer was removed from the test (right) side only while blocking buffer was allowed to stay on control

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66 (left) side. The 1 antibody was then applied to te st side and allowed to incubate for 30 minutes. The 1 antibody was titrated to 5g/ml concentr ation for this assay. Fo llowing incubation with the 1 antibody the slides were washed with PB S three washes of 5 minutes each. The slides were next incubated with the biotinylated 2 antibody for 30 minutes, followed by three washes of PBS for 5 minutes each. The s lides were then allowed to incubate with the AB enzyme reagent for 30 minutes, again followed by three wash es of PBS for 5 minutes each. The substrate was now applied and allowed to incubate for 10 minutes. Following incubation with the substrate the slides were dehydrated as follows: Dehydration dH2O-1 minute 50% ETOH-1 minute 75% ETOH-1 minute 95% ETOH -1 minute 100% ETOH-1 minute Xylene-2 minutes Xylene-2 minutes After dehydration the samples were mounted wi th Permount (Fisher Scientific, USA) and cover slip applied. After all immunostaining was complete Ze iss Axiovision microscope was used to take photographs of sample at varying magnifications. After all photographs were taken they were analyzed using Image J68 to quantify the level of growth factor staining.

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67 Estradiol EIA Kit The estrogen concentration of each sample was assayed using Estradiol EIA Kit (Cayman Chemicals, Ann Arbor, Michigan, USA). Prior to beginning the assay all solutions and buffers were made as follows: EIA Buffer Dilute the contents of one vial of EIA buffer c oncentrate (Vial #4) with 90 ml of Ultra Pure water Wash Buffer Dilute the contents of the vial (5 ml) of wash buffer concentr ate (vial #5) to a volume of 2 liters with Ultra Pure water, then add 1 ml of Tween 20 (vial 5a) Estradiol Standard A pipette tip was equilibrated with ethanol, using the equi librated pipette tip 100l of Estradiol Standard (vial #3) was placed into a clean test tube then diluted with 900l Ultra Pure water. Bulk standard concentration will be 10ng/ml Eight clean test tubes numbered 1 through 8 were labled 900l EIA Buffer was aliquoted into tube #1 500l EIA buffer was aliquoted into tubes #2 through #8 100l of bulk standard (10ng/ml) was transf erred into tube #1 and mixed thoroughly Serial dilutions were made by removing 500l from tube #1 and placing it in tube #2, mixed thoroughly 500l was removed from tube #2 and placed into tube #3, mixed thoroughly The process was repeated for tubes #4 through #8 Estradiol Acetylcholinesterase Tracer 100 dtn estradiol tracer (vial #2) was reconstituted with 6ml of EIA buffer Estradiol Antiserum 100 dtn estradiol antiserum (vial #1) was reconstituted with 6 ml EIA buffer Plate Set Up See table 3-1

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68 Performing the Assay EIA Buffer 100l EIA buffer was added to Non Specific Bi nding (NSB) wells and 50l EIA buffer to Maximum Binding (B0) wells Estradiol Standard 50l from tube #8 was added to both of the lowest standard wells (S8) 50l from tube #7 was placed in the next lowest standard wells (S7) This process was continued until all the standard wells were aliquoted The same pipette tip was used to aliquot all of the samples, the tip was equilibrated in each standard prior to pipetting that standard Samples 50l of sample was added to corresponding wells Estradiol Acetylcholinesterase Tracer 50l of estridiol tracer was added to each well except the Total Activity (TA) and Blank (Blk) wells Estradiol Antiserum 50l of estradiol antiserum was added to each well except the Total Activity (TA), the Non Specific Binding (NSB), and the Blank (Blk) wells After all wells were filled the plate was covered with plastic film and allowed to incubate for one hour at room temperature. During the one hour incubation 100dtn E llmans Reagent (vial #8) was reconstituted with 20ml of Ultra Pure wa ter. Since Ellmans Reagent is unstable once it is prepared it was protected from the light. Wh en the one hour incubation was finished the wells were emptied and washed five times with wash buffer, followed by the addition of 200l of Ellmans to each well and 5l of tracer to the Total Activity (TA) wells. The plate was then

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69 covered with plastic film and allowed to incuba te in the dark for one hour. After this final incubation the plate was read using Wallac 1420 multilabel counter.

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70Table 3-1. Plate 1 set up for Estradiol EIA 1 2 3 4 5 6 7 8 9 10 11 12 A Blank Blank Blank S5 S5 S5 04-00-01 04-00-01 04-00-01 09-00-03 09-00-03 09-00-03 B NSB NSB NSB S6 S6 S6 05-00-01 05-00-01 05-00-01 09-00-04 09-00-04 09-00-04 C B0 B0 B0 S7 S7 S7 06-00-01 06-00-01 06-00-01 10-00-01 10-00-01 10-00-01 D TA TA TA S8 S8 S8 06-00-02 06-00-02 06-00-02 11-00-01 11-00-01 11-00-01 E S1 S1 S1 01-00-01 01-00-01 01-00-01 07-00-01 07-00-01 07-00-01 11-00-02 11-00-02 11-00-02 F S2 S2 S2 02-00-01 02-00-01 02-00-01 08-00-01 08-00-01 08-00-01 11-00-03 11-00-03 11-00-03 G S3 S3 S3 02-00-02 02-00-02 02-00-02 09-00-01 09-00-01 09-00-01 12-00-01 12-00-01 12-00-01 H S4 S4 S4 03-00-01 03-00-01 03-00-01 09-00-02 09-00-02 09-00-02 12-00-02 12-00-02 12-00-02 Table 3-1a. Plate 2 set up for Estradiol EIA 1 2 3 4 5 6 7 8 9 10 11 12 A 13-00-01 13-00-01 13-00-01 19-00-02 19-00-02 19-00-02 26-00-01 26-00-01 26-00-01 34-00-01 34-00-01 34-00-01 B 14-00-01 14-00-01 14-00-01 20-00-01 20-00-01 20-00-01 27-00-01 27-00-01 27-00-01 35-00-01 35-00-01 35-00-01 C 15-00-01 15-00-01 15-00-01 21-00-01 21-00-01 21-00-01 28-00-01 28-00-01 28-00-01 36-00-01 36-00-01 36-00-01 D 16-00-01 16-00-01 16-00-01 22-00-01 22-00-01 22-00-01 29-00-01 29-00-01 29-00-01 37-00-01 37-00-01 37-00-01 E 17-00-01 17-00-01 17-00-01 22-00-02 22-00-02 22-00-02 30-00-01 30-00-01 30-00-01 38-00-01 38-00-01 38-00-01 F 17-00-02 17-00-02 17-00-02 23-00-01 23-00-01 23-00-01 31-00-01 31-00-01 31-00-01 39-00-01 39-00-01 39-00-01 G 18-00-01 18-00-01 18-00-01 24-00-01 24-00-01 24-00-01 32-00-01 32-00-01 32-00-01 40-00-01 40-00-01 40-00-01 H 19-00-01 19-00-01 19-00-01 25-00-01 25-00-01 25-00-01 33-00-01 33-00-01 33-00-01 12-00-03 12-00-03 12-00-03

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71 CHAPTER 4 RESULTS AND DISCUSSION The normal estrogen levels of pre-menopausal women vary greatly during the menstrual period (Table 4-1). Although, estrogen levels decline as women get older, post menopausal women still produce estrogen albeit at a much lo wer amount. Men are also producers of small amounts of estrogen. Estrogens and androgens are s ynthesized from the neut ral lipid cholesterol. Dehydroepiandrosterone (DHEA), the precursor of both androgens and estr ogens, is synthesized from cholesterol as a result of a series of tr ansformations. It is subsequently converted to testosterone via either andros tenedione (ADione) or androstene diol (ADiol). The estrogens estrone and 17 -estradiol are respectively synthesized from ADione and testosterone by the action of the enzyme aromatase.69 As far as was known there was only one pati ent on hormone replacement therapy (HRT) and one patient on an estrogen inhibitor. Both of these patients were from the non burned injured group so that their estradiol levels were not affected by burn injury. The patient on HRT was taking Estratest (Solvay Pharmaceuticals, Inc.) wh ich is a mixture of the sodium salts of the sulfate esters of the estrogenic substances. The primary source of estrogen in normally cycling adult women is the ovarian follicle and estrad iol (E2) is the princi pal intracellular human estrogen and is substantially more potent than it s metabolites, estrone and estriol, however after menopause most endogenous estrogen is produced by conversion of androstenedione, secreted by the adrenal cortex, to estrone by peripheral tissues. Therefore, estrone and the sulfate conjugated for estrone sulfate are the most abunda nt circulating estrogens in post menopausal women.70 The patient on estrogen inhibitor was ta king Femara (Novartis). In post menopausal women estrogens are mainly derived from the action of the aromatase enzyme, which converts adrenal androgens to estrone and estridiol. Fe mara (Letrozole) is a nonsteroidal competitive

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72 inhibitor of the aromatase enzyme system; bl ocking conversion of androgens to estrogens.71 The difference in estradiol levels of each of these individuals can be s een in Figure 4-1. Each of these patients were considered to be post menopausal, th e patient on estrogen inhi bitor is a 57 year old female and was taking the estrogen inhibitor be cause of a diagnosis of breast cancer and the patient taking the estrogen replacement al though only 39 years old was status post total hysterectomy. Although showing a large difference in range both of these va lues lie within the normal range for a pre menopausal female in mid cycle (Table 4-1) Although, on estrogen inhibitor, this 57 year old patient, in this st udy exhibited an estradiol level above what is considered normal for post menopausal females. This could be due to continued estrogen synthesis by the liver, adrenals, or other pe ripheral organs including fat and muscle tissue. Something of interest that was noted was the es tradiol levels of the female with the highest percentage of total body surface area burned (TBSA) and the male with the highest TBSA. The male had a TBSA that was almost twice as much as the female that coincided with an estradiol level that was almost twice that of the female This led to the thought that maybe TBSA had something to do with the amount of estrogen pro duced following injury (Figure 4-1), however no literature could be found to support this idea and the rest of the data di d not support this trend. The estradiol levels of the male and post menopausal patie nts showed a significant increase above normal values (Figure 4-2). This is consiste nt with studies that sh ow increased estradiol levels in men and post menopausal women following trauma such as burns.72 Other situations that could raise estradiol levels among these gr oups include other forms of trauma, sepsis, how over weight an individual is as fat cells produce more estrogen, and how much alcohol some one consumes.72,73 However, the pre menopausal group had es tradiol levels that were with in the normal range. This could be because the male and post menopausal groups had a significantly

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73 lower estradiol level prior to injury and due to the trauma of burn injury and sepsis following injury they produced more estradiol than the pre menopausal group who al ready had a significant amount of estradiol on board at time of injury an d therefore did not need to produce as much. An ANOVA statistical analysis was performed (p=0.9) wh ich indicates that there is not a statistical difference in these three groups. These initial estradiol levels were taken at time of the first surgical debridement following burn injury the average days for each group are liste d in Figure 4-2. There were 8 patients that received a second surgical debrid ement (Figure 4-3) of these 8 pa tients, 5 were male and 3 were female. A two-tailed T-test was performed (p =0.8) which indicated that there was not a significant difference in male and female estrad iol levels at the time of the second surgical debridement. The females were not separated into pre or post menopausal because the total number of females receiving a second debridemen t was so small. There were only 2 females and no males that received a third debridement. Figur e 4-4 shows the decline in estradiol levels of females at the first, second, and third debr idements. An ANOVA statistical analysis was performed on this data (p=0.6) which indica tes no significant diffe rence among these groups. There was not a decrease in estrad iol levels from the second to third debridements. The sample size of females receiving a second and third debridement being so small, n=3 and n=2 respectively, could skew the re sults and give a false correlation between estradiol levels following injury and time since injury. Previously mentioned was the fact that the ma le patient with the hi ghest percentage of TBSA also had an estradiol level twice that of the female with the highest TBSA. Figure 4-5 shows a break down of estradiol levels based on percentage TBSA for males, females, and normal non burn injured males and females. An ANOVA statistical an alysis was performed

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74 (p=0.9) which indicates no signi ficant difference among these gr oups. The largest proportion of patients fall into a category of TBSA 19 percent, which is consis tent with data from the American Burn Association1. The estradiol levels except for one or two outliers all fall between 100 and 600 pg/ml which is noted to be the normal range for pre menopausal females.74 Something unusual that was noted was the estrad iol levels of the normal non burned males, both of which were well above the normal expect ed values for adult males. There could be a number of reasons for this, a closer look at body mass index (BMI), medications (prescribed and OTC), alcohol intake, illegal drug use as well as other parameters could gi ve a better explanation for these two values being so much higher than expected.73,72 The estradiol levels are also listed according to age (Figur e 4-6). Again, all values range between 100 and 600 pg/ml which is consistent with the estradiol levels from Figure 4-5, and again an ANOVA statistical analys is was performed (p=0.4) whic h again indicates no significant difference among groups. What is interesting is that there is no clear cut difference among age groups. What would be interesting to see would be estradiol levels from these same patients prior to injury to compare estradiol le vels prior to in jury and after injury a nd see which group actually had the most increase? Another piece of informa tion that would have been interesting to know would have been asking the pre menopausal females where they were in their menstrual cycle as the estradiol levels peak as they get closer to mid cycle and then start to decline again. Another good question would be, following thermal burn in jury, do estradiol levels go up more when women are in the early, late, or mid cycle? It is probably not probable th at all pre menopausal women were near mid cycle as the reason that their estradiol levels did not show as a dramatic an increase as the post menopausal and male groups.

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75 Estrogens increase TGFsecretion by dermal fibroblast in vitro regardless of age.75 In fact there are estrogen recep tors on all the major cell type s involved with wound healing suggesting that estrogen may directly modulate the function of these cells.54 In adult human skin CTGF is normally not expressed unless induced, TGFinduces CTGF expression.3 Therefore, we did immunohistochemistry staining for TGFand for CTGF to look for localization of these growth factors in relation to estradiol levels. The first task after immunostaining was comple te was to grade the level of staining based on a visual analog scale (VAS) of 0-5. This wa s accomplished by a two tester grading system. Each grader was blinded to whether the slide bein g graded was from a male or female or pre or post menopausal female. After each grader comple ted all slides, the numbers were averaged together to come up with a final grade for each slide. These grades were then used to select slides from each category; male, pre menopausal, and post menopausal female. Photographs of these slides were made using the Zeiss Axiovision mi croscope and staining intensity quantified using computerized digital image analysis by Image J.68 After comparisons were made from the VAS, image analysis using Image J68 was used to verify the accuracy of the VAS for the TGFstained slides. This was done by normalizing the tissue sample by dividing the percentage of stai ning by the total area of the sample. What was noted from this was that the post menopausal fe males had a lower normalized amount of staining as compared to the pre menopausal and males gr oups, this was consistent with data from the VAS (Figure 4-7). The post-menopa usal staining intensity was very similar to the staining intensity of the normal donor site tissue. A P earsons correlation test was performed (r=0.5) which indicated a weak positive correlation betw een the VAS and the computerized digital image analysis by Image J.68 This was further verified by a two tailed T-test (p=0.003) which

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76 indicated that r is significantly greater than 0. The in laid bar graph (Figure 4-7) shows the averages of the individual scores versus the normalized values, this gives a more dramatic presentation of how much more staining intensity there is among the burn injured tissue over the normal non injured donor site tissue. A Mann-Whitney statistical test was performed and the p value comparing male burn tissue to male donor ti ssue was 0.02 which is a significant difference, the p value comparing pre-menopausal female burn tissue to pre-menopausal donor tissue was 0.08 which is considered not significant, and th e p value comparing post-menopausal burn tissue to post-menopausal donor tissue was 0.01 which is considered very signif icant. Even though the difference between the pre-menopausal burn an d donor tissue was not significant the trend remains the same with the burn injured tissue showing a greater stai ning intensity for TGFamong all three groups, and among the three gro ups the post-menopausal females showing the lowest intensity for staining for burn injured tissue. After the data was collected from the TGFslides the process was repeated for the CTGF stained slides (Figure 4-8). A Pearsons correlati on test was again used to verify the relationship between the VAS and the computerized digita l image analysis. However, this time the correlation showed an increase in the r valu e indicating a good positive correlation (r=0.6). A two tailed T-test was performed (p<0.0001) wh ich confirmed the resu lts of the Pearsons correlation test indicating that r is significantly greater than 0. The trend for the CTGF staining intensity is very similar to the TGFstaining intensity. However, a Mann-Whitney test performed on male burn tissue versus male donor tissue, pre-menopausal burn tissue versus premenopausal donor tissue, and post-menopausal burn tissue versus post-menopausal donor tissue determined that there was not a significant diffe rence among these three groups with p values of 0.15, 0.08, and 0.12 respectively. Although, there was not a significant difference among these

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77 groups the trend continues to be that the burn in jured tissue has a higher st aining intensity for the growth factors TGFand CTGF with the post-menopausal group having the lowest intensity of staining among all the burn injured tissue. There are two types of estrogen receptors, ER and ER These receptors are found on all the major cell types involved in wound hea ling. These receptors are co-expressed with ER playing a dominant role over ER .76 Ankrom et al77 showed that the ER increased with increasing age and Haczynski et al76 showed in an experiment using only post menopausal women that the ER out numbered the ER In a review by Pelletier78 the ER did not immunolocalize to any specific stru cture of human skin. However, ER was found to be highly expressed in the epidermis. These ER localization studies have shown that ER is widely expressed in human skin a nd its appendages, whereas ER is only found in very few subsets of structures.78 It is the therefore concluded that if in the presence of estrogen certain cells are stimulated to produce TGFin response to wound healing, that this is accomplished through the ER and that with increasing numbers of ER such as in post menopausal females, there is a decrease in the amount of TGFproduced by these cells. This would explain why the amount of staining of TGF, as seen by the blinded two grader VAS and by the computerized digital image analysis, was lowest among the post menopausal women (Figure 4-7). The fact that TGFstimulates the production of CTGF in response to injury would explain why the post menopausal women also had the lowest amount of staining fo r CTGF. An interesting addition to this would have been to separate young male s from aged males to determine if there was a staining intensity difference such as between the pre and post menopausal women. It has already been establishe d that estrogen levels are incr eased as a response to injury, and this was confirmed by the estradiol levels obtained after thermal burn injury which showed

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78 elevated estradiol levels among male and post-me nopausal female thermal burn injured victims. It is difficult to say whether the pre-menopausal females had elevated estr adiol levels because all values fell with in a normal range. Imm unohistological photographs confirm that TGFstaining intensity after thermal burn injury increases as seen in Figure 4-9 comparing the thermal burned injured tissue and donor site tissu e of a male burn victim. Note the difference in intensity between the thermal injured tissu e compared to the donor site ti ssue. Even though the donor site tissue is from a burned injured pa tient its staining pattern is si milar to that of a normal non burn injured patient (Figure 4-12). This same staining pattern is seen among the pre and post menopausal women where the burn injured tissue shows increased staining intensity while the donor site tissue shows a staining pattern similar to that of normal non burn injured females (Figures 4-10, 4-11, 4-13). This w ould indicate that the response to injury is localized to the area of injury and not a systemic response. CTGF is expressed in many different tissues and organs and stimulat es proliferation and chemotaxis of fibroblast directly. Most interesti ngly, it is a potent inducer of extracellular matrix proteins, such as collagen type I and fibronectin and their integrin recep tors, and it acts as a mediator of TGFin these processes.79 However, CTGF is not normally expressed unless it is induced by TGF.3 Experiments by Igarashi et al80 show the presence of TGFand CTGF in wound tissue. TGFwas present at the earliest times following injury with a peak at day 3 followed by a rapid decrease, however, CTGF exhibi ted a much different expression pattern with a peak in levels at day 9. Levels of CTGF continue to accumulate for up to 24 hours after exposure to TGF.80 Immunohistological photographs of CTGF stained tissue show s staining to both the burn injured and donor site tissue (Figure 4-14) of a burn injured male victim with dermal and

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79 epidermal involvement. This same pattern is also seen in a pre menopausal female burn injured victim (Figure 4-15), however, the post menopa usal pattern of stai ning (Figure 4-16) is noticeably decreased to both the dermis and epidermi s. This remains consistent with the previous mentioned blinded two grader VAS and computeri zed digital image analysis (Figure 4-8) which indicated a much lower intensity of CTGF staining among the post menopausal females compared to the pre menopausal females and ma le groups. In normal non burn injured tissue CTGF is not normally expressed 80, this is demonstrated in th e immunohistological photographs of normal non burn injured tissue from male (Figure 4-17) and pre and post menopausal females (Figure 4-18) in which there is no or very little evidence of CTGF staining noted. In summary, we know that estradiol levels in crease in response to the trauma of thermal burn injury, We know that all of the cells involved in wound hea ling have estrogen receptors and that there are two isofor ms of these receptors, ER and ER It is known that these receptors are co expressed and that ER is dominate over ER however ER levels seem to increase with age. It is our belief that it is the ER that is responsible for stim ulating wound healing cells to produce TGFfollowing thermal burn injury which in turn stimulates CTGF allowing wound healing to occur. We know that males and pr e menopausal females had increased intensity of staining of TGFand CTGF over post menopausal females. What is still left unanswered is why the pre menopausal females did not increase estrad iol levels as drastically as the males and post menopausal females in response to thermal burn injury? Why did both of the normal non burn injured males have such high estradiol levels? We believe that most unanswered questions could be answered by taking a closer more in depth me dical history and medication list. Some things that are left for future studies would be perf orming extraction assays to compare quantitative levels of TGFand CTGF to estradiol levels and hist ological stainings. Also, it would be good

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80 to do some immunostaining for TGFreceptors. A final thought w ould be to set up a knock out assay to verify that it is truly the ER that is responsible for si gnaling the cell to produce TGF.

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81 Table 4-1. Estradiol levels for adult males and females during each phase of the menstrual cycle and after menopause74 Total Estradiol (pg/ml) Adult Male 10-50 Adult Female Pre Menopausal Early Follicular 20-150 Late Follicular 40-350 Mid-Cycle 150-750 Luteal 30-450 Post Menopausal 20

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82 Figure 4-1. The Effects of Hormone Replacem ent, Hormone Inhibitor, and Percent Burn on Estadiol Levels (n=1). (A)Estradiol le vels of a 39 y/o female non burn injured patient on estrogen replacement compared to a 57 y/o female non burn injured patient on estrogen inhibitor. (B) The estr adiol levels of the female with the highest percentage burn injury compar ed with the male with the highest percentage burn injury.

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83 Figure 4-2. Estradiol Levels of Male s vs. Preand Post-Menopausal Females at First Debridement, ( +/)The average estradiol levels of all males compared to all pre menopausal and post menopausal females at the time of the first debridement following burn injury. Average days from time of injury until first debridement are listed for each group. An ANOVA test was performed (p=0.9) which indicated that there was no significant difference among these groups.

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84 Figure 4-3. Estradiol Levles of Male s vs. Females at Second Debridement, (+/). The average estradiol levels for all males and females undergoing a second debridement are listed with the aver age days since injury until second debridement listed. A two-tailed T-test wa s performed (p=0.8) which indicated no significant difference among these two groups.

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85 Figure 4-4. Female Estradiol Levels at Debridements 1-3, ( +/). The average estradiol levels for all females at the first debr idement, all females undergoing a second and third debridement along with the average da ys since time of injury for each group. An ANOVA was performed (p=0.6) which i ndicated that there is no significant difference among these groups.

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86 Figure 4-5. Estradiol Levels vs. Percent Burn for males and Females Following Burn injury. The estradiol levels of male and female burned injured patients broken down by percent total body surface area (TBSA) burned compared to the estradiol levels of non burned normal tissue. An ANOVA test was performed; single values could not be used therefore were left out of the statistical analysis. Results among the remaining groups returned a p value of 0.9 which indicates that there is no significant difference among these groups. Ho rizontal lines indicate the mean for each group.

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87 Figure 4-6. Estradiol Levels vs. Age for Males and Females Following Burn injury. The average estradiol levels of male, pr e menopausal, and post menopausal broken down by age. An ANOVA was performed; single values could not be used therefore were left out of the statisti cal analysis. Results among the remaining groups returned a p value of 0.4 which i ndicates that there is no significant difference among these groups. Horizontal bars indicate the mean for each group.

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88 Figure 4-7. Quantitative Image An alysis vs. Visual Analog Scale for TgF. The individual scores from two masked graders using a visual analog scale versus computerized digital image analysis to determine staining intensity of TGFin human skin tissue samples following thermal burn inju ry. The in laid bar graph shows the averages of these scores. A Pearsons co rrelation test was performed and a weak positive correlation was determined (r=0.5) The coefficient of determination (r2) was determined to be 0.3, and a two tailed T-test (p=0.003) determined that r is significantly greater than 0. Also, a Mann-Whitney test was performed to determine significance of staining intensity of TGFbetween burned injured tissue and normal donor site tissue. Co mparing male burn tissue to male donor site tissue the p value was 0.02 which is determined to a significant difference. Comparing pre-menopausal female burn tissue to pre-menopausal female donor site tissue the p value was 0.08 which is not a significant difference. Comparing post-menopausal female burn tissue to pos t-menopausal donor site tissue the p value is 0.01 which is a very significant difference.

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89 Figure 4-8. Quantitative Image Analysis vs Visual Analog Scale fo r CTGF. The individual scores from two masked graders using a visual analog scale versus computerized digital image analysis to determine stai ning intensity of CTGF in human skin tissue samples following thermal burn inju ry. The in laid bar graph shows the averages of these scores. A Pearson s correlation test was performed and a positive correlation was identified (r=0.6). The coefficient of determination (r2) was determined to be 0.3, and a two tailed T-test (p<0.0001) determined that r was significantly greater than 0. Also, a Mann-Whitney test was performed to determine significance of staining intens ity of CTGF between burned injured tissue and normal donor site tissue. Co mparing male burn tissue to male donor tissue the p value was 0.2 which is no t significant. Comparing pre-menopausal burn tissue to pre-menopausal donor tissu e the p value was 0.08 which is not significant. Comparing post-menopausal burn tissue to post-menopausal donor tissue the p value was 0.12 wh ich is not significant.

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90 Figure 4-9. Immunohistology for Tgfin human burned injured and donor site tissue (males). (A) Burn injured control sample, omission of the 1 Ab was used as the control. (B) Burned injured test sample imm unohistochemistry staining for TGF, sample taken from burned injured area. Significant struct ures are labeled. (C) Donor site control sample, omission of the 1 Ab was used th e control. (D) Donor site test sample immunohistochemistry staining for TGF, sample taken from donor site prior to donor tissue harvest. Significant structures are labeled. Note differences in dermal staining among burn injured tissue and donor site tissue. (100x magnification) A B C D epidermis dermis dermis hair follicle dermis epidermis destroyed by thermal injury hair follicle dermis epidermis destroyed by thermal injury burn injured control sample burned injured test sample donor site control sample donor site test sample

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91 Figure 4-10. Immunohistology for Tgfin human burned injured a nd donor site tissue (premenopausal females). (A) Burn injured control sample, omission of 1 Ab was used as the control. (B) Burn injured test sa mple immunohistochemist ry staining for TGF, sample taken from burn injured area. Significant structures are labeled. Although the dermal tissue appears to have some stai ning in the control sample, it is important to note the difference in the staining intens ity of the epidermis among the control and test sample. (C) Donor site control sample omission of the 1 Ab was used as the control. (D) Donor site test sample immunohistochemistry staining for TGF, sample taken from donor site prior to donor tissue harvest. Signifi cant structures are labeled. Note the differences in dermal staining intensity among the burn injured and donor site tissues. (100x magnification) A B C D epidermis destroyed by thermal injury dermis epidermis destroyed by thermal ij dermi epidermi s dermis epidermis burned injured control sample burned injured test sample donor site control sample donor site test sample

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92 Figure 4-11. Immunohistology for Tgfin human burned injured and donor site (postmenopausal females). (A) Burn injured control sample, omission of the 1 Ab was used as the control. (B) Burn inju red test sample immunohistochemistry staining for TGFsample taken from burn injured area. Significant structures are labeled. (C) Donor site control sample, om ission of the 1 Ab was used as the control. (D) Donor site test sample immunohistochemistry staining for TGF, sample taken from donor site prior to donor tissue harvest. Si gnificant structures are labeled. Note differences in dermal staining intensity among burn injured and donor site tissue. (100x magnification) A B C D blood vessel collagen fibers in dermis blood vessel collagen fibers in dermis epidermis dermis epidermis dermis burned injured control sample burn injured test sample donor site control sample donor site test sample

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93 Figure 4-12. Immunohistology for Tgfin human normal tissue (males). (A) Normal tissue control sample from pa tient #31, omission of 1 Ab was used as the control. (B) Normal tissue test sample i mmunohistochemistry staining for TGFfrom patient #31, significant structur es are labeled. This sample was taken from elective plastic surgery site. (C) Normal tissue c ontrol sample from patient #33, omission of 1 Ab was used as the control. (D) Normal tissue test sample immunohistochemistry staining for TGFfrom patient #33, signifi cant structures are labeled. This sample was taken from electiv e plastic surgery site. Patient #31 showing slight increase in dermal staining compar ed to patient #33 many variables could be responsible including pre morbid conditions. A closer look at pre morbid conditions and medications would be helpful in future studies. (100x magnification) epidermis dermal papilla (papillary dermis) epidermal ridge dermis (reticular dermis) epidermis dermal papilla (papillary dermis) epidermal ridge dermis (reticular dermis) sweat gland (eccrine) epidermis Blood vessel dermis Normal tissue control sample (pt. 31) No r mal tissue test sample (pt. 31) Normal tissue control sample (pt. 33) Normal tissue test sample (pt. 33) A B C D

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94 Figure 4-13. Immunohistology for Tgfin human normal tissue (preand post-menopausal females). (A) Normal tissue control sample from a pre menopausal female, omission of 1 Ab was used as the cont rol. (B) Normal tissue test sample immunohistochemistry staining for TGFfrom pre menopausal female, significant structures are labeled. This sample was ta ken from elective plastic surgery site. (C) Normal tissue control sample from a pos t menopausal female, omission of 1 Ab was used as the control. (D) Normal tissue test sample immunohistochemistry staining for TGFfrom post menopausal female, si gnificant structures are labeled. This sample was taken from elective plas tic surgery site. ( 100x magnification) A B C D epidermis dermis Sweat gland (eccrine) blood vessel epidermis dermis epidermis dermis sweat gland (eccrine) epider mis dermis Normal pre-menopausal control sample Normal pre-menopausal test sample Normal post-menopausal control sample Normal post-menopausal test sample

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95 Figure 4-14. Immunohistology for CTGF in human burned injured and donor site tissue (male). (A) Burn tissue control sample, om ission of the 1 Ab was used as the control. (B) Burn tissue test sample immunohistochemistry staining for CTGF, the sample was taken from the burn injured ar ea. Significant structures are labeled. (C) Donor site control sample, omission of the 1 Ab was used as the control. (D) Donor site test sample immunohistochemist ry staining for CTGF, the sample was taken from the donor site prior to donor tis sue harvest. Signifi cant structures are labeled. (100x manafication) A B C D sebaceo us gland excretory duct blood vessel dermal papilla external root sheath burned injured control sample burned injured test sample donor site control sample donor site test sample epidermis destroyed by thermal injury derm is epidermis destroyed by thermal injury derm is epidermi s dermis

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96 Figure 4-15. Immunohistology for CTG in human burn injured and donor site tissue (premenopausal females). (A) Burn injured control sample, omission of the 1 Ab was used as the control. (B) Burn inju red test sample immunohistochemistry staining for CTGF; the sample was taken from the burn injured area. Significant structures are labeled. (C) Donor site cont rol sample, omission of 1 Ab was used as the control. (D) Donor site test sa mple immunohistochemi stry staining for CTGF; the sample was taken from the donor site prior to don or tissue harvest. Significant structures are la beled. (100x magnification) A B C D burned injured control sample burned injured test sample dermis collagen fibers in dermis dermis epidermis dermis donor site control sample donor site test sample

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97 Figure 4-16. Immunohistology for CTGF in human burn injured and donor site tissue (postmenopausal females). (A) Burn tissue control sample, omission of the 1 Ab was used as the control. (B) Burn tissue test sample, the sample was taken from the burn injured area. Signifi cant structures are labele d. (C) Donor site control sample, omission of the 1 Ab was used as the control. (D) Donor site test sample immunohistochemistry staining for CTGF, the sample was taken from the donor site prior to donor tissue harvest. Sign ificant structures are labeled. (100x magnification) A B C Dburned injured control sample burned injured test sample donor site control sample donor site test sample dermis dermi s epidermis dermis epidermis note cornefied outer layer and dermal papilla with dermal ridges dermis

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98 Figure 4-17. Immunohistology for CTGF in hu man normal tissue (males). (A) Normal tissue control sample from patient #31, omission of the 1 Ab was used as the control. (B) Normal tissue test samp le immunohistochemi stry staining for CTGF in patient #31, the sample was taken from elective plastic surgery site area. Significant structures are labeled. (C) Normal tissue control sample from patient #33, omission of the 1 Ab was used as the control. (D) Normal tissue test sample immunohistochemistry staini ng for CTGF in patient #33, the sample was taken from elective plastic surgery si te. Significant structures are labeled. Note the lack of staining in patient # 33 which is what would be expected since CTGF is not normally expressed in nor mal tissue. However, patient #31 does show some slight increase staining intensity which could be indicative of on going pre morbid condition since patien t #31 also had increase in TGFstaining. (100x magnification) A B C D Normal tissue control sam p le Normal tissue test sam p le Normal tissue control sam p le Normal tissue test sam p le (p t. e p idermi blood vessel dermis e p idermi blood vessels e p idermis e p idermal rid g e dermal p a p illa dermi e p idermis e p idermal rid g e dermal p a p illa dermis

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99 Figure 4-18. Immunohistology for CTGF in human normal tissue (pre-and post-mneopausal females). (A) Normal tissue control sample in pre menopausal female, omission of the 1 Ab was used as the c ontrol. (B) Normal tissue test sample immunohistochemistry staining for CTGF in pre menopausal female. Tissue sample was taken from elective plastic surg ery site. Note the very little staining intensity which is consistent with n ormal tissue not e xpressing CTGF unless induced to. Significant struct ures are labeled. (C) Norma l tissue control sample in post menopausal female, omission of the 1 Ab was used as the control. (D) Normal tissue test sample immunohist ochemistry staining for CTGF in post menopausal female, tissue sample was take n from elective plastic surgery site. Significant structures have been labeled. Note the almost non existent staining for CTGF in the post menopausal female, this is consistent with normal tissue not expressing CTGF unless induced to, also consistent with post menopausal females having less staining intensity than pre menopausal females or men per visual analog scale and computerized digital imaging analysis. (100x magnification) A B C D Normal pre-menopausal control sample Normal pre-menopausal test sample epider mis dermis epidermis dermal papilla reticular dermis blood vessel (capillaries) epidermi s dermis epidermi s dermis

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100 APPENDIX A PROTOCOL #480-2005 1. Project Title: Analysis for CTGF and TGFlevels in deep partial thickne ss thermal burn injury tissue versus normal tissue. 2. Investigator(s): David W. Mozingo, M.D. Winston Richards, MD Gregory Schultz, Ph.D. Lyle L. Moldawer, Ph.D. Brent Seagle, M.D. Wayne Lee, MD Donald McCurry, P.T. Karen Perrin, MSN, ARNP Tera Thigpin 3. Abstract: Hormones are chemical signaling molecules pro duced in one site of the body that then travel to another site to have an effect In this way one cell can communicate with distantly located cells. The ovaries produce the steroid hormones, estrogen and progesterone, that are responsible for the development of seconda ry sexual characteristics and develop and maintain the reproductive function in th e female. The normal development and maturation of the female is dependent on es trogens. Besides stimulating development of the reproductive structures and secondary se xual characteristics, estrogens are also necessary for the health of the skin a nd vascular system, and bone homeostasis.(7) Estrogen has been shown to play a crucial ro le in the wound healing process in both men and women.(1,2) Premenopausal women are shown to have a faster rate of healing following injury, while postmenopausal women and men heal slower but have decrease scar tissue formation.(2) Estrogen has been shown to preven t a decrease in collagen levels and to increase levels of mucopolysaccharid s and hyaluronic acid keeping the skin thick and moist, respectively. The lack of estrogen has been shown to improve the quality of scar formation.(4) Transforming growth factor (TGF) and connective tissue grow th factor (CTGF) are involved in the wound healing process. TGFinduces synthesis and inhibits degradation of extracellular matrix (ECM) and stimulates granulation tissue fo rmation and collagen deposition(2). CTGF is a major autocrine growth s timulator for connective tissue cells,

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101 and its production is regulated by TGF.(3) Since estrogen is known to play a crucial role in wound healing and the growth factors TGFand CTGF are also involved in the wound healing process; this study will focus on the relationship between TGF, CTGF levels and the hormone estrogen. 4. Specific Aims: The purpose of this study is to investigate the relationship between estrogen and CTGF and TGFlevels in pre/postmenopausal wome n and men of varying ages following thermal burn injury versus the CTGF and TGFlevels in pre/postmenopausal women and men of varying ages w ith normal tissue. To better understand these relationships, these questions will be addressed: How do es trogen levels correlate with healing rates and quality of scar tissue followi ng a thermal burn injury? How do TGFand CTGF levels vary with estrogen levels? 5. Background and Significance: Each year in the United States, trauma is the leading cause of death for people between the ages of 1 and 24.(6) One type of trauma injury is a burn. A burn is defined as tissue damage caused by a variety of agents, such as heat, chemicals, elec tricity, sunlight, or nuclear radiation. The most common burns are caused by scalds, building fires, and flammable liquids and gases.(6) Burns often lead to infection, due to damage to the skins protective layer. The larger the burn, the more increased chance of infection. In the United States, 10,000 people die every year of burn related infections. Therefore, it is the goal of health care providers to reduce the si ze of a burn wound as quickly as possible in order to decrease the chance of such infections. One of the main problems involved with wounds caused from a burn injury is the formation of scar tissue. Disfigurement fr om scars and burned tissue can affect self concept, body image, comfort in interpers onal situations, and acceptance from peers in the work place. Severe burns are more lik ely to result in long term quality of life problems with both physical and psychosocial aspects.(8) Muscle contractures can develop from protective posturing to decrease pa in. Contractures lead to loss of range of motion and require physical therapy for stretc hing program and serial casting. Severe contractures may require surgical release to restore range of motion.(9) Increased levels of estrogen have been shown to increase the rate of wound healing(1,2), however, increased estrogen levels incr ease scar tissue formation.(2) Twenty years ago, a burn that covered 50% of the body was routinely fata l; today, thanks to advances in wound cleaning, people with burns c overing 90% of their body can survive (but often with permanent impairments). While the goal is to reduce the size of the burn as quickly as possible in order to decrease th e chance for infection, it is hoped that in the future this can be done without increasing scar tissue formation.

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102 6. Research Plan: It is projected that we will enroll 80 patients in to this research that will meet the criteria below: Inclusion Criteria: 1. Patient meets one of the following criteria: a). Any patient admitted to the Acute Care Surgery Service following thermal burn injury that requires one or more surgical debridements. b). Any patient which will be undergoing an elective surgical procedure by the Plastics Reconstructive Surgery Se rvice that will involve excision of normal skin that would normally be discarded. 2. Male or Female 18 years of age or older. 3. Patient must be willing to comply with protocol and pr otocol procedures. Exclusion Criteria: 1. Pregnant and nursing mothers. 2. Patients suffering from terminal disease 3. Patients with chemical or electrical burns. 4. Patients who have received systemic co rticosteroids within 30 days prior to surgical procedure, unless they are receiving them for acquired adrenal insufficiency during their thermal burn care. The Principal Investigator or his designated re search staff will perform informed consent. After informed consent has been obtained the patient will be separated into two categories, patients with thermal burn injury and patients without thermal burn injury. Thermal Burn Injury Tissue Patients Samples will be collected during surgical pro cedures up to four times for a possible total of eight tissue samples. After the patient has been anesthetized and the procedure has begun, a 6mm punch biopsy will be taken from th e injured area prior to debridement as well as a 6 mm punch biopsy from normal skin prior to donor site harvesting. Normal Tissue Patients Patients undergoing elective plastic/reconstruc tive surgeries will have a one time 6 mm biopsy performed from the normal tissue that has been excised and will be discarded. Patients in both groups will have blood take n during their surgical procedure from existing intravenous lines when available. One teaspoon of blood will be collected in a lavender/EOTA tube prior to the biopsy procedure. These blood samples will be used to test for plasma hormone and growth factor levels.

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103 Tissue samples collected from thermal injure d and non-thermally injured patients will be divided with 50% being placed in form alin and 50% being placed in RNAlater. These samples will be used for molecular analysis. Also, a database will be set up to look at specific sub groups. The subgroups will include but are not limited to males grea ter than 50 years of age, male s less than 50 years of age, premenopausal women, and postmenopausal women. 7. Potential Health Risks: The risks or discomforts associated with part icipation in this study are the same as those associated with the medical and surgical care subjects receive as part of the standard of care for treatment of their medi cal condition at this facility. The risks of drawing blood from a vein incl ude discomfort at the site of puncture; and, uncommonly, faintness from the procedure. 8. Potential Health Benefits: While there are no immediate dire ct individual patient benefits from participation in this study, patients with thermal burn injuries ma y in time directly benefit from the new knowledge generated from the tissue collected. 9. Potential Financial Risks: None 10. Potential Financial Benefits: None 11. Conflict of Interest: There is no conflict of interest involved w ith this study beyond th e professional benefit from academic publication or presentation of the results. 12. References 1. Ashcroft, Gillian. Stuart Mills, Keji an Lei, Linda Gibbons, Moon Jin Jeong, Marisu Taniguchi, Matthew Burow, Mi chael Horan, Sharon Wahl, Toshinori Nakayama. Estrogen modulates cutane ous wound healing by down regulating macrophage migration inhibitory factor The Journal of Clinical Investigation. 2003. 111:1309-1318.

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104 2. Ashcroft, Gillian. Jason Ashworth. Pot ential Role of Estrogens in Wound Healing American Journal of Clinical Dermatology. 2003; 4(11): 737-743. 3. Atsuyuki, Igarashi, Okochi Hitoshi, Douglas Bradham, Gary Grotendorst. Regulation of Connective Tissue Growth Factor Gene Expression in Human Skin Fibroblasts and During Wound Repair Molecular Biology of the Cell. 1993. Vol.4,637-645. 4. Kovacs, EJ, TP Plackett, PL Witte. Estrogen Replacement, Aging, and Cell Mediated Immunity After Injury Journal of Leukocyte Biology. 2004. 76(1):3641. 5. Shah, MG, HI Maibach. Estogen and Skin, an Overveiw American Journal of Clinical Dermatology 2001. 2(3):143-150. 6. Burn facts figures www.nigms.nih.gov/news/facts/tr aumaburnfactsfigures.html., July 31, 2007. 7. Hormones, www.e-Hormones.com, July 31, 2007. 8. Weed, Roger Debra Berans. Basics of Burn Injury: Implications for Case Management and Life Care Planning Lippincotts Case Management. 2005; 10 (1): 22-29 9. Brown, Melissa Phala Helm. Life Care Pl anning for the Burn Patient Life Care Planning and Case Management Handbook (2nd Ed, p 247-262) Boca Raton, Fl: CRC Press, LLC. 10. Burn Surgery, /www.burnsurgery.org July 31, 2007

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105 APPENDIX B CASE REPORT FORMS Case Report Forms Oct 21, 2005 Patient Initials: Patient ID: Screening Date: __ __ __ Inclusion Criteria: Y N [ ] [ ] Any patient admitted to the Acute Care Surgery Service following thermal burn injury that requires one or more surgical debridements. or [ ] [ ] Any patient which will be undergoing an elective surgical procedure by the Plastics Reconstr uctive Surgery Service that will involve excision of rmal skin th at would normally be discarded. [ ] [ ] Male or Female 18 years of age or older. [ ] [ ] Patient willing to comply with protocol and protocol Procedures. Exclusion Criteria: Y N N/A [ ] [ ] [ ] Pregnant and nursing mothers. [ ] [ ] Patients suffering from terminal disease. [ ] [ ] Patients with chemical or electrical burns. [ ] [ ] Patients who have received systemic corticosteroids within 30 days prior to surgical procedur e, unless they are receiving them for acquired adrenal insufficiency during their normal burn care.

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106 Case Report Forms Oct 21, 2005 Patient Initials: Patient ID: General Patient Information: Male or Female: __________________________________________________________ Age: ___________________________________________________________________ Menopausal Status: _______________________________________________________ Hormone Replacement Therapy, Yes / No Explain: ________________________________________________________________ Medical History: _________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ____________________________________________________________ Burn Assessment: Date of Burn: __ __ __ Location of Burn(s): _______________________________________________________ Degree of Burn: __________________________________________________________ Percentage of TBSA of Burn: _______________________________________________ Debridement Site #1: ______________________________________________________ Date of Biopsy #1: ________________________________________________________ Biopsy Site of Thermal Burn Tissue and Sample Number: ________________________ ________________________________________________________________________ Biopsy Site of Normal Donor Tissue and Sample Number: ________________________ Blood Sample Collection, Yes / No Blood Sample Number: ____________________________________________________ Is Patient on Antibiotics, Yes / No Explain: ________________________________________________________________

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107 Debridement Site #2: ______________________________________________________ Date of Biopsy #2: ________________________________________________________ Biopsy Site of Thermal Burn Tissue and Sample Number: _________________________ ________________________________________________________________________ Biopsy Site of Normal Donor Tissue and Sample Number: ________________________ ________________________________________________________________________ Blood Sample Collection, Yes / No Blood Sample Number: ____________________________________________________ Is Patient on Antibiotics, Yes / No Explain: ________________________________________________________________ Debridement Site #3: ______________________________________________________ Date of Biopsy #3: ________________________________________________________ Biopsy Site of Thermal Burn Tissue and Sample Number: ________________________ ________________________________________________________________________ Biopsy Site of Normal Donor Tissue and Sample Number: ________________________ ________________________________________________________________________ Blood Sample Collection, Yes / No Blood Sample Number: ____________________________________________________ Is Patient on Antibiotics, Yes / No Explain: ________________________________________________________________

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108 Debridement Site #4: ______________________________________________________ Date of Biopsy #4: _______________________________________________________ Biopsy Site of Thermal Burn Tissue and Sample Number: _________________________ ________________________________________________________________________ Biopsy Site of Normal Donor Tissue and Sample Number: ________________________ ________________________________________________________________________ Blood Sample Collection, Yes / No Blood Sample Number: ____________________________________________________ Is Patient on Antibiotics, Yes / No Explain: ________________________________________________________________ Normal Tissue Collection: Date of Surgery/Biopsy: ___________________________________________________ Type of Surgery: _________________________________________________________ Biopsy Site of Normal Tissu e and Sample Number: ______________________________ ________________________________________________________________________ Blood Sample Collection, Yes / No Blood Sample Number: ____________________________________________________ Is Patient on Antibiotics, Yes / No Explain: ________________________________________________________________

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109 Case Report Forms Oct 21, 2005 Patient Initials: Patient ID: Study Completion Date: __ __ __ Study Completion: Yes No Did the subject complete all study related activities? [ ] [ ] If no, explain why: ________________________________________________________ Were there any protocol deviations or violations reported? [ ] [ ] If yes, please comment: ____________________________________________________ Were there any Serious Adverse Events reported? [ ] [ ] If yes, please describe: _____________________________________________________ Did the subject decease during the study period? [ ] [ ] If yes, when? __ __ __ Is the patient evaluable? [ ] [ ] Additional Comments: _____________________________________________________ __________________________ __ __ __ Principal Investigator Signature Date

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110 APPENDIX C INFORMED CONSENT IRB# 480-2005 Informed Consent to Participate in Research and Authorization for Collection, Use, and Disclosure of Protected Health Information You are being asked to take part in a research study. This form provides you with information about the study and seeks your au thorization for the collection, use and disclosure of your protected health information necessary for the st udy. The Principal Invest igator (the person in charge of this research) or a repr esentative of the Principal Investigat or will also describe this study to you and answer all of your que stions. Your participation is entirely voluntary. Before you decide whether or not to take part, read the in formation below and ask questions about anything you do not understand. If you choo se not to participate in this study you will not be penalized or lose any benefits to which you would otherwise be entitled. 1. Name of Participant ("Study Subject") ________________________________________________________________________ 2. Title of Research Study Analysis for CTGF and TGFlevels in deep partial thickne ss thermal burn injury tissue versus normal tissue. 3. Principal Investigator and Telephone Number(s) David W. Mozingo, M.D. (352) 273-5667

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111 4. Source of Funding or Other Material Support University of Florida 5. What is the purpose of this research study? The purpose of this study is to investigate th e relationship between estrogen and healing in pre/postmenopausal women and men of different ages following burn injury versus the healing in pre/postmenopausal women and me n of different ages with normal skin. 6. What will be done if you ta ke part in this research study? The Principal Investigator or his research staff will perform informed consent. After informed consent has been obtained, you will be separated into two groups, subjects with burn injury and subjects without burn injury. Subjects With Burn Injury Tissue samples will be collected during your nor mal standard of care surgical procedure. After you have been sedated for your surgical procedure, the surge on will perform a 6mm punch biopsy (about the size of a pencil eraser) from the injured area and a sample of normal skin will be taken from your de signated donor site. This procedure could be performed up to four times depending on your need for more surg eries as part of your standard of care. Subjects Without Burn Injury A tissue sample will be collected once during your elective surgical pr ocedure. You have been selected to participate in this research study because yo ur surgery requires the removal of excess skin that would normally be discar ded. From this discarded skin a 6mm punch biopsy (about the size of a pencil eraser) will be taken. Both groups will have blood drawn while they ar e in surgery through an existing intravenous line if possible. This blood will be te sted for hormones and growth factors. The tissue sample collected from both groups will be split in half and saved. These tissues will have tests performed on them at a later date A database will be set up to track tissue, tissue test results and your basic demographic information such as age, sex, premenopausal and postmenopausal status. Your confidential in formation, such as name and birth date, will not be included in this database. If you have any questions now or at any time during the study, you may contact the Principal Investigator listed in #3 of this form. 7. If you choose to participate in this study, how long will you be expected to participate in the research?

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112 For those patients that do have thermal burn inju ries participation will last up to the time of your fourth or final surgical pr ocedure, whichever comes first. For those patients that do not have a thermal bur n injury participation in this study will end after the one time tissue sample is collected. 8. How many people are expected to participate in this research? Approximately 80 individuals are antici pated to participate in this study. 9. What are the possible discomforts and risks? The risks or discomforts associated with part icipation in this study are the same as those associated with the medical and surgical care su bjects receive as part of the standard of care for treatment of their medical condition at this facility. The risks of drawing blood from a vein include discomfort at the site of puncture; possible bruising and swelling around the puncture s ite; rarely, an infect ion; and, unc ommonly, faintness. This study may include risks th at are unknown at this time. Participation in more than one research study or project may further incr ease the risks to you. Please inform the Principal Investigator (liste d in #3 of this consent form) or the person reviewing this consent with you before enrolli ng in this or any other research study or project. Throughout the study, the researchers will notif y you of new informa tion that may become available and might affect your d ecision to remain in the study. If you wish to discuss the information above or any discomforts you may experience, you may ask questions now or call the Principal Investigator or contact person liste d on the front page of this form. 10a. What are the possible benefits to you? There are no benefits to you if you ag ree to participate in this study. 10b. What are the possible benefits to others? The information gained from th is study may someday improve healing standards and practice in burn care.

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113 11. If you choose to take pa rt in this research study, will it cost you anything? Taking part in this study will not cost you anyt hing. The sponsor of this study will perform the blood and tissue tests and these tests will not be billed to you. Costs for routine medical care procedures will be charged to you or your insurance company. 12. Will you receive compensation for taking part in this research study? You will receive no money or other compen sation for participation in this study. 13. What if you are injure d because of the study? If you experience an injury that is directly cau sed by this study, only professional medical care that you receive at the University of Florida Health Science Center will be provided without charge. However, hospital expens es will have to be pa id by you or your insurance provider. No other compensation is offered. Please contact the Principal Investigat or listed in Item 3 of this form if you experience an injury or have any questions about an y discomforts that you experience while partic ipating in this study. 14. What other options or treatme nts are available if you do no t want to be in this study? The alternative to being in this study is to do nothing and not sign this Consent Form. Participation in this study is entirely voluntary. You are free to refuse to be in the study, and your refusal will not influence current or future health care you receiv e at this institution. 15a. Can you withdraw from this research study? You are free to withdraw your consent and to stop participating in this research study at any time. If you do withdraw your consent, there will be no pena lty, and you will not lose any benefits you are entitled to. If you decide to withdraw your consent to participate in this research study for any reason, you should contact David W. Mozi ngo, MD at (352) 273-5667. If you have any questions regarding your right s as a research subjec t, you may phone the Institutional Review Board (IR B) office at (352) 846-1494. 15b. If you withdraw, can information abou t you still be used and/or collected? If you decide to stop your partic ipation in this study, we will not continue to collect any more information from you. We would ask that you a llow us to maintain the information we had already collected.

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114 15c. Can the Principal Investigator with draw you from this research study? You may be withdrawn from the study without your consent for the following reasons: the study doctor thinks it is neces sary for your health or safety; if any significant side effect occurs; you participate in another i nvestigational study that skew s data being collected for this research study; the Principal Investig ator stops the study; or administration reasons 16. If you agree to participate in this research study, the Principal In vestigator will create, collect, and use private informat ion about you and your health. Once this information is collected, how will it be kept secret (confidential) in or der to protect your privacy? Information collected about you and your health (called protecte d health information), will be stored in locked filing cabinets or in computers with security passwords. Only certain people have the legal right to review these research records, and they w ill protect the secrecy (confidentiality) of these reco rds as much as the law allo ws. These people include the researchers for this study, certain University of Florida officials, the hospital or clinic (if any) involved in this research, and the Instituti onal Review Board (IRB; an IRB is a group of people who are responsible for looking after the rights and welfare of pe ople taking part in research). Otherwise your research record s will not be released without your permission unless required by law or a court order. If you participate in this research study, th e researchers will collect use, and share your protected health information with others. Item s 17 to 26 below describe how this information will be collected, used, and shared. 17. If you agree to participate in this research study, what protected health information about you may be collected, us ed and shared with others? Your protected health informatio n may be collected, used, and shar ed with others to determine if you can participate in the stud y, and then as part of your participation in the study. This information can be gathered from you or your pa st, current or future health records, from procedures such as physical examinations, x-rays blood or urine tests or from other procedures or tests. This information will be created by receiving study treatments or participating in study procedures, or from your stud y visits and telephone calls. If you agree to be in this resear ch study, it is possible that so me of the information collected might be copied into a "limited data set" to be used for other research purposes. If so, the limited data set may only include information that does not directly identify you. For example, the limited data set cannot include your name, address, telephone number, social

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115 security number, or any other photographs, numbers, codes, or so forth that link you to the information in the limited data set. If used, limited data sets have legal agreements to protect your identity and confidentiality and privacy. 18. For what study-related purposes will your protected health information be collected, used, and shared with others? Your protected health informatio n may be collected, used, and shar ed with others to make sure you can participate in the research, through your participation in the research, and to evaluate the results of the research study. 19. Who will be allowed to coll ect, use, and share your protected health information? Your protected health inform ation may be collected, used, and shared with others by: the study Principal Investigator, Davi d Mozingo, M.D. and his/her staff other professionals at the University of Fl orida or Shands Hospital that provide studyrelated treatment or procedures the University of Florida Institutional Review Board 20. Once collected or used, who may your prot ected health information be shared with? Your protected health inform ation may be shared with: the study sponsor, the University of Florida United States and foreign governmental agen cies who are responsible for overseeing research, such as the Food and Drug Admi nistration, the Department of Health and Human Services, and the Office of Human Research Protections Government agencies who are responsible for overseeing publ ic health concerns such as the Centers for Disease Control and Fede ral, State and local health departments) 21. If you agree to participate in this rese arch, how long will you r protected health information be used and shared with others? Your protected health information will be coll ected until the end of the study. This information will be used and disclosed foreve r since it will be stored for an indefinite period of time in a secure database. If you withdraw your permi ssion fro the use and shar ing of your protected health information, then your informa tion will be removed form the database. 22. Why are you being asked to allow the collectio n, use and sharing of your protected health information? Under a new Federal Law, researchers cannot collec t, use, or share with others any of your protected health information for research unless you allow them to by si gning this consent and authorization.

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116 23. Are you required to sign th is consent and authorization and allow the researchers to collect, use and share with others yo ur protected health information? No, and your refusal to sign will not affect your treatment, paym ent, enrollment, or eligibility for any benefits outside this research study. However, you cannot participate in this research unless you allow the collection, use and sharing of your protected health information by signing this consent/authorization. 24. Can you review or copy your protected health informatio n that has been collected, used or shared with others under this authorization? You have the right to review and copy your pr otected health informati on. However, you will not be allowed to do so until after the study is finished. 25. Is there a risk that your pr otected health information coul d be given to others beyond your authorization? Yes. There is a risk that information received by authorized persons could be given to others beyond your authorization and not covered by the law. 26. Can you revoke (cancel) your au thorization for collection, use and sharing with others of your protected health information? Yes. You can revoke your authorization at any time before, during, or after your participation in the research. If you revoke, no new inform ation will be collected about you. However, information that was already co llected may still be used and shared with ot hers if the researchers have relied on it to complete and pr otect the validity of the research. You can revoke your authorization by giving a written requ est with your signature on it to the Principal Investigator. 27. How will the researcher(s) benefi t from your being in this study? In general, presenting research results helps the ca reer of a scientist. Therefore, the Principal Investigator may benefit if the re sults of this study are presented at scientific meetings or in scientific journals.

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117 28. Signatures As a representative of this study, I have explaine d to the participant the purpose, the procedures, the possible benefits, and the risks of this research study; the alternatives to being in the study; and how the participants protected h ealth information will be collected, used, and shared with others: ____________________ ____________________ ______ _______ ______________ Signature of Person Obtaining Consent & Authorization Date Consenting Adults. You have been informed about this studys purpose, procedures, possible benefits, and risks; the alternatives to bei ng in the study; and how your protected health information will be collected, used an d shared with others. You have received a copy of this Form. You have been given the opportunity to ask questions before you sign, and you have been told that you can ask other questions at any time. Adult Consenting for Self. By signing this form, you voluntarily agree to participate in this study. You hereby authorize the collection, use and sh aring of your protected health information as described in sections 17-26 above. By signing this form, you are not waiv ing any of your legal rights. ____________________ ____________________ ______ _______ ______________ Signature of Adult Consenting & Authorizing for Self Date Parent/Adult Legally Rep resenting the Subject. By signing this form, you voluntarily give your permission for the person named below to participate in this study. You hereby authorize the collection, use and sharing of protected hea lth information for the person named below as described in sections 17-26 above. You are not wa iving any legal rights for yourself or the person you are legally representing. After your signature, please print your name and your relationship to the subject. ____________________ ____________________ ______ _______ ______________ Consent & Authorization Signature Date of Parent/Legal Representative _________________________________________________________________________Print: Name of Legal Representative of and Relationship to Participant:

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118 APPENDIX D IHC PROTOCOL FOR CTGF Tissue Should NEVER Dry Out Deparafinizing Xylene gets cloudy when us ing > 15 slides, chan ge this out after every 25 slides and wash container Prepare blocking solution (1% Human serum) before starting, make a liter and keep @ 40C Prepare 10 Ab (keep on ice)(prepare in 1% Human serum)(10 g/ml CTGF) Deparafinize Xylene 5 Min Change after every 25 slides and wash container Xylene 5 Min Change after every 75 slides and wash container 100% ETOH 5 Min 95% ETOH 5 Min 75% ETOH 5 Min 25% ETOH 5 Min dH2O 5 Min Record diagram of slide in not ebook (control on left)(test on right) Make wax circles around samples, try to make circ les the same size, use paper circle sticky as a guide Add blocking buffer, Record time of first sample Block 3 hours (this reduces variability between samples) No need to wash at this step Only remove Blocking buffer from test (right side of slide) Add same volume of 10 Ab Incubate in humidity cham ber overnight (Record Time!) Keep In Mind, Next Time You Complete Experi ment, It Has To Be The EXACT Same Time For Everything! Collect the 10 Ab on the test side only, cover with PBS (record starting and stopping time) Once all slides have 10 Ab removed then place into a tub of PBS PBS 5 Min PBS 5 Min Then place in tub of 20 Ab (1% Human Serum) for 1 hour

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119 Wash again in tub of PBS PBS 5 Min PBS 5 Min PBS 5 Min Tub Ready of 1 2 3 system (same lot #, same time)

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120 Substrate (vector red)(same lot #)(levam isol)(make lots of substrate buffer)

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121 APPENDIX E TGFIHC PROTOCOL Tissue should never dry out DeparafinizingXylene gets cloudy when using >15 slides, change this out after every 25 slides and wash container. Prepare blocking solutions (1% FBS in PBS) before starting, make a Liter, filter sterilize and keep at 4 C Prepare 1 Ab (keep on ice)(prepa re in blocking so lution)(1:150 TGF) use 1 Ab sparingly. Deparafinize Xylene 2 min Change after every 25 slides and wash container Xylene 2 min Change after every 25 slides and wash container 100% ETOH 1 min 95% ETOH 1 min 75% ETOH 1 min 25% ETOH 1 min dH2O 1 min Record diagram of each slide in notebook (c ontrol on left)(test on right) along with a designation for each slide Quickly make wax circle around samples, try to make circles the same size, beware not to press immuno pen too deeply-this can cause wax to run KEEP SAMPLES HYDRATED! Incubate samples 10 min in 1% H2O2 diluted in PBS Wash in PBS twice for 5 min each Incubate for 1 hour in blocking serum Remove blocking serum from test side only Keeping control side hydrated with blocking seru m, incubate test side 30 min with 1 Ab Wash with three changes of PBS for 5 min each Incubate 30 min with biotinylated 2 Ab Wash with three changes of PBS for 5 min each Incubate 30 min with AB enzyme reagent Wash with three changes of PBS for 5 min each

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122 Incubate in 1-3 drops of peroxidase substrate dH2O 5 min 25% ETOH 1 min 75% ETOH 1 min 95% ETOH 1 min 100% ETOH 1 min Xylene 2 min Xylene 2 min Mount with permount and add cover slip

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123APPENDIX F PATIENT DATA Patient Reference # Sample # Sex Age Date of Biopsy Menopausal Status % Burn Site of Biopsy Diabetic Receiving Antibiotics Hormone Replacement 01 01-01-R M 62 11/21/2005 N/A 15 Buttock Yes Yes No 01-01-F 02-01-R Back 02-01-F 00-01-B 02 01-01-R M 27 11/28/2005 N/A 20 Forearm No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 01-02-R 12/9/2005 Back 01-02-F 02-02-R Back 02-02-R 00-02-B 03 01-01-R F 40 12/2/2005 Pre 3 Hand No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 04 01-01-R M 19 1/3/2006 N/A 5 Leg No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 05 01-01-R M 22 1/11/2006 N/A 7 Leg No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 06 01-01-R M 18 1/10/2006 N/A 32 Thigh No Yes No 01-01-F

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124Patient Reference # Sample # Sex Age Date of Biopsy Menopausal Status % Burn Site of Biopsy Diabetic Receiving Antibiotics Hormone Replacement 02-01-R Thigh 02-01-F 00-01-B 01-02-R 1/27/2006 Thigh No 01-02-F 02-02-R Abd 02-02-F 00-02-B 07 01-01-R M 52 1/15/2006 N/A 7 Leg No No No 01-01-F 02-01-R Leg 02-01-F 00-01-B 08 01-01-R F 70 1/30/2006 Post 35 Thigh No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 09 01-01-R F 79 2/10/2006 Post 35 Chest No Yes No 01-01-F 02-01-R Chest 02-01-F 00-01-B 01-02-R 2/15/2006 Chest Yes 01-02-F 02-02-R Thigh 02-02-F 00-02-B 01-03-R 2/27/2006 Chest Yes 01-03-F 02-03-R Thigh 02-03-F 00-03-B 01-04-R 3/9/2006 Arm Yes 01-04-F 00-04-B

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125Patient Reference # Sample # Sex Age Date of Biopsy Menopausal Status % Burn Site of Biopsy Diabetic Receiving Antibiotics Hormone Replacement 10 01-01-R M 25 2/13/2006 N/A 25 Abd No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 11 01-01-R F 72 2/13/2006 Post 25 Chest No No No 01-01-F 02-01-R Abd 02-01-F 00-01-B 01-02-R 2/27/2006 Chest Yes 01-02-F 02-02-R Thigh 02-02-F 00-02-B 12 01-01-R F 19 3/1/2006 Pre 30 Chest No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 01-02-R 3/6/2006 Arm Yes 01-02-F 02-02-R Thigh 02-02-F 00-02-B 01-03-R 3/13/2006 Abd Yes 01-03-F 02-03-R Thigh 02-03-F 00-03-B 13 01-01-R F 32 5/5/2006 Pre 13 Leg Yes Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 14 01-01-R M 57 5/5/2006 N/A 3 Hand No Yes No

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126Patient Reference # Sample # Sex Age Date of Biopsy Menopausal Status % Burn Site of Biopsy Diabetic Receiving Antibiotics Hormone Replacement 01-01-F 02-01-R thigh 02-01-F 00-01-B 15 01-01-R F 42 5/24/2006 Pre 11 Arm No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 16 01-01-R M 21 5/26/2006 N/A 27 Leg No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 17 01-01-R M 64 6/1/2006 N/A 15 Chest No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 01-02-R 6/19/2006 Hand No 01-02-F 02-02-R Thigh 02-02-F 00-02-B 18 01-01-R M 79 6/21/2006 N/A 18 Leg No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 19 01-01-R M 35 6/28/2006 N/A 66 Chest No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 01-02-R 7/14/2006 Shoulder Yes 01-02-F

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127Patient Reference # Sample # Sex Age Date of Biopsy Menopausal Status % Burn Site of Biopsy Diabetic Receiving Antibiotics Hormone Replacement 02-02-R Abd 02-02-F 00-02-B 20 01-01-R F 65 7/14/2006 Post 13 Chest No No No 01-01-F 02-01-R Abd 02-01-F 00-01-B 21 01-01-R M 21 7/21/2006 N/A 2 Foot No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 22 01-01-R M 50 9/6/2006 N/A 15 Arm No No No 01-01-F 02-01-R Hip 02-01-F 00-01-B 01-02-R 9/13/2006 Leg No 01-02-F 02-02-R Hip 02-02-F 00-02-B 23 01-01-R F 41 9/15/2006 Pre 6 Leg No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 24 03-01-R F 43 9/20/2006 Pre N/A Breast No No No 03-01-F 00-01-B 25 03-01-R F 55 9/29/2006 Post N/A Breast No No No 03-01-F 00-01-B 26 01-01-R M 43 10/18/2006 N/A 13 Leg No No No 01-01-F

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128Patient Reference # Sample # Sex Age Date of Biopsy Menopausal Status % Burn Site of Biopsy Diabetic Receiving Antibiotics Hormone Replacement 02-01-R Thigh 02-01-F 00-01-B 27 03-01-R F 56 10/26/2006 Post N/A Arm No No No 03-01-F 00-01-B 28 01-01-R F 69 12/1/2006 Post 10 Shoulder No Yes No 01-01-F 02-01-R Groin 02-01-F 00-01-B *29 03-01-R F 57 12/4/2006 Post N/A Abd No No No 03-01-F 00-01-B 30 01-01-R F 74 1/17/2007 Post 9 Arm Yes No No 01-01-F 02-01-R Abd 02-01-F 00-01-B 31 03-01-R M 55 1/19/2007 N/A N/A Eyebrow Yes No No 03-01-F 00-01-B 32 03-01-R F 34 1/19/2007 Pre N/A Eyelid No No No 03-01-F 00-01-B 33 03-01-R M 23 1/22/2007 N/A N/A Sacrum No Yes No 03-01-F 00-01-B **34 03-01-R F 39 1/25/2007 Post N/A Abd No No Yes 03-01-F 00-01-B 35 03-01-R F 60 1/29/2007 Post N/A Thigh Yes No No 03-01-F 00-01-B 36 03-01-R F 22 1/31/2007 Pre N/A Neck No No No 03-01-F

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129Patient Reference # Sample # Sex Age Date of Biopsy Menopausal Status % Burn Site of Biopsy Diabetic Receiving Antibiotics Hormone Replacement 00-01-B 37 01-01-R M 19 2/3/2007 N/A 15 Leg No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 38 01-01-R F 30 2/14/2007 Pre 3.5 Arm No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 39 01-01-R M 48 2/14/2007 N/A 8 Leg No Yes No 01-01-F 02-01-R Thigh 02-01-F 00-01-B 40 01-01-R M 53 2/21/2007 N/A 3 Hand No No No 01-01-F 02-01-R Thigh 02-01-F 00-01-B

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130The first set of two numbers = patient 01-40 The second set of two numbers = site of biopsy 01 = Sample taken from deep partial thickness thermal burn injury tissue 02 = Sample taken from non thermal burn injured donor site 03 = Sample taken of normal tissue taken from non burn injured patient The third set of two numbers = number of biopsy 01 = First biopsy 02 = Second biopsy 03 = Third biopsy F = Formalin R = RNAlater B = Blood Example of patient and sample number: 01-01-01-R, refers to patient #1, sample taken from burn injured tissue, from the first debridement, placed in RNAlater solution. Patient is on estrogen inhibitor, Femara ** Patient is on estrogen replacement, Estratest

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131 LIST OF REFERENCES 1. Burn Incidence and Treatment in the US 2007 Fact Sheet .Chicago, IL: American Burn Association; available from http:// www.ameriburn.org/resources_factsheet. Internet; accessed 20 Jan. 2005. 2. Hakvoort, T., Altun, V., van Zuijlen, P. P. and et al., Transforming growth factor-beta(1), -beta(2), -beta(3), basic fibrobl ast growth factor and vascular endothelial growth factor expression in keratinocytes of burn scars. Eur. Cytokine Netw.2000;11:233-239. 3. Leask, A. and Abraham, D. J., TGF-beta signaling and the fibrotic response. FASEB J.2004;18:816-827. 4. Igarashi, A., Nashiro, K., Kikuchi, K. and et al., Connective tissue growth factor gene expression in tissue sections from localized sc leroderma, keloid, and other fibrotic skin disorders.J. Invest Dermatol.1996;106:729-733. 5. Tobin, D. J., Biochemistry of human skin--our brain on the outside.Chem. Soc. Rev.2006;35:52-67. 6. Wysocki, A. B., Skin anatomy, phys iology, and pathophysiology.Nurs. Clin. North Am.1999;34:777-97.. 7. Rook, A, Wilkinson, DS, and Ebling, FJG, Textbook of Dermatology.; Massachussets:Blackwell Science. 1998p1-368: 8. Kierszenbaum, A, Histology and Cell Bi ology: An Introduction to Pathology. Missouri: Mosby Inc; 2002 p51-75 9. Goldsmith, LA, Physiology, Biochemistr y, and Molecular Biology of the Skin.; New York: Oxford; 1991 p. 20-80. 10. Wysocki, A, Mustoe, T, and Schultz, G, Skin Molecular Cell Biology of. In. Meyers, R editor, Encyclopedia of Mol ecular Cell Biology and Molecu lar Medicine. California: Wiley-VCH; 2006, vol l3 p. 217-250: 11. Krause, K. and Foitzik, K., Biology of the hair follicle: the basics.Semin. Cutan. Med. Surg.2006;25:2-10. 12. Moore, K. A. and Lemischka, I. R., Stem cells and their niches.Science3-312006;311:1880-1885.

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136 72. Schroder, J., Kahlke, V., Staubach, K. H. et al., Gender differences in human sepsis.Arch. Surg.1998;133:1200-1205. 73. Kovacs, E. J., Faunce, D. E., and Messi ngham, K. A., Ethanol a nd burn injury: estrogen modulation of immun ity.Alcohol2004;33:209-216. 74. DeGroot, L and Jameso n, J, Endocrinolgy.2006;3:3613-5 75. Ashcroft, G. S. and Ashworth, J. J., Pote ntial role of estrogens in wound healing.Am. J. Clin. Dermatol.2003;4:737-743. 76. Haczynski, J., Tarkowski, R., Jarzabek, K. et al., Human cultured skin fibroblasts express estrogen receptor alpha and be ta.Int. J. Mol. Med.2002;10:149-153. 77. Ankrom, M. A., Patterson, J. A., d'Avis, P. Y. et al., Age-related changes in human oestrogen receptor alpha func tion and levels in osteoblasts.Biochem. J.8-1-1998;333 ( Pt 3):787-794. 78. Pelletier, G. and Ren, L.,P,Localization of sex steroid receptors in human skin.Histol. Histopathol.2004;19:629-636. 79. Werner, S. and Grose, R., Regula tion of wound healing by growth factors and cytokines.Physiol Rev.2003;83:835-870. 80. Igarashi, A., Okochi, H., Bradham, D. M. et al. Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair.Mol. Biol. Cell1993;4:637-645.

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137 BIOGRAPHICAL SKETCH When I was in high school, college was the last thing on my mind. I did not have any direction or path. Halfway through my senior year in high school I got a job at a local textile plant, I started out packing boxes and eventually moved up to internal qualit y control inspector (just fancy words to say that I l ooked at cloth all night). I stayed at this job for three after high school, still had no direction or anything that I particularly wa nted to do. However, during the three years at the textile plant, I was laid off three times and finally on the third time the plant closed for good. I was twenty years old with ju st a high school diploma and the only skill I had was looking at cloth. I really was in no hurry to look for another job, until one day an Army recruiter called me. Just for the heck of it I asked him about the Army college fund. Things happened fairly quickly from then, in less than a month I was Private McCurry, U.S. Army! I spent 3 years in the Army stationed with a fiel d artillery unit based at Fort Hood, Texas. During my time in Texas I never really had the desire to make the military a care er, but I did start to think about school. I even took a few classes at Central Texas College. I was released from the Army in May of 1990, but by the fall I was recalled back to active duty because of Operation Desert Storm. I started college at South Carolina State Univer sity of Florida, in fall 1991, after Desert Storm was over. I started there as a biology major because by this time I decided that I wanted to be a physical therapist and the requirements for PT school closely paralleled the degree requirements for a biology degree at SCSU. I conti nued to work as a rehab tech, nursing assistant and EMT while continuing to pursue my degree in biology. I finally gr aduated in May of 1994 with a cum laude degree in biology from South Carolina State Universi ty. This occasion was bitter sweet because my father passed away just 2 months prior and was never able to see this accomplishment in my life.

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138 Why did I choose physical therapy? Well, I wa s in pretty good shape from being in the military and I thought what a cool job where you can get paid to show people how to work out. I enrolled at the Medical University of South Caro lina in the summer of 1995. I continued to work weekends in the emergency room as an ER tech and graduated in June 1997 with a degree in physical therapy. I passed the South Carolina St ate Board that October and worked the next seven years as an acute care physical therapist at The Regiona l Medical Center in Orangeburg, South Carolina. Now, you ask, how did I get involved in w ound care? There are many areas of physical therapy and by working in an acute care setting I got to see a litt le bit of a lot of things, wound care being one. The popular ar eas of PT such as orthopedics to me quickly became a little boring. Then one day I met a nurse, Marie Gehling. She wanted to start a clinic to follow up on wound patients that were released from the hospita l. Over time this clinic grew from patients lined up in a small closet like room and Marie on the floor to now one of the largest wound clinics in the state of South Carolina. Marie even tually recruited (either willingly or unwillingly) Dr. John Samies to be the medical director of the wound center. It is these two people that I credit for where I am today as far as my career goes. As a physical therapist, I worked closely with Marie and Dr. Samies and would perfor m modalities and dressi ng changes on the wound clinic patients. These two people have a passion for patient care that is unequal in anyone that I have ever met. They have taught me a great deal professionally; however there was part of me that wanted more. One day I was placing a dressing on a patien ts foot after whir lpool therapy, I dont remember this patients name, but his question to me I will always remember. He asked how come you are using that particul ar dressing. I honestly could not answer his question. I knew it

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139 was a good dressing choice, I used many times befo re with good results, but I could not explain how it worked. This made me feel very foolish, I was a professional, I should know this kind of thing. My sister and I brought my mom to Shands at the University of Florida for a second opinion on a medical condition. My mom was in the hospital for two weeks and there is only so much sitting around watching someone sleep that I could do, so I went for a walk. I went up this hall, down that hall and by pure luck or divine in tervention I saw the sign that would change my life; The Wound Healing Institute. I walked in and introduced my self and met with Dr. Gregory Schultz, who I would later find out is pretty famous in the wound healing community. He explained to me what was required for graduate school and when I went home to South Carolina I applied to graduate school and a few months later I was accepted. I started graduate school at the University of Florida in the fall of 2004. I applied for the masters program, which is a two year program. By the time I am finished it will have taken me three years to complete this program because I ha ve only been able to attend on a part time basis. I work at North Florida Regional Medical Center as a wound care phys ical therapist, so need less to say the last three year s of my life have been pretty hectic. I am hoping to graduate this fall, August 2007, and looking forward to be ing part of the Gator Nation! My plans for the future right now are a littl e up in the air, but I do plan on taking my wound care certification through the Academy fo r the Advancement of Wound Care (AAWC) once things calm down a little. I feel that my time here at UF will make me a better clinician and more confident in being able to answer any question that patients through my way.