______________________________________________________________________________

KERATIN BIOMATERIAL TREATMENTS FOR BURN INJURY AND

MECHANISMS OF TISSUE SURVIVAL

BY

DEEPIKA RANI PORANKI

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

in the Molecular Genetics and Genomics Program

August 2012

Winston-Salem, North Carolina

Approved by

Mark Van Dyke, PhD., Advisor

Michael Tytell, PhD., Chair

Mark O. Lively, PhD

Joseph Molnar, M.D., Ph.D., F.A.C.S

Mark Willingham, PhD

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ACKNOWLEDGEMENTS

My time at Wake Forest had been an incredible learning experience. This thesis is the

culmination of my work at Wake Forest but the experiences and knowledge I gained here will

stay with me for a lifetime. My work and my personal transformation would not have been

possible without the invaluable support of my excellent mentors and amazing colleagues.

First and foremost, I am indebted to my mentor, Dr. Mark Van Dyke, for his constant help,

expert guidance, enormous support and committed mentorship. I feel extremely fortunate to have

worked with the “best mentor“, anyone can hope for. My gratitude goes to him for always

putting the students first. Mark’s personal attributes of dedication, commitment and poise will

always be something that I wish to strive for.

As program director and committee member, Dr. Mark Lively was always very helpful with his

meticulous nature and timely advice. I am also grateful to my other committee members: Dr.

Michael Tytell, Dr. Joseph Molnar and Dr. Mark Willingham for their guidance, advice and

willingness to serve on my committee amidst their busy schedules.

I am thankful to Carmen Gaines for her support and friendship at WFIRM and after. It was a

great experience working with her on the wound healing projects. Her detail-oriented nature and

thoroughness have been a great source of help for our project(s). It seemed like yesterday that

Heather Coan taught me the nuances of tissue culture work, when I just started working in the

lab. She moved on but the techniques that I learnt from her helped me for the major part of my

thesis.

Thanks to many others, who helped me at different stages of this work. I cannot forget the help

of Christina Ross and Mary Ellenberg during the hectic animal study. Renae Hall, Cynthia

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iii

Miller, Sandy Sink and Tammy Cockerham from the surgery core helped us complete the project

without any issues. I am appreciative of Dr. Richard Clark for hosting us in his lab at Stony

Brook and training us in the intricacies of histological analysis; Dr. Wei Du for continuing on

that training at Wake Forest and answering the questions we had in histology and Bridgette Jones

for helping with the sectioning. If it was not for Barbara Summerlin, Deborah McLaughlin and

Susan Pierce working behind the scenes, my graduate student life would have been so much

tougher.

The past and present members of the Keratin group: Paulina Hill, Mária Bahawdory, Bailey

Fearing, Lauren Pace, Julie Gupton, Fiesky Nunez, Roche de Guzman, Jill Richter, Olga

Roberts, Matt Stern, Alana Sampson and Arthur J.Allen have been a constant source of

knowledge and support to me.

I am very grateful to my parents, Satyaprasad Poranki and Annapurna Poranki, for teaching the

value of honesty and integrity and always being there for me –no matter what. I am also grateful

to my in-laws, Prabhakara Raju Vinukonda and Sri Lakshmi Vinukonda, for their commitment to

higher education and wholeheartedly encouraging me. Thanks to other family members: Uday,

Priya, Ravi, Rama, Suresh, Sruthi and their kids for all their love and support. There was also a

great deal of support we received from family friends in Winston-Salem.

Last, thanks to my husband, Naveen Vinukonda and daughter, Asmithaa Vinukonda, without

whom, I wouldn’t have come this far. My loving, encouraging and patient husband has always

been the strongest source of support, right from the beginning of this worthwhile journey. My

daughter was a few-month old baby, when I started preparing for GRE. Today she is a 6 yr. old

getting ready to graduate from kindergarten. If not for her bringing joy and happiness, this

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journey would have been so much more strenuous. All along my daughter saw her mom struggle

to fulfill a dream and I hope this stays with her as an inspiration.

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DEDICATION

Dedicated to

Daddy & Amma

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TABLE OF CONTENTS

LIST OF FIGURES AND TABLES .......................................................................................... vii

ABBREVIATIONS ....................................................................................................................... x

ABSTRACT ................................................................................................................................ xiii

CHAPTER 1 - INTRODUCTION ............................................................................................... 1

CHAPTER 2- A KERATIN BIOMATERIAL PROMOTES SKIN REGENERATION

AFTER BURNS IN VIVO AND RESCUES CELLS AFTER THERMAL

STRESS IN VITRO ............................................................................................... 37

CHAPTER 3 - GAMMA KERATOSE MAINTAINS CELL VIABILITY IN VITRO

AFTER THERMAL STRESS BY REGULATING THE EXPRESSION OF

CELL DEATH PATHWAY SPECIFIC GENES ................................................ 78

CHAPTER 4 - DEVELOPMENT OF A PORCINE DEEP PARTIAL THICKNESS BURN

MODEL ................................................................................................................. 119

CHAPTER 5 - ASSESSMENT OF DEEP PARTIAL THICKNESS BURN TREATMENT

WITH KERATIN BIOMATERIAL HYDROGELS IN A SWINE MODEL 146

CHAPTER 6 - DELAYED TREATMENT OF PORCINE DEEP PARTIAL THICKNESS

BURNS SUBJECT TO THERAPEUTIC KERATIN BIOMATERIALS ...... 170

CHAPTER 7 - SUMMARY, CONCLUSIONS AND PROSPECTIVE RESEARCH ........ 200

SCHOLASTIC VITA ................................................................................................................ 208

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vii

LIST OF FIGURES AND TABLES

CHAPTER 1 - INTRODUCTION ............................................................................................... 1

Figure 1: Three Zones in Burn Wounds: ..................................................................................... 3

Figure 2: Schematic diagram of Wool fiber ................................................................................ 7

Figure 3: Apoptotic Pathways ................................................................................................... 14

Figure 4: Autophagy and its inhibitors ...................................................................................... 16

Table 1: Gene Glossary ............................................................................................................. 18

CHAPTER 2 - A KERATIN BIOMATERIAL PROMOTES SKIN REGENERATION

AFTER BURNS IN VIVO AND RESCUES CELLS AFTER THERMAL

STRESS IN VITRO ............................................................................................... 37

Figure 1. Mouse Chemical Burn Wound Area .......................................................................... 65

Figure 2. Digital Images from Mice Chemical burn study ........................................................ 66

Figure 3. Swine Thermal Burn Wound Area ............................................................................. 67

Figure 4. Digital Images from Swine thermal burn study ......................................................... 68

Figure 5. In Vitro Thermal Stress Model .................................................................................. 69

Figure 6. Percent Cell Viability Curve ..................................................................................... 70

Figure 7. Keratose SDS PAGE .................................................................................................. 71

Supplementary Table 1: Band # 1, Molecular Weight ~110 KDa ............................................ 72

Supplementary Table 2: Band # 2, Molecular Weight ~60 KDa .............................................. 73

Supplementary Table 3: Band # 3, Molecular Weight ~50-40 KDa ......................................... 74

Supplementary Table 4: Band # 4, Molecular Weight ~30 Kda ............................................... 75

Supplementary Table 5: Band # 5, Molecular Weight ~20-15 Kda .......................................... 76

Supplementary Table 6: Band # 6, Molecular Weight ~8-10 KDa ........................................... 77

CHAPTER 3 - GAMMA KERATOSE MAINTAINS CELL VIABILITY IN VITRO

AFTER THERMAL STRESS BY REGULATING THE EXPRESSION OF

CELL DEATH PATHWAY SPECIFIC GENES ................................................ 78

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viii

Figure 1: In vitro Heat shock Model ....................................................................................... 105

Table 1: RNA Quantification .................................................................................................. 106

Table 2: Gene expression of gamma keratose treated cells at 12hr......................................... 107

Table 3: Gene expression of gamma keratose treated cells at 18hr......................................... 109

Table 4: Gene expression of growth media treated cells at 18hr............................................. 110

Table 5: Gene expression of gamma keratose treated cells at 24hr......................................... 112

Table 6: Gene expression of growth media treated cells at 24 hr............................................ 113

Table 7: Total number of genes significantly expressed in gamma keratose treated cells and

growth media treated cells with p<0.05 .................................................................................. 114

Supplementary Table 1: Genes included in the PCR array ..................................................... 115

CHAPTER 4 - DEVELOPMENT OF A PORCINE DEEP PARTIAL THICKNESS BURN

MODEL ................................................................................................................. 119

Figure 1. Differences in healing of deep partial thickness scald burns due to inconsistent

burning. .................................................................................................................................... 135

Figure 2. Burns with non-uniform severity created with brass block heated in boiling water

along the right flank of the animal .......................................................................................... 136

Figure 3. 360 grade, 3 cm diameter brass block used for burn wound creation ...................... 137

Figure 4. Burn device operation. ............................................................................................. 138

Figure 5. Heated brass blocks with temperature sensors. ........................................................ 139

Figure 6. Contact angle analysis using water and PEG:H2O azeotropic mixture to determine

surface wetting. ........................................................................................................................ 140

Figure 7. Healing progression of wounds with and without islands ....................................... 141

Figure 8. Visually consistent burn wounds ............................................................................. 142

Figure 9. Histological scoring data from day 1 wounds .......................................................... 143

Figure 10. Tissue sections stained with Gomori trichrome stain ............................................ 144

Figure 11. Percent dermal tissue damage ................................................................................ 145

CHAPTER 5 - ASSESSMENT OF DEEP PARTIAL THICKNESS BURN TREATMENT

WITH KERATIN BIOMATERIAL HYDROGELS IN A SWINE MODEL 146

Figure 1: Visual wound assessment ........................................................................................ 164

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Figure 2: Burn depth ................................................................................................................ 165

Figure 3: Early time points rates of re-epithelialization .......................................................... 166

Figure 4. Rates of re-epithelialization during later stages of wound healing .......................... 167

Figure 5: Thickness of granulation tissue ................................................................................ 168

Table 1: Percent increase in burn depth from day 3 to 6 ......................................................... 168

Table 2. Rates of re-epithelialization at day 3 and day 6 ....................................................... 169

Table 3. Rates of re-epithelialization between days 9 and 15. ................................................ 169

Table 4. Days to wound closure .............................................................................................. 169

CHAPTER 6 - DELAYED TREATMENT OF PORCINE DEEP PARTIAL THICKNESS

BURNS SUBJECT TO THERAPEUTIC KERATIN BIOMATERIALS ...... 170

Figure 1. Schematic of randomized placement of treatments ................................................ 191

Figure 2. Extent of burn wound size reduction over time ...................................................... 192

Figure 3. Degree of wound contracture as a function of time ................................................ 193

Figure 4. H&E images of deep partial thickness burn depth at Day 1 ................................... 194

Figure 5. H&E images of deep partial thickness burns at Day 6............................................ 194

Figure 6. H&E images of deep partial thickness burns at Day 12.......................................... 195

Figure 7. Initial rates of re-epithelialization in deep partial thickness burns ......................... 196

Figure 8. Rates of re-epithelialization in second degree burns during late stages of healing 197

Figure 9. H&E images of deep partial thickness epithelium at Day 12 .................................. 198

Figure 10. Degree of reepithelialization at Day 30 post-burn ................................................. 198

Table 1. Initial rates of reepithelialization between Days 0 and 6 ......................................... 199

Table 2. Rates of reepithelialization between Days 9 and 15 ................................................. 199

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ABBREVIATIONS

µm: Micrometer

ACUC: Animal Care and Use Committee

APC: Activated protein C

cm: Centimeter

DE: Dermal-epidermal

DI: Deionized water

DoD: Department of Defense

EGF: Epidermal growth factor

FGF2: fibroblast growth factor 2

GM-CSF: Granulocyte monocyte colony stimulating factor

H&E: Hemotoxylin and Eosin

h: Hour

H2O: Water

HBSS: Hanks balanced salt solution

hr: hour

IPA: Isopropyl alcohol

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KAP: Keratin associated proteins

KDa: Kilo dalton

kg: Kilograms

KOS: Keratose

mcg/h: Micrograms per hour

mg: Milligrams

min: Minutes

MKH: Modified keratose hydrogel

mL: Milliliters

MRad: Mega radiation absorbed dose

ºC: degrees celsius

PBS: Phosphate buffer saline

PCR: Polymerase chain reaction

PEG: Polyethylene glycol

RCF: Relative centrifugal force

rpm: Revolutions per minute

r-TPA: Recombinant tissue type plasminogen

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s: Seconds

SD: Standard Deviation

sec: Second

SIRC: Statens Seruminstitut Rabbit Cornea

SSD: Silver sulfadiazine

TBSA: Total body surface area

TCP: Tissue culture plastic

w/v: weight/volume

WFIRM: Wake Forest Institute for Regenerative Medicine

WFU IACUC: Wake Forest University Institutional Animal Care and Use Committee

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ABSTRACT

Deepika Rani Poranki

KERATIN BIOMATERIAL TREATMENTS FOR BURN INJURY AND

MECHANISMS OF TISSUE SURVIVAL

Dissertation under the direction of

Mark Van Dyke, Ph.D., Assistant Professor

Wake Forest Institute for Regenerative Medicine

Thermal burns typically display an injury pattern dictated by the transfer of the thermal energy

into the skin and underlying tissues. There are often three zones of injury represented by a

necrotic zone of disrupted cells and tissue, an intermediate zone of injured and dying cells, and a

distant zone of stressed cells that will recover with proper treatment. There are currently no

approved clinical therapies that target the zone of stasis and can reduce the need for excision and

grafting by salvaging potentially viable cells and tissue and thereby contributing to spontaneous

healing. In this project, we endeavored to investigate the potential of keratin biomaterials to

mediate such healing. Pilot studies testing treatment of chemical (mouse model) and thermal

(swine model) burns showed that a native mixture (termed “crude keratose” throughout this

thesis) of keratose was able to promote wound healing by stabilizing the size of the burn,

suggesting that cell survival was being facilitated in the zone of stasis. These results led us to

hypothesize that the interaction of the keratose biomaterial with cells and tissue promoted

survival and reduced the total body surface area burned. As crude keratose is a heterogeneous

mixture of proteins representing alpha and gamma protein fractions, we determined the

differential activity on cell survival associated with these fractions by using an in vitro thermal

stress model and showed that gamma keratose contributed to cell survival while alpha keratose

did not. In additional studies, the specific mechanism of cell survival associated with the gamma

keratose fraction was investigated. These results showed that gamma keratose was able to down

regulate the genes involved in cell death pathways. Later, an in vivo porcine deep partial

thickness burn model was developed to study the wound healing properties of a modified

keratose hydrogel (MKH) that contained a reduced amount of gamma keratose, reflecting the

same concentrations used in in vitro thermal stress model. The MKH showed a faster re-

epithelialization rate and wound closure compared to other treatments. Finally, to investigate the

potential clinical relevance to actual burn treatment, a delayed treatment thermal burn study was

performed. These results showed that MKH was able to promote faster wound healing compared

to other treatments but not with the same efficacy compared to more immediate post-burn

treatment. This work suggested the potential benefit of using keratin biomaterial in burn therapy

and provided informative data for developing second generation keratin biomaterial treatments.

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CHAPTER 1

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INTRODUCTION

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Introduction:

Skin is the largest organ of the body and acts primarily as a homeostatic tissue

maintaining body temperature and fluid balance. There are two main layers of the skin:

epidermis and dermis. Cells in the basal layer of the epidermis give rise to proliferating

keratinocytes that differentiate as they move up the strata [1, 2]. As they reach the surface of the

skin, they undergo programmed cell death [3] and these flattened, enucleated cells form the outer

stratum corneum, a heavily keratinized layer of highly cross-linked proteins that provide a

structural barrier to keep pathogens out and fluid in. This entire process is relatively fast and the

epidermis turns over every 2 weeks. The dermis consists of blood vessels, nerves, hair follicles,

smooth muscle, lymphatic tissue, and elastin fibers, along with loose connective tissue collagen.

Fibroblasts, adipocytes and macrophages are the main cell types found in dermis. The renewing

property of the epidermis helps in burn healing or any other skin injury.

Burns:

Burns are defined as injuries caused by heat, friction, electricity, radiation or chemicals.

They are classified into first degree (1°), second degree (2°), third degree (3°) and fourth degree

(4°) depending on the depth of the burn. First degree burns mainly involve the epidermal layer of

the skin. Second degree burns are further divided to superficial or deep partial thickness burns.

Superficial partial thickness burns do not extend entirely through the dermis and leave behind

epithelial-lined dermal appendages including sweat glands, hair follicles and sebaceous glands.

Deep partial thickness burns extend to the lower layers of dermis and usually heal in 3-9 weeks.

Third degree burns are full thickness burns that involve all layers of the dermis and subcutaneous

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adipose tissue. Fourth degree burns involve all the layers of dermis, subcutaneous tissue and the

underlying fascia or muscle.

According to Douglas Mac G Jackson, intensity of the burn is characterized by 3

concentric zones – the Zones of hyperemia, stasis and coagulation [4] (Figure 1). The outer zone

of hyperemia is characterized by the presence of blood circulation and active metabolism. The

intermediate zone of stasis is where the blood circulation is not reduced but metabolism is

diminished. If proper treatment is provided, the viable cells in this zone will survive. The central

zone of coagulation is the zone where injury occurred and is characterized by complete

destruction of the blood vessels. The cells in this zone are completely necrosed [4].

Figure 1: Three Zones in Burn Wounds: The central zone of coagulation is where injury

occurred and cells are completely necrosed, intermediate zone of stasis is where the blood

circulation is not reduced but metabolism is diminished and outer zone of hyperemia is

characterized by the presence of blood circulation and active metabolism.

(www.indiasurgeons.com)

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The majority of burn wounds are healed either by re-epithelialization, scar formation,

contraction, skin grafting, or combinations thereof. Re-epithelialization occurs mainly in first and

second degree burns where remnants of epithelium are present. Epithelial cells migrate from the

wound edges and regions around hair follicles to the wound bed and help in resurfacing the

residual dermis [4-7].

In scar formation, collagen deposition occurs in the wounded region. Scarring can be

divided into 3 phases – 1) Inflammatory phase where high number of inflammatory cells are

present in the wounded region, 2) Proliferative/collagen phase where there is rapid increase in

the collagen content, and 3) Maturation phase where the tensile strength of the skin increases but

there is no increase in the collagen content [8]. Contraction of the wound is mainly due to

myofibroblasts that express α-actin. The last form of healing is skin grafting which can be

divided into 3 phases – 1) Phase of imbibition where skin absorbs nutrients from the wound bed,

2) Phase of neovascularization where blood vessels invade the graft, and 3) Phase of maturation

where collagen bridges are formed between the wound bed and the graft [9].

Some burns are superficial at presentation and do not require extensive treatment and re-

epithelialize on their own, while some are full thickness burns and are best treated with excision

and grafting. However, many burns are indeterminate with mixed burn depth which may require

grafting. A typical protocol for burn care is to allow the wound to re-epithelialize if it can occur

by itself, preventing further tissue injury and avoiding infection of the wounded area.

As a standard of care, the patient is topically treated with antimicrobial agents like silver

sulfadiazine cream or silver impregnated dressings [10]. However, silver sulfadiazine has been

shown to inhibit re-epithelialization [11, 12]. In burns that are superficial and re-epithelializing,

other antimicrobial agents like topical antibiotic creams are preferred [13].

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Skin grafts are used in treating partial thickness and full thickness burns. Autografts are

the best skin grafts and are harvested from appropriate areas of the patient’s unburned skin.

When large areas of the patient are burned, harvesting sufficient autograft can be a major

challenge, particularly when unburned areas are inaccessible or undesirable (e.g. soles of the feet

or palms). In these cases there are temporary skin substitutes or permanent skin substitutes

available in the market.

Temporary skin substitutes provide transient physiologic wound coverage, help to

control pain and can absorb modest amounts of wound exudate. These include human allograft,

human amnion, xenografts and synthetic membranes. These are usually applied on a cleaned,

debrided wound. Other than human allograft, temporary skin substitutes do not vascularize but

provide effective temporary wound coverage and control pain. Human allograft is the most

often used temporary biological dressing. Allograft can either be viable with live cells

(keratinocytes, fibroblasts, endothelial cells and langerhans cells) or non-viable without any live

cells and usually is glycerolized or gamma irradiated, freeze dried or ethylene oxide treated [14-

16]. Human amnion is derived from the amniotic membrane. It acts as a temporary dressing for

clean wounds like partial thickness burns, donor sites or full thickness wounds [17-19]. One of

the main drawbacks of amnion is the difficulty of screening for viral diseases. Xenograft is

porcine derived and is used as temporary coverage for clean superficial second degree burns and

donor sites [20]. Synthetic membranes are semi-permeable membrane dressings that provide a

barrier for bacteria, vapor and also control pain until the underlying wound heals [21, 22].

Biobrane is a two layer nylon mesh that helps in fibrovascular in-growth and has an outer silastic

layer that act as vapor and bacterial barriers. One type of hydrocolloid dressings is a three

layered structure that has a porous inner layer, absorbent methyl cellulose middle layer, and semi

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permeable outer layer. This provides a moist environment for the wound to heal and absorbs

wound exudate [23, 24].

Permanent skin substitutes are also available but are not ideal. Epicel, Integra and

Alloderm are marketed as permanent skin substitutes because some or all of their components

become integrated into the healing wound. Epicel is cultured epidermal autograft. Epithelial cells

are isolated from a small skin biopsy and are cultured in vitro until there are enough cells to form

a dressing of useful size [25, 26]. These epithelial cell sheets are attached to petroleum coated

gauze which acts as a carrier and then applied on burn wounds [27]. Integra is the first dermal

substitute approved by the US Food and Drug Administration. Integra is also known as artificial

skin [28]. It contains an inner bovine collagen fibrous layer and outer silicone layer with vapor

transmission characteristics similar to normal epithelium. Once full thickness wounds are

covered with Integra and become vascularized, the outer silicone layer is replaced with thin

epithelial autograft 2-3 weeks later [29]. Alloderm is an acellular human dermal allograft. It is

devoid of epidermis and must be covered by a split thickness autograft at the time of initial

operation. It helps in reducing post operative scarring [30, 31].

Keratin Biomaterial:

Keratins are intermediate filament proteins that provide mechanical stability and integrity

for epithelial cells and tissues [32]. Mammalian keratins are classified into two groups – hard

keratins and soft keratins based on their structure, function and regulation. Hard or trichocytic

keratins, have high sulfur content and are found in a number of external appendages such as

wool and hair fibers. These have ordered arrays of intermediate filaments embedded in a matrix

of cysteine rich proteins (Figure 1). Soft or cyto keratins, are characteristic of epithelial cells

with low sulfur content [33-36]. Hair keratins and cyto keratins consist of obligate heterodimers:

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type I and type II proteins with non-helical N-terminal and C-terminal domains and a central

coiled rod domain. Type I and II chains differ in their molecular weight [36].

The hair follicle is a remarkably proliferative and regenerative organelle. To produce new

hair, the follicle undergoes a series of cycles – anagen (growth), catagen (regression) and telogen

(rest) [37]. During anagen follicles produce the entire hair shaft. During catagen and telogen,

stem cells are activated; the lower cycling portion of the hair follicle regresses in growth and is

in a resting phase. Stem cells activated in this phase trigger the new hair growth cycle again.

More than 30 growth factors and cytokines are involved in the hair cycling process, and have

also been shown to play a role in the regeneration of other tissues [38].

Figure 2: Schematic diagram of Wool fiber: Human hair has a similar superstructure to wool

fiber with 80% alpha- to 20% gamma-keratin. Alpha-keratins are the high molecular weight (40-

60kDa), low sulfur containing proteins that are mainly composed of the right-handed, alpha-

helical proteins. Gamma-keratins are the low molecular weight (10-25kDa), high sulfur

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containing proteins that hold the keratin intermediate filaments together (Used with permission

from Elsevier, Plowman JE, Proteomic database of wool fibers. J Chromatogr B Analyt Technol

Biomed Life Sci. 2003 Apr 5;787(1):63-76) .

Hair fiber consists of 3 components – cuticle, cortex and medulla (Figure 2). Cuticle is

the thin outer surface of the fiber and consists of β-keratins that help to protect the hair fiber from

external physical and chemical damage. Cortex is composed of spindle shaped cells with keratin

filaments contained within them. Type I (acidic) and Type II (basic to neutral) intermediate

filament (IF) keratins form coiled coil obligatory heterodimers through the interaction of their α-

helical rod domains [39]. Two dimers assemble in a staggered anti-parallel fashion to make a

tetramer. Two tetramers form an octamer and four octamers join to form a cylindrical unit length

filament (ULF). Individual ULF join to form short filaments which fuse end to end followed by a

compaction phase that leads to the formation of 10nm diameter IF [40]. The hair keratins have

high sulfur content and are further reinforced by highly cross linked matrix proteins called

keratin associated protein (KAPs) [32, 41].Medulla is present occasionally in the center of the

hair fiber. The many different structures and keratin sub-types lead to the complexity of the hard

keratin protein family.

Keratins Classification: Keratins constitute two classes of the intermediate filament (IF)

family proteins – type I acidic keratins and type II basic keratins [42, 43]. Type I keratins are

smaller in size with acidic isoelectric point (pI) and type II keratins are larger in size with neutral

to slightly basic pI. Genome analyses demonstrated that there are a total of 54 functional keratin

genes in humans – 28 type I keratins on chr 17q21.2 and 26 type II keratins on chr 12q13.13 [44-

46]. In 2006, Schweizer et al., published a new consensus nomenclature for mammalian keratins

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by dividing the keratins into 3 categories: 1) epithelial keratins/genes 2) hair keratins/genes, and

3) keratin pseudogenes..

The cortical proteins in the hair fiber can be divided into two groups – a cystine poor

fraction called α-keratin and cystine rich fraction called γ-keratins. The α-keratins are the main

structural component of the hair with average molecular weight 40-60KDa [47]. In the

terminology of past wool literature, the cystine rich fraction is termed γ-keratins with a

molecular weight range of 10-25KDa [47]. However, more recent publications state that the

cysteine rich fraction is more appropriately termed keratin associated proteins (KAP) and that

these matrix proteins in human hair are coded by 85 genes [41]. To add to the nomenclature

confusion, γ-keratin is often used to describe an acid-soluble fraction isolated after extraction of

oxidized keratin proteins, or so-called “keratose”. KAP can account for as much as 20-30% of

hair fiber proteins. The α-keratins assemble together to form keratin intermediate filaments

(KIF’s) that impart toughness to the fiber. The matrix proteins functions as disulfide cross linkers

that hold the KIF’s together. In humans, there are total 17 hair keratin genes – 11 type I and 6

type II [46] and more than 85 KAP genes [41].

Using chemical methods, soluble keratin proteins are extracted from the hair fiber by

breaking the disulfide crosslinks. If an oxidant is used cystine is converted to cysteic acid

derivatives and the product is termed “keratoses”, and if a reductant is used cystine is converted

to cysteine and the product is termed “kerateines”. Keratoses are hygroscopic, non-disulfide

cross linkable, water soluble, and susceptible to hydrolytic degradation at extreme of pH due to

polarization of the backbone caused by the electron withdrawing properties of cysteic acid.

These characteristics cause relatively quick degradation (days to weeks) of keratoses in vivo.

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Kerateines are less polar, more stable at extremes of pH and can be re-crosslinked through

oxidative coupling of cysteine groups. These can persist in vivo for weeks to months.

Hydrogel Treatments for Burns:

A burn dressing should serve three principle functions: comfort, metabolic and protective

[48]. They act as a protective barrier, absorb excess wound exudate, and maintain a moist

environment, which in turn helps in pain reduction. A moist wound environment is known to

accelerate wound healing [49-51], and there is abundant literature on various hydrogel

treatments. Collagen and chitosan hydrogels, and a combination of these two with other

hydrogels like alginate or dextran are the commonly used treatments. Collagen sheets were

introduced commercially in the 1960’s for the covering of large excised wound areas[52].

Research and variations soon followed, with one study showing that modified collagen

membrane was permeable to topical antibiotics with no significant antigenicity and was superior

to homograft and heterograft in autograft uptake [53]. In vitro experiments have shown that

human keratinocytes were able to grow extensively on human dermal collagen and differentiate

within a few days into columnar conformation [54]. Porous collagen sponges were able to

provide three dimensional matrix for tissue infiltration in vivo and also cell growth in vitro [55].

Collagen sheets were shown to promote proliferation and attachment of neonatal rat

keratinocytes in vitro and promote faster re-epithelialization when these sheets were used to

cover full thickness burns on the dorsum of rats [56]. More recently, collagen hydrogel has been

used synergistically with other hydrogel systems in the treatment of burns. Collagen and chitosan

porous scaffolds cross linked with glutaraldehyde have shown an increase in proliferation in

vitro, and when tested in animals have shown that the scaffold was able to support and accelerate

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11

fibroblast cell infiltration from surrounding tissue, suggesting its use as a potential dermal

equivalent [57]. Chitosan –collagen hydrogel incorporated with lysostaphin was shown to control

methicillin-resistant Staphylococcus aureus infection in third degree burn patients and promoted

healing [58].

Chitosan is a de-acetylated derivative of chitin that has been shown to facilitate burn

wound healing in vivo [59-67] and cell adhesion and viability in vitro in primary rat fibroblast

cells [59]. Full thickness burn wounds that were treated with high molecular weight, high de-

acetylated chitosan showed faster re-epithelialization and wound closure in rats compared to 2%

Fucidin treatment [68]. Chitosan hydrogel was also used as a carrier for biopharmaceuticals,

antimicrobials and growth factors. A chitosan gel formulation with epidermal growth factor

(EGF) was shown to significantly increase cell proliferation and had a faster re-epithelialization

rate in second degree burns in rats [60, 67]. It was also used as a fibroblast growth factor 2

(FGF2) carrier that induced angiogenesis and collateral blood circulation in impaired diabetic

mice [69]. In Wistar-Albino rats, Chitosan gels with controlled slow release granulocyte-

monocyte colony stimulating factor (GM-CSF) assisted burn wound healing with better collagen

fibril organization [70]. Chitosan acetate bandages were shown to control the growth of

Pseudomonas aeruginosa and Pseudomonas mirabilis bacteria, and control the development of

systemic sepsis in third degree burns in mice [64]. A hydrogel sheet composed of alginate,

chitosan and fucoidan was shown to promote wound healing in healing impaired rats [71].

Dextran hydrogel scaffolds were shown to promote early inflammatory cell infiltration in full

thickness burns, which led to the degradation of the scaffold and in turn promoted the infiltration

of endothelial cells into the wounded region. This enhanced neo-vascularization and restoration

of hair fibers and epidermal morphology similar to normal mouse skin [72].

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12

Alginates are polysaccharides derived from sea weed and there are a wide variety

available commercially that have been shown to promote wound healing [73-78]. Alginate

dressings are typically made from calcium alginate, which has cytotoxicity and immune

reactions associated with it. [79]. Development of alginate dressing with less calcium ions

reduced the cytotoxicity and foreign body reaction in both in vitro and in vivo experiments [79,

80]. Hydrocolloid dressings are used in partial thickness and small burns where the wound

exudate is comparatively less [81, 82]. Compared to hydrocolloid dressings, alginate dressings

remain gelled longer as the presence of calcium ion crosslinks decreases the biodegradability of

alginate gels [83]. The antimicrobial properties of the above mentioned alginate dressings have

been increased by the addition of silver ions [84-90].

Cell Death Pathways:

In burns, one of the main reasons for the increase in total body surface area (TBSA)

burned is the increase in cell death after the thermal insult. Apoptosis and Necrosis are the two

main modes of cell death that occur when the tissues are exposed to heat. There are two

apoptotic pathways – extrinsic or death receptor pathway, and intrinsic or mitochondrial pathway

(Figure 3). In both these pathways, cysteine aspartyl-specific proteases (caspases) are activated,

which further leads to the morphological and biochemical changes associated with apoptosis.

The death receptors usually belong to the tumor necrosis factor super family, comprising a

subfamily with a characteristic intracellular death domain [91]. When the ligands of the tissue

necrosis factor (TNF) family bind to their respective death receptors such as CD-95 and TRAIL-

R1, they are activated and attract intracellular Fas-associated death domain (FADD), which

further recruits caspases [92]. Caspase 8 and 10 function as initiator caspases that are recruited to

the death inducing signaling complex (DISC) [93]. The DISC cleaves the procaspase 8 and 10

______________________________________________________________________________

13

and yield active initiator caspases 8 and 10 [94]. Active caspase 8 cleaves BH3 interacting

domain (BID), a BCL2 family protein which translocates to mitochondria and initiates the

intrinsic pathway [95, 96]. Activation of mitochondria leads to the release of Cyt C into the

cytosol, which binds to apoptotic protease activating factor 1(APAF1) to form the apoptosome.

Apoptosome, along with TP, cleave procaspase 9 and activate it. Any of the BCL2 family

proteins: BAD, BID, BIM can activate the intrinsic pathway of apoptosis. Once the initiator

caspases are activated, they cleave and activate executioner caspases 3, 6 and 7, which cleave the

cellular substrates downstream [92]. Once the phosphatudylserine are exposed on the cell

membrane, the remains of the cell are engulfed by phagocytosis [97].

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14

Figure 3: Apoptotic Pathways: The 2 major pathways of apoptosis:the extrinsic (Fas and other

TNFR superfamily members and ligands) and the intrinsic (mitochondria-associated) pathways.

Both pathways lead to activation of caspase-3, giving rise to apoptotic cell death.(Used with

permission from Seminars from Arthritis and Rheumatism, Schultz DZ, Apoptosis: Programmed

cell death at a molecular level. Semin Arthritis Rheum. 2003 Jun;32(6):345-69)

There are some other cell death pathways that are not well defined and do not require caspase

activation [98-100]. Apoptotic pathways can be regulated at the initial receptor level, by

inhibiting caspase activation, or by influencing the permeability of the mitochondrial membrane.

Receptor level regulation is performed by FLIPs (FADD-like interleukin-1 -converting

enzyme-like protease (FLICE/caspase-8)-inhibitory proteins) [101]. The members of BCL2

family proteins regulate apoptosis at the mitochondrial level and may be either pro-apoptotic or

anti-apoptotic [95, 96]. Inhibitor of Apoptotic Proteins (IAPs) bind and inhibit activation of

caspases [102]. IAPs are inhibited by SMAC/DIABLO (second mitochondria-derived activator

of caspase/direct IAP binding protein with low pI), which is released from mitochondria along

with cyt c [103, 104].

Necrosis used to be considered a disorganized mode of cell death, especially when the

cells are exposed to severe physiochemical conditions. But recently it was shown that necrosis

occurs during normal physiological development [105, 106]. There are published in vitro studies

showing that even though apoptosis is completely inhibited, cell death still occurs and is usually

independent of caspases, following a specific signaling pathway where the cell morphology

looks more like necrotic cell death [107-111]. FADD (Fas-associated death domain) plays a

crucial role in propagating apoptotic or necrotic signals as it has both a death domain that can

trigger necrosis and a death effector domain that can propagate apoptosis [112, 113]. When

______________________________________________________________________________

15

caspase activation is impeded, FADD is not recruited when the TNF ligand binds to the death

receptor TRAIL-R1 [114-116]. Instead, a complex 1 is formed at the plasma membrane

consisting of TNF-R1, TRAF2 and RIP1 that activates NF-κB and MAPKs [117, 118]. After

receptor endocytosis, a second complex with TRADD that recruits FADD and procaspase 8 is

formed [119, 120]. If complex 1 is not able to induce sufficient anti-apoptotic proteins, then

caspase 8 is activated leading to caspase induced apoptosis [121]. If the anti-apoptotic proteins

block the caspases, then necrosis occurs through the caspase independent pathway [109]. Holler

et al. have shown that RIP is required for necrotic cell death induced by TNF and TRAIL, and

that it has its own kinase activity that activates the cell death pathway when phosphorylated

[122]. In Rip null (Rip–/–) mouse fibroblast cells, it is shown that RIP is required for TNF-

induced activation of the MAPKs extracellular-signal-related kinase (ERK), p38 and c-Jun

amino-terminal kinase (JNK) [123].

Autophagy refers to a catabolic process involving degradation of the cell’s own

components and is usually activated by oxidative, nutritional or toxic stresses. Macroautophagy

is an evolutionarily conserved, genetically controlled multi-step process in which intracellular

organelles are sequestered within characteristic double or multi-membrane autophagic vacuoles

termed autophagosome, and finally delivered to lysosomes for bulk degradation [124] (Figure 4).

Embryonic fibroblasts from double knock out Bax-/-

Bak-/-

mice are resistant to apoptotic

inducers. When treated with etoposide, the cells failed to undergo apoptosis and instead

manifested a massive autophagy followed by cell death [125]. But knocking down the autophagy

related gene (Atg) products beclin-1 and Atg5 reduced etoposide induced cell death [125]. Using

other apoptotic inhibitors did not trigger the autophagy cell death pathway, indicating that Bax

and Bak absence played an important role in autophagy activation [125] . In other cell types like

______________________________________________________________________________

16

L929 mouse fibrosarcome, Human Jurkat T lymphoma and U937 monocytoid cells, inhibition of

cystein proteases (caspases) was shown to induce autophagy [126, 127]. In stress conditions like

nutrition depletion or loss of growth factors, autophagy gets activated by the inhibition of

apoptosis and promotes cell survival [128, 129]. If autophagy is inhibited in cell stress conditions

then the cells usually die through apoptosis. This cell death can be postponed by inhibiting BAX

or BAK or caspases [130]. In vitro and in vivo experiments have shown induced apoptotic cell

death by the inactivation of Atg genes – Atg 5 or 7, beclin-1[131-134].

Figure 4: Autophagy and its inhibitors: Autophagy starts with the stepwise engulfment of

cytoplasmic material (cytosol and/or organelles) by the phagophore (also called isolation

membrane), which sequesters material in double-membraned vesicles named autophagosomes

(also called autophagic vacuoles). There are 4 regulatory process: (1)De‑repression of the

______________________________________________________________________________

17

mTOR Ser/Thr kinase (2)Activation of mammalian Vps34 in the initial steps of vesicle

nucleation (3)Two ubiquitin-like conjugation systems that are part of the vesicle elongation

process (4)Autophagosomes maturation by the fusion with lysosomes to create autolysosomes.

(Used with permission from Nature Publishing Group, Maiuri MC, Zalckvar E, Kimchi A,

Kroemer G Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev

Mol Cell Biol. 2007 Sep;8(9):741-52)

Bcl2 and Bcl-XL are regulators of beclin-1. Lamp2,lysosome-associated membrane

glycoprotein-2. Autophagy may develop as a primary response to stress stimuli, which then

triggers either apoptosis or necrosis [135, 136]. There is crosstalk between all the cell death

pathways, apoptosis, necrosis and autophagy, depending on the external and internal conditions

to which the cells are exposed.

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18

Table 1: Gene Glossary

Abbreviation Full Form Function

CD-95 Fas ligand

Cytokine that binds to TNFRSF6/FAS, a receptor

that transduces the apoptotic signal into cells.

TRAIL-r1

tumor necrosis

factor receptor

superfamily Receptor for the cytotoxic ligand TNFSF10/TRAIL.

FADD

Fas-associated death

domain

Apoptotic adaptor molecule that recruits caspase-8

or caspase-10 to the activated Fas (CD95) or TNFR-

1 receptors.

BID

BH3-interacting

domain death

agonist

This gene encodes a death agonist that

heterodimerizes with either agonist BAX or

antagonist BCL2.

Cyt C Cytochrome C

Electron carrier protein. Plays a role in apoptosis.

Suppression of the anti-apoptotic members or

activation of the pro-apoptotic members of the Bcl-2

family leads to altered mitochondrial membrane

permeability resulting in release of cytochrome c

into the cytosol. Binding of cytochrome c to Apaf-1

triggers the activation of caspase-9, which then

accelerates apoptosis by activating other caspases

APAF1

Apoptotic protease

activating factor 1

This gene encodes a cytoplasmic protein that

initiates apoptosis. This protein contains several

copies of the WD-40 (beta transducin)domain, a

caspase recruitment domain (CARD), and an

ATPase domain (NB-ARC).

BAD

BCL2-associated

agonist of cell death

Promotes cell death. Successfully competes for the

binding to Bcl-X(L), Bcl-2 and Bcl-W, thereby

affecting the level of heterodimerization of these

proteins with BAX. Can reverse the death repressor

activity of Bcl-X(L), but not that of Bcl-2 (By

similarity). Appears to act as a link between growth

factor receptor signaling and the apoptotic pathways

BIM BCL2-like 11 Induces apoptosis.

Caspases

3,6,7,8,10

cysteinyl aspartate

proteases

Caspases are involved in the signal transduction

pathways of apoptosis, necrosis and inflammation.

Initiator caspases include Capases 1,-4,-5,-8,-9,-10,-

11,-12 and effector caspases include Caspase -3,-6,-7

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19

FLIP

FADD-like

interleukin-1 -

converting enzyme-

like protease

(FLICE/caspase-8)-

inhibitory proteins

Apoptosis regulator protein which functions as a

crucial link between cell survival and cell death

pathways in mammalian cells.

SMAC,

DIABLO

second

mitochondria-

derived activator of

caspase/direct IAP

binding protein with

low pI

Promotes apoptosis by activating caspases in the

cytochrome c/Apaf-1/caspase-9 pathway.

TRAF2

TNF receptor-

associated factor 2

Regulates activation of NF-kappa-B and JNK and

plays a central role in the regulation of cell survival

and apoptosis.

RIP1

receptor (TNFRSF)-

interacting serine-

threonine kinase

Serine-threonine kinase which transduces

inflammatory and cell-death signals (necroptosis)

following death receptors ligation.

NF-kB

nuclear factor

kappa-light-chain-

enhancer of

activated B cells

NF-kappaB (nuclear factor-kappa B) is a rapidly

acting primary transcription factor involved in

cellular responses to stimuli such as cytokines and

stress and plays a key role in regulating the immune

response to infection.

TRADD

TNFRSF1A-

associated via death

domain

Adapter molecule for TNFRSF1A/TNFR1 that

specifically associates with the cytoplasmic domain

of activated TNFRSF1A/TNFR1 mediating its

interaction with FADD. Overexpression of TRADD

leads to two major TNF-induced responses,

apoptosis and activation of NF-kappa-B

TNF

Tumor Necrosis

Factor

Cytokine that binds to TNFRSF1A/TNFR1 and

TNFRSF1B/TNFBR.

ERK

MAPKs

extracellular-signal-

related kinase

Involved in both the initiation and regulation of

meiosis, mitosis, and postmitotic functions in

differentiated cells by phosphorylating of

transcription factors

P38, JNK,

MAPK9

p38, c-Jun amino

terminal kinase,

Mitogen activated

rotein kinase

Three major MAPKs include ERKs (Extracellular

signal-Regulated Kinases), JNKs (c-Jun NH(2)-

terminal protein Kinases), and p38 Kinases. These

are activated by environmental stresses and

inflammatory cytokines

Beclin-1 autophagy-related Plays a central role in autophagy.

______________________________________________________________________________

20

ATG5

autophagy related 5

homolog

Required for autophagy. Its expression is a relatively

late event in the apoptotic process, occurring

downstream of caspase activity.

ATG7

autophagy related 7

homolog

Functions as an E1 enzyme essential for multi

substrates such as ATG8-like proteins and ATG12.

BAX, BAK1

Bcl-2-associated X

protein

Accelerates programmed cell death by binding to,

and antagonizing the apoptosis repressor BCL2.

Bcl2 B-cell lymphoma-2

Potent inhibitor of cell death. Inhibits activation of

caspases.

Bcl-XL

B-cell lymphoma-

extra large Positive regulator of apoptosis

Lamp2

Lysosomal-

associated

membrane protein 2 Implicated in tumor cell metastasis.

______________________________________________________________________________

21

Rationale for the hypothesis that the interaction of keratin biomaterial with burned tissue

promotes cell survival and will reduce total body surface area (TBSA) burned:

Previous work in our group has focused on testing the wound healing capacities of keratin

biomaterials in animals. Two wound healing burn studies were performed – a mouse chemical

burn study and a swine thermal burn study. Both of the animal studies have shown that keratin

biomaterials promote tissue salvage after burn injury and speed healing. Generally, hydrogel

treatments are able to provide a moist environment, help control exudation of wound fluid, which

in turn reduces pain. As previously discussed, there are many hydrogel treatments available for

burn patients, but none of those treatments were able to show that they facilitate cell survival and

tissue salvage by any of the aforementioned pathways. From our previous wound healing studies,

it was shown that a hydrogel made from a “crude” preparation of keratose (i.e. not otherwise

refined or purified) was able to reduce TBSA burned and accelerate wound healing. As crude

keratose is a heterogeneous mixture of alpha, gamma and KAPs, we hypothesized that separating

the different fractions and testing their cell salvage abilities would be helpful in the development

of better keratin formulations for the treatment of burns. To test this hypothesis, three specific

aims were undertaken and form the basis of this thesis project:

Specific Aim 1: To develop an in vitro thermal injury model that mimics the Jackson model [4]

of burn injury and determine the effect of keratin subtype on cell survival and recovery from

thermal injury.

Specific Aim 2: To understand the molecular mechanism by which keratose biomaterial

promotes cell survival.

Specific Aim 3: To test the wound repair capabilities of meta-keratose in a partial thickness pig

burn injury model.

______________________________________________________________________________

22

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CHAPTER 2

_____________________________________________________________________________________

A KERATIN BIOMATERIAL PROMOTES SKIN REGENERATION

AFTER BURNS IN VIVO AND RESCUES CELLS AFTER THERMAL

STRESS IN VITRO

Poranki Da, Whitener W

a, Howse S

a, Mesen T

a, Howse B

a, Burnell J

a, Greengauz-Roberts O

a,

Molnar Jb, Van Dyke M

a

aWake Forest School of Medicine, Wake Forest Institute for Regenerative Medicine

bDepartment of Plastic and Reconstructive Surgery

Wake Forest School of Medicine, Medical Center Boulevard, Winston Salem, NC 27157

The following manuscript has not yet been submitted for publication. Deepika Poranki prepared

the manuscript. Dr. Mark Van Dyke acted in an advisory and editorial capacity.

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38

Abstract:

Thermal burns typically display an injury pattern dictated by the transfer of the thermal

energy into the skin and underlying tissues. There are often three zones of injury represented by

a necrotic zone of disrupted cells and tissue, an intermediate zone of injured and dying cells, and

a distant zone of stressed cells that will recover with proper treatment. Burn treatment is directed

toward removal of clearly necrotic skin and therapeutic support for remaining tissue that may

spontaneously heal without complication. Excised areas of the wound then require grafting.

Decisions regarding when and how much damaged tissue to excise are largely determined

empirically, and there is substantial debate about the relative merits of early or late excision and

grafting. Early grafting carries the risk of removing too little tissue, as cells in the intermediate

zone continue to die up to 10 days after injury. Late excision can expose the patient to many

inflammatory mediators produced by the dead and dying tissue. Strategies to facilitate the

survival of cells and tissue in the intermediate zone of burn injury may be capable of mediating

spontaneous healing, or reducing the overall extent of excision and grafting, but to date no such

therapies are available for clinical use.

The wound healing capabilities of a keratin biomaterial hydrogel were studied in two

pilot studies, one using a chemical burn model in mice and the other a thermal burn model in

swine. In both studies, an oxidized form of keratin biomaterial called “keratose” was shown to

prevent enlargement of the initial wound area and promote faster wound closure. This keratose

hydrogel was provided as a heterogeneous mixture of proteins representing an “alpha” fraction

and a “gamma” fraction. These different fractions of keratin have disparate properties including

molecular weights, amino acid content, and solubility characteristics. They can be easily

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39

separated by techniques such as dialysis or isoelectric precipitation. To determine if there was a

differential wound healing activity associated with these two keratose fractions, an in vitro heat

stress model mimicking the different zones of burn injury was developed. Treating thermally

stressed dermal fibroblast with growth media containing alpha, gamma or the native mixture

(termed “crude keratose” throughout this manuscript) showed that gamma keratose was able to

maintain cell viability and promote cell proliferation. By the third day of treatment, the cell

viability for gamma keratose-treated cells was 125% ± 15.6, whereas with other treatments the

cell viability was 65% ± 20.7 (crude), 46% ± 32.5 (alpha) and 37% ± 20 (fibroblast media).

Electrophoretic separation of the gamma keratose fraction revealed sub-fractions of

different molecular weights ranging from 110kDa to 8kDa. Mass spectrometry analysis

identified keratins 81, 83, 85, 86, 31, 33A, 33B, 34 in the excised bands. Epithelial keratins 1 and

10 were identified in the excised bands with molecular weights ~60 kDa and ~8 kDa,

respectively. Keratins 81, 83, 85, 86, 31, 33A, 33B and 34 were also identified with lower than

expected molecular weights, suggesting that these were degraded alpha keratose peptides.

Keratin-associated protein 19-5, galectin-7, calmodulin-like protein 3, thioredoxin, and cystatin

A were also identified in the ~8 kDa excised band.

These results suggest that the gamma fraction of keratose is comprised essentially of

degraded alpha keratose proteins, and that these peptides may facilitate the rescue of cells from

thermal injury. Treatment of burns with gamma keratose may therefore represent a potential

therapy for wounds with an intermediate zone of damaged tissue that has the potential to

contribute to spontaneous healing.

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40

Keywords: keratin, biomaterial, burn, wound, heat shock, hydrogel, keratose, alpha keratin,

gamma keratin, proteomics, mass spectrometry

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41

1. Introduction:

Burn wound repair is a complex process with a series of overlapping phases:

Inflammation; matrix deposition; and tissue remodeling. The duration of these individual phases

varies depending on the intensity and depth of the burn. Most burn dressing treatments aim to

facilitate these stages of wound healing by providing a moist environment, control excessive

exudate buildup, and protect against infection that would perturb normal healing. It has been

suggested that a burn dressing should serve three principle functions: Provide comfort by

protecting the wound surface from external air currents; absorb wound secretions and reduce

water loss from exposed burns; and protect from microorganisms [1]. In recent years, hydrogel

wound dressings have drawn increased attention as they serve all of these purposes. They act as a

protective barrier, absorb excess wound exudate, and maintain a moist environment, which in

turn helps in pain reduction. A moist wound environment is known to accelerate wound healing

[2-4].

There is abundant literature on various hydrogel treatments, among them chitosan,

alginate, collagen and hydro-colloid dressings that have been shown to promote burn wound

healing. Chitosan is a de-acetylated derivative of chitin that has been shown to promote burn

wound healing in vivo [5-13]. Chitosan did not appear to have a detrimental effect on primary rat

fibroblast viability in vitro [5]. In another study, full thickness burn wounds treated with high

molecular weight, highly de-acetylated chitosan showed faster re-epithelialization and wound

closure in rats compared to 2% Fucidin treatment. [14]. Chitosan hydrogel has also been used as

a carrier for biopharmaceuticals, antimicrobials and growth factors. Chitosan gel formulation

with epidermal growth factor (EGF) was shown to significantly increase cell proliferation and to

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42

promote faster re-epithelialization in second degree burns in rats [6, 13]. It was also used as a

fibroblast growth factor 2 (FGF2) carrier that induced angiogenesis and collateral blood

circulation in impaired diabetic mice [15]. Another study using pluronic/chitosan hydrogels with

recombinant human EGF enhanced epidermal cell differentiation in second degree burns in mice

with impaired wound healing [16]. Also, chitosan gels with controlled slow release granulocyte-

monocyte colony stimulating factor (GM-CSF) assisted in better collagen fibril organization in

Wistar-Albino rats [17]. Chitosan acetate bandages were shown to control the growth of

Pseudomonas aeruginosa and Pseudomonas mirabilis and to prevent the development of

systemic sepsis in mice with third degree burns [10]. Despite these positive findings, chitosan

has no apparent utility beyond providing a moist wound healing environment and a physical

barrier, and typically is combined with another therapeutic to enhance its efficacy.

Wound dressings containing alginate, a polysaccharide derived from sea weed, have also

been investigated. In 1987, Barnett and coworkers showed that a calcium alginate dressing was

an effective hemostat and facilitated wound repair [18]. A wide variety of alginate dressings are

available commercially that promote wound healing by providing a moist environment [19-24].

However, like chitosan, alginates have no intrinsic capacity for facilitating wound healing

beyond their physical attributes. Moreover, a disadvantage of alginate dressings containing

calcium alginate is that some cytotoxicity and immune reactions have been noted [25].

Development of alginate dressings containing less calcium has reduced the cytotoxicity and

foreign body reaction in experiments in vitro and in vivo [25, 26], but there are no market-

leading burn dressings based on alginate biomaterials.

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43

Dressings containing collagen have also been used in the treatment of burns. They

provide a moist wound environment and act as a barrier to microorganisms. Collagen dressings

have been shown to promote faster re-epithelialization, early granulation tissue formation [27-

29], and when used as a composite wound dressing along with other biomaterials or antibiotics,

were shown to promote faster wound healing with a reduced rate of infection [30-32].

Lyophilized bovine collagen has been shown to help in the management of pain and faster

epithelialization of skin graft donor sites [33]. However, these findings do not differ substantially

than those for other hydrogel biomaterials and have no specific efficacy for burn wound healing.

An additional limitation to the use of collagen dressings for burn wounds is that the environment

of a burn is highly proteolytic, and protein-based dressings such as collagen can breakdown

rapidly [34].

Hydrocolloid dressings are primarily used in partial thickness burns and small burns with

less wound exudate [35, 36]. In their intact state hydrocolloid dressings are impermeable to water

vapor and provide an effective barrier to transepidermal moisture loss, whereas in presence of

wound exudate the dressings absorb the liquid and form gel, making them permeable to vapor

[36]. Hydrocolloid dressings along with silver sulfadiazine have been shown to promote faster

wound closure and also help in pain management [37]. The principal benefit of using these

dressings for burns is pain management.

Recognizing the limited functionality of the above mentioned dressings, additional

enhancements have been made by commercial vendors of these products. Prominent among them

is the addition of antimicrobial properties by the addition of silver ions [37-43]. However, the

underlying limitation of all of these products is that they have no particular efficacy for burn

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44

wounds and the complex physiology of skin that has been damaged by the application of external

energy (thermal and electrical) or agents (chemical). The opportunity therefore exists to develop

new treatments that can specifically address the pathophysiology of burns.

Keratin biomaterial hydrogels may represent a burn-specific wound dressing technology.

In addition to possessing the physical attributes for a wound dressing (facilitates a moist

environment, protects the wound, absorbs excess wound fluid, etc.), keratins may facilitate the

survival of thermally and chemically damaged cells. Keratins represent a class of biomaterials

that have been used in a variety of biomedical applications, but the understanding of how keratin

proteins function in these applications is only at its nascent stages.

The major proteins contained in human hair fibers are classified as alpha keratins [44],

beta keratins and keratin-associated proteins (KAP) [45]. They can be extracted using oxidative

or reductive chemical techniques, and separated using various methods into purified fractions

with different molecular weights, amino acid content, and solubility characteristics [46]. Another

fraction derived from the oxidative extraction of these proteins from hair fibers is often referred

to in the literature as “gamma” keratin [47].

The general biocompatibility and utility of keratin biomaterials in potential medical and

biotechnology applications has been demonstrated in numerous studies showing proof of

principle for cell culture, wound healing, bone and nerve regeneration, drug delivery, hemostasis,

corneal tissue engineering, cardiac repair, and wound healing [48-61]. Recently, keratin

dressings were shown to enhance wound healing by the activation of keratinocytes in a porcine

partial thickness burn model [54]. However, no data was provided on the type of keratin used in

these dressings, or the underlying mechanism of wound repair. Given this body of evidence, we

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45

hypothesized that keratose hydrogels would facilitate both chemical and thermal burn repair, and

that there may be differential efficacy among the different keratose fractions present in a mixed

protein hydrogel. To test this postulate, we utilized a mouse chemical burn model and a swine

thermal burn model to conduct pilot studies using a hydrogel prepared from an un-purified

sample of crude keratose extract. We then investigated the differential effect of alpha and gamma

keratose fractions on cell survival in an in vitro model of thermal insult.

2. Materials and Methods:

2.1. Keratin Biomaterial Extraction: Keratin biomaterial was extracted from hair using an

oxidative protocol described by de Guzman et al. [62]. This heterogeneous mixture of

cortical proteins is referred to as crude keratose. For crude keratin hydrogel preparations,

the extract solution was dialyzed against endotoxin free water using a 5kDa spiral wound

cartridge (Millipore, Billerica, MA) for 5 volume washes. Finally, the solution was

concentrated to minimal volume, adjusted to pH 7.4 and lyophilized.

2.2. Separation of Alpha and Gamma Keratose fractions: Crude keratose was further

separated into alpha and gamma keratose fractions by isoelectric precipitation.

Concentrated hydrochloric acid was added to the crude keratose solution with stirring

until the pH dropped to 4.2, facilitating the precipitation of the insoluble alpha keratose.

The soluble gamma keratose was separated by fixed angle centrifugation at 1,500 rpm

(252 RCF) for 15min at 4ºC and filtered. After neutralizing the gamma solution to pH

8.5, it was dialyzed against endotoxin free water using a 5kDa spiral wound cartridge

(Millipore) for 5 volume washes. Finally, the solution was concentrated to minimal

volume, adjusted to pH 7.4 and lyophilized. The precipitated alpha keratose was

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46

reconstituted to a concentrated solution using a minimum of 0.1M sodium hydroxide and

then dialyzed against endotoxin free water using a 30kDa spiral wound cartridge

(Millipore) for 5 volume washes. Finally the solution was concentrated to minimal

volume, adjusted to pH 7.4 and lyophilized.

2.3. Hydrogel Preparation: Crude keratin hydrogel was prepared by reconstituting the

lyophilized powder at 15 percent by weight with normal saline. It was not sterilized

prior to use. Chitosan (Sigma-Aldrich) was similarly prepared by reconstituting the dry

powder at 9 weight percent with saline. Chitosan also was not sterilized prior to use.

Gels were loaded into syringes so that they could be accurately applied to the wounds.

2.4. Mouse Chemical Burn Model: Forty eight CD1 mice were used for this study under a

protocol approved by the Wake Forest University Institutional Animal Care and Use

Committee (WFU IACUC). A few minutes prior to the surgery and under general

anesthesia (2-3% halothane by nose cone inhalation), hair at the dorsal mid-line and

below the shoulders of the mice was removed with a commercial depilatory cream. The

area of exposed skin was washed with soap and water, then disinfected with iodine and

cleaned with saline soaked gauze. Chemical burns were created with 90% phenol in a 1

cm x 2 cm area of skin exposed for 60 sec. Phenol was removed with copious amounts

of phosphate buffered saline. Twenty minutes after creation of the phenol burns, the

treatments were applied, covered with a commercial adhesive bandage, and the animals

placed in a protective jacket to keep the dressings in place. Treatments included crude

keratose hydrogel, chitosan hydrogel, and saline only. Dressings were changed every 2

days for 20 days. Digital pictures were taken with a ruler in the field and wound size

measurements made using digital image analysis software (ImageJ, NIH, Bethesda,

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47

MD). Wound size values for each treatment group were averaged (e.g. n=12 on day 0,

n=11 on day 2, n=10 on day 4, etc.) and reported as mean ± standard error of the mean

(SEM).

2.5. Swine Thermal Burn Model: Four Yorkshire swine were used for this study under a

protocol approved by the WFU IACUC. Anesthesia was introduced intramuscularly with

ketamine (20mg/kg), xylazine (2.2mg/kg), acepromazine (1mg/kg) and then maintained

by nose cone with 2-3% halothane throughout the surgery and during dressing changes.

Several minutes prior to producing the burn, the animals were shaved and washed with

soap and water. Thermal burns were created using 3.5 cm diameter heated brass blocks

with a contact time of 12 sec [63]. Sixteen such wounds were created, eight on each side

of the dorsal mid-line between the shoulders and hip. Treatments were applied within 30

minutes after burn creation and included crude keratose hydrogel, chitosan hydrogel or

saline soaked gauze. Treatment areas were covered with dry gauze followed by an

occlusive dressing (adhesive Ioban), a cotton stocking, a plastic saddle over the back,

and a nylon jacket to keep all dressings in place. Dressing changes were performed on

anesthetized swine every 3 days. Digital pictures were taken with a ruler in view and

wound size measurements made using digital image analysis software (ImageJ). Wound

size values for each treatment group were averaged (e.g. n=16 on days 0, 3, 6, and 9;

n=12 on days 12 and 15; n=8 on day 18; n=4 on days 21, 24, and 27) and reported as

mean ± SEM.

2.6. In Vitro Thermal Injury Model:

2.6.1. Primary Mouse Skin Fibroblast Isolation: Primary mouse fibroblasts were

isolated from adult CD1 mouse ear pads. The ear pads were incubated in Dispase II

______________________________________________________________________________

48

overnight and then the dermis was peeled off using forceps and minced before

incubating in 0.35% type I collagenase reconstituted in DMEM high glucose for 30

min at 37˚C on a shaker. The suspension was filtered using a 100 micron mesh filter

and cells pelleted by centrifugation. The cell pellet was washed once with Hank’s

balanced salt solution (HBSS) to remove the residual collagenase. Finally, the

fibroblasts were seeded in a 150 mm tissue culture plastic (TCP) dish at 2.5 x 106

cells/mL in fibroblast media [DMEM high glucose plus 10% Fetal Bovine Serum

(Hyclone® Laboratories, Inc., Logan,Utah) and 1% Antibiotic/Antimycotic solution

(Hyclone® Laboratories, Inc.)].

2.6.2. In Vitro Thermal Stress Method: Primary mouse fibroblasts from passages 3 to

6 were used for these experiments. Fibroblasts were seeded in the range of 1.83 x

105

to 2.36 x 105cells/mL in a 100 mm TCP dish. The cells were grown in fibroblast

media until 95% confluent. Media changes were performed every 3 days. For a

thermal injury experiment, a media change was performed one day prior to heating

the cells. On the day of the experiment, fibroblasts were moved from a 37˚C, 5%

CO2 incubator to a 44˚C, 5% CO2 incubator for 60 minutes. After the thermal stress,

culture dishes were moved back to a 37˚C, 5% CO2 incubator to allow for

reattachment of the cells and for the media temperature to equilibrate. Six hours

after heat exposure, various treatments were applied in fresh media. Treatments

included 0.01mg/mL crude keratose, 0.001mg/ml gamma keratose, 0.001mg/ml

alpha keratose and fibroblast media. All keratose samples were sterilized by gamma

irradiation at 1 MRad before adding to the media. This experiment was performed

______________________________________________________________________________

49

with three replicates. All the data were normalized to 0 hours (before heat shock)

and reported as mean ± standard error of the mean (SEM).

2.6.3. Trypan Blue Assay: Equal amounts of cell suspension and 0.4% trypan blue

(Invitrogen, Gibco, Grand Island, NY) were mixed together and stained cells were

counted using a hemocytometer. Live cells with intact cell membrane excluded the

trypan blue dye whereas dead cells retained the dye and were blue.

2.7. Detection and Identification of Gamma Keratose proteins: Proteins associated with

the gamma keratose fraction were separated by sodium dodecyl sulfate polyacrylamide

gel electrophoresis (SDS-PAGE) and stained protein bands were recovered and analyzed

by mass spectrometry as previously described [62].

2.8. Statistical Analysis: Two-way analysis of variance (ANOVA) and Bonferroni post-hoc

analyses were performed using Graph pad Prism at 95% confidence intervals for both

the in vivo and in vitro studies.

3. Results:

3.1. Mouse Chemical Burn Study: Compared to the other treatments, keratose hydrogel

treated burns showed no increase in wound size over the first four days post-injury and

faster wound closure. There was a statistically significant reduction in wound area with

keratose treatment relative to either chitosan or saline at days 4 through 16 (Figure 1).

Digital pictures of representative burn wounds at day 0, 8 and 16 showed faster wound

healing progression and closure (Figure 2). A well-defined eschar (sloughed skin) was

observed early in the keratose treated wound at day 8 compared to day 16 for the

chitosan and saline soaked gauze treatments (occlusive dressing).

______________________________________________________________________________

50

3.2. Swine Thermal Burn Pilot Study: Compared to the other treatments, keratose hydrogel

treatment again showed no increase in burn wound size and resulted in faster wound

closure (Figure 3). There was a statistically significant reduction in wound area at days

3, 6, and 12 in the keratose treated group compared to both chitosan and saline. Digital

pictures of representative burn wounds at day 3, 9 and 24 shows wound healing

progression and faster wound closure (Figure 4).

3.3. In Vitro Thermal Stress Model: Control cultures were examined for their ability to

mimic the phases of heat induced cell damage. Immediately after heat stress, 40-50%

cell death was noted by cell counting using trypan blue staining. Further cell death was

evident in the following 24 hours if proper treatment was not provided. After 7 days,

cultures grew back to a confluent state (Figure 5). Using this in vitro heat injury model,

different keratose treatments were compared to each other and to control treatment (i.e.

fibroblast growth media containing serum). Fibroblasts that received a single treatment

of gamma keratose at 0.001mg/mL dissolved in the fibroblast media and were able to

maintain their viability, whereas all other treatments resulted in additional cell death

between the time of treatment (6 hours) and 24 hours. Even by day 3, the other

treatments were only able to maintain their post-treatment cell numbers [65% ± 20.7

(crude), 46% ± 32.5 (alpha) and 37% ± 20 (fibroblast media)], while the gamma keratose

treated fibroblasts had 125% ± 15.6 cell viability (Figure 6). The gamma keratose

treatment resulted in statistically significant cell growth compare to all other treatments

at day 3, and compared to the alpha keratose treatment at day 5.

3.4. Identification and separation of gamma keratose proteins: Electrophoretic separation

of gamma keratose revealed proteins of different molecular weights ranging from

______________________________________________________________________________

51

110kDa to 8kDa. Protein bands at 110 kDa, 40-60 kDa, ~30 kDa, ~15-20 kDa, and ~8-

10 kDa that were excised for mass spectrometry analysis were identified as containing

keratins 81, 83, 85, 86, 31, 33A, 33B, 34 in all the excised regions. Epithelial keratins 1

and 10 were identified in the excised regions with molecular weight 60kDa and 8kDa.

The excised region at ~110 kDa band was identified as alpha keratin heterodimers,

which were previously reported by de Guzman et al [62]. Keratin-associated protein 19-

5, galectin-7, calmodulin-like protein 3, thioredoxin, and cystatin A were identified in

the band at ~8 kDa (Figure 7). Mass spectrometry analysis of the excised bands with

protein identities, molecular weights and protein scores is provided as supplementary

data (Tables 1-6).

4. Discussion:

Hydrogels are gaining more interest as wound dressings, especially for the treatment of

burns, ulcers, and other necrotic wounds [64]. They have inherent properties such as the ability

to absorb wound exudate and provide a moist wound environment that enhances healing [3, 64].

Keratin proteins provide a readily available, inexpensive biomaterial hydrogel that has been

shown to be beneficial for regenerating tissues [51, 54, 56-58, 65-67]. Recently, a keratin

biomaterial extracted from wool was shown to promote faster re-epithelialization in deep partial

thickness burns [54]. Moreover, keratose, the oxidized form of keratin protein extracted from

hair and wool fibers, was previously purported to be beneficial for wound healing [68].

However, neither of these accounts attempts to describe a plausible mechanism for improved

wound healing, nor do they describe the keratin in sufficient detail to provide much insight into

its structure or composition. The ability of a hydrogel made from a structural protein such as

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52

keratin to provide beneficial wound healing is not surprising, given the general nature of

hydrogels and the proven efficacy of providing a protected wound environment where moisture

can be controlled. However, in this study, we sought to demonstrate the particular efficacy of a

keratose biomaterial hydrogel for the treatment of chemical and thermal burns, then investigate a

potential mechanism of cell rescue that was tied to a specific fraction of keratin biomaterial that

has until now, been uncharacterized in the literature. In both pilot animal studies, a crude form of

keratose was used that contained both gamma and alpha fractions. While this mixture of proteins

was efficacious in both models, we were not able to determine if either of these fractions

individually or synergistically produced differential effects on the damaged tissues. To better

understand the relative contributions of these different forms of keratin biomaterial, a heat stress

model that mimics thermal injury in vivo was developed.

The injury to tissue that results from thermal insult was first described by Jackson [69].

According to Jackson’s burn model, burn injury is characterized by three zones: 1) the inner zone

of coagulation, which is characterized by necrotic tissue, 2) an intermediate zone of stasis, which

is characterized by damaged cells that may live or die depending on treatment and other factors,

and 3) an outer zone of hyperemia where cells are stressed but will likely survive if the wound

does not get infected and standard treatment is given [69]. Similar to Jackson’s model, the heat

stress culture system was optimized to produce a population of cells that died from necrosis

during the heat treatment phase, a second population of cells that died from apoptosis in the

hours following thermal injury, and a third population that recovered and could re-establish the

culture. Using this model, we were able to show that gamma keratose was better able to maintain

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53

cell viability compared to crude keratose, alpha keratose, and fibroblast growth media. By day 5,

gamma keratose treated fibroblast growth was so robust that the culture was over-confluent.

Heat stress models have been used previously to isolate cells into a more simplistic

system and study molecular mechanisms of damage and survival [70]. In this study we not only

showed that a biomaterial made from oxidized keratin protein can improve fibroblast survival,

but that this phenomenon is associated only with the gamma keratose fraction. As this is the first

study to show that this keratose fraction has particular efficacy for cell salvage after heat stress,

we performed mass spectrometry analysis to understand the composition of proteins present in

the gamma keratose fraction. Mass spectrometry analysis of the excised bands showed that they

contain keratins 81, 83, 85, 86, 31, 33A, 33B, and 34. Because molecular weights between 26

and 46 kDa do not correspond to any known, intact human hair alpha keratin or KAP, and

because there are no alpha keratin proteins with molecular weights below 46kDa, we conclude

that all proteins identified by mass spectroscopy in the 30kDa, 15-20kDa, and 8kDa

electrophoretic bands must represent fragments of degraded alpha keratose proteins. In much of

the early published literature on keratins, the gamma fraction has been described as being

derived from the cortical matrix [71], and this concept has become dogma in the current keratin

biomaterials field. However, given the oxidation and extraction conditions employed in our

protein isolation method, it is not surprising that the generation of peptide fragments occurs,

especially when considering recent evidence that shows a number of weak bonds in alpha keratin

proteins [72].

The concept that fragments of larger proteins have a biological function different than

that of the parent molecule is not new. Extra cellular matrix (ECM) derived from various tissues

______________________________________________________________________________

54

contain growth factors that promote tissue remodeling and angiogenesis [73]. Badylak et al.

showed that bone marrow derived stem cells actively participated in the remodeling of ECM

scaffolds in vivo. They also suggested that portion of these bone marrow derived stem cells

might have participated in angiogenic response [74]. In another study, it was shown that a low

molecular weight peptide fraction of ECM (5-16kDa) has chemoattractant properties for mature

endothelial cells both in in vitro and in vivo assays. This low molecular weight peptide fraction

was derived from small intestine sub mucosa ECM. The ECM containing structural proteins

(collagens, proteoglycans, glycoproteins, glycosylaminoglycans) and growth factors was reduced

to particulate form and further separated into seven fractions based on molecular weight. These

biologically active peptides were not purified or characterized completely but have been shown

to attract endothelial cells that might have promoted angiogenesis when ECM scaffolds were

implanted in vivo [75].

It has been shown that proteolytic degradation exposes cryptic sites on resulting peptide

fragments, imparting biological function that was not possessed by the intact protein [76].

Understanding more about the composition and structural nature of gamma keratose and the

active peptide region(s) that may be responsible for promoting survival in thermally stressed

cells may assist in the development of better keratin-based treatments for burn injuries.

5. Conclusions:

Pilot burn studies in mice and pigs have identified the potential for a keratose biomaterial

to spare burned tissue and facilitate skin regeneration. A closer examination of this observation

has revealed that gamma keratose, identified through proteomic techniques as peptide fragments

cleaved from the structural alpha keratins during extraction, were primarily responsible for this

______________________________________________________________________________

55

phenomenon. The gamma keratose fraction was better able to rescue cells after thermal insult in

an in vitro model system and therefore may represent a potential therapy for burn injury.

Conflict of interest statement

Mark Van Dyke, Ph.D. holds stock and is an officer in the company, KeraNetics LLC, which has

provided partial funding for this research. Wake Forest Health Sciences has a potential financial

interest in KeraNetics, LLC through licensing agreements.

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56

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Figures:

Figure 1. Mouse Chemical Burn Wound Area

Digital Image analysis shows that crude keratose hydrogel treatment of chemical burns results in

a significant difference in wound area compared to saline and chitosan hydrogel control

treatments at days 4 through 16 (indicated by asterisks).

0 2 4 6 8 10 12 14 16 18 200.0

0.2

0.4

0.6

0.8

1.0

1.2

Saline

Chitosan

Keratose* ****

Day

Norm

alized W

ound S

ize

______________________________________________________________________________

66

Figure 2. Digital Images from Mice Chemical burn study

Digital pictures of representative burn wounds at day 0, 8 and 16 show enhanced wound healing

and closure with keratose treatment compared to chitosan or saline (occlusive dressing).

______________________________________________________________________________

67

Figure 3. Swine Thermal Burn Wound Area

Digital Image analysis shows that crude keratose hydrogel treatment of thermal burns results in a

significant difference in wound area compared to saline and chitosan hydrogel control at days 3,

6 and 12.

0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0

1.2

Saline

Chitosan

Keratose* *

*

Day

Norm

alized W

ound S

ize

______________________________________________________________________________

68

Figure 4. Digital Images from Swine thermal burn study

Digital pictures representing burn wounds at day 3, 9 and 24 shows wound healing progression

and faster wound closure.

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Figure 5. In Vitro Thermal Stress Model

An in-vitro heat shock model was developed that produced three populations of cells: necrotic,

apoptotic, and survivable. This graph represents the growth curve of the survivable cells treated

with fibroblast growth media after heat treatment at 44oC for 60 minutes.

0

20

40

60

80

100

120

140

160

0hr 8hr 1d 3d 5d 7d

% C

ell

Via

bil

ity

Time Point

% Cell Viability

Average

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Figure 6. Percent Cell Viability Curve

Gamma keratose treated cells did not show a characteristic drop in viability at 24hrs and were

able to grow to 125% ± 15.6 confluency by day 3, whereas with other treatments the fibroblasts

grew to only 65% ± 20.7 (crude), 46% ± 32.5 (alpha) and 37% ± 20 (Fibroblast Media) of pre-

treatment levels. P<0.001 - ***, P<0.01- **, P<0.05 - *

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Figure 7. Keratose SDS PAGE

Mass spectrometry analysis of gamma keratose identified keratin 81, 83, 85, 86, 31, 33A, 33B,

34 in all the excised regions. Epithelial keratins – 1 and 10 were identified in the excised regions

with molecular weight 60 kDa and 8 kDa. Keratin-associated protein 19-5, galectin-7,

calmodulin-like protein 3, thioredoxin, and cystatin A were also identified in the ~8 kDa excised

band.

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Supplementary Mass Spectrometry Analysis Data:

Supplementary Table 1: Band # 1, Molecular Weight ~110 KDa

UniProtKBAccession Protein/Gene ID Score Molecular Weight #pep/cover%

P13645 Keratin, type I KRT10 1897 59703 82/48.7

P04264 Keratin, type II KRT1 1878 66149 87/37.4

P78385 Keratin, type II KRT83 1709 55928 107/36.5

Q14533| Keratin, type II KRT81 1704 56875 110/35.6

O43790 Keratin, type II KRT86 1672 55120 109/37.0

Q15323 Keratin, type I KRT31 1619 48693 99/49.5

P78386 Keratin, type II KRT85 1598 57306 90/33.7

Q14525 Keratin, type I KRT33B 1527 47325 73/50.0

O76009 Keratin, type I KRT33A 1481 47166 77/50.0

P35908 Keratin, type II KRT2 1420 66110 57/38.4

P35527 Keratin, type I KRT9 1175 63320 39/38.0

O76011 Keratin, type I KRT34 1042 50818 48/35.8

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Supplementary Table 2: Band # 2, Molecular Weight ~60 KDa

UniProtKBAccession Protein/Gene ID Score Molecular Weight #pep/cover%

O43790 Keratin, type II KRT86 1816 55120 276/42.0

Q14533| Keratin, type II KRT81 1794 56875 274/37.0

P78385 Keratin, type II KRT83 1762 55928 254/37.9

P78386 Keratin, type II KRT85 1720 57306 187/33.9

P13645 Keratin, type I KRT10 1711 59703 128/37.4

Q15323 Keratin, type I KRT31 1489 48693 160/48.1

P04264 Keratin, type II KRT1 1475 66149 132/35.1

O76009 Keratin, type I KRT33A 1316 47166 115/44.8

Q14525 Keratin, type I KRT33B 1283 47325 104/49.8

P35908 Keratin, type II KRT2 1092 66110 76/31.8

P35527 Keratin, type I KRT9 1088 63320 61/36.4

O76011 Keratin, type I KRT34 962 50818 66/35.1

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Supplementary Table 3: Band # 3, Molecular Weight ~50-40 KDa

UniProtKBAccession Protein/Gene ID Score Molecular Weight #pep/cover%

Q15323 Keratin, type I KRT31 2013 48693 143/50.5

P78386 Keratin, type II KRT85 1953 57306 109/35.7

Q14525 Keratin, type I KRT33B 1811 47325 118/48.0

Q14533| Keratin, type II KRT81 1802 56875 118/37.2

P78385 Keratin, type II KRT83 1795 55928 114/38.1

O43790 Keratin, type II KRT86 1816 55120 115/38.7

O76009 Keratin, type I KRT33A 1659 47166 123/49.0

O76011 Keratin, type I KRT34 1537 50818 70/43.3

P04264 Keratin, type II KRT1 1314 66149 30/30.0

P13645 Keratin, type I KRT10 1169 59703 28/38.6

Q92764 Keratin, type I KRT35 808 51635 33/23.7

P35908 Keratin, type II KRT2 776 66110 21/22.5

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Supplementary Table 4: Band # 4, Molecular Weight ~30 Kda

UniProtKBAccession Protein/Gene ID Score Molecular Weight #pep/cover%

P78386 Keratin, type II KRT85 2038 57306 113/42.2

Q15323 Keratin, type I KRT31 1810 48693 108/48.1

Q14533| Keratin, type II KRT81 1761 56875 121/42.8

Q14525 Keratin, type I KRT33B 1758 47325 78/48.0

O43790 Keratin, type II KRT86 1757 55120 118/44.4

P78385 Keratin, type II KRT83 1709 55928 116/41.4

P04264 Keratin, type II KRT1 1467 66149 44/40.2

O76011 Keratin, type I KRT34 1448 50818 61/41.1

O76009 Keratin, type I KRT33A 1399 47166 82/42.8

P13645 Keratin, type I KRT10 1248 59703 44/41.0

P35527 Keratin, type I KRT9 771 63320 18/37.2

O76015 Keratin, type I KRT38 690 52054 22/19.7

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Supplementary Table 5: Band # 5, Molecular Weight ~20-15 Kda

UniProtKBAccession Protein/Gene ID Score Molecular Weight #pep/cover%

P78386 Keratin, type II KRT85 2252 57306 171/42.2

O43790 Keratin, type II KRT86 2132 55120 199/44.2

Q14533| Keratin, type II KRT81 2088 56875 194/39.2

P78385 Keratin, type II KRT83 2080 55928 194/40.2

Q15323 Keratin, type I KRT31 1847 48693 146/49.0

Q14525 Keratin, type I KRT33B 1587 47325 107/50.5

O76009 Keratin,type I KRT33A 1570 47166 126/48.0

O76011 Keratin, type I KRT34 1413 50818 86/42.2

P04264 Keratin, type II KRT1 959 66149 38/28.9

P13645 Keratin, type I KRT10 723 59703 23/19.4

P35908 Keratin, type II KRT2 600 66110 35/25.6

O76015 Keratin, type I KRT38 579 52054 18/18.9

P47929 Galectin-7 331 15123 8/36.0

P27482 Calmodulin-like protein 3 310 16937 15/30.2

P15090 Fatty acid-binding protein 273 14824 19/34.1

P10599 Thioredoxin 221 12015 8/31.4

P01040| Cystatin A 220 11000 5/48.0

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Supplementary Table 6: Band # 6, Molecular Weight ~8-10 KDa

UniProtKBAccession Protein/Gene ID Score Molecular

Weight

#pep/cover%

P78385 Keratin, type II KRT83 1301 55928 35/31.0

Q14533| Keratin, type II KRT81 1271 56875 36/32.3

O43790 Keratin, type II KRT86 1271 55120 37/33.5

P04264 Keratin, type II KRT1 1240 66149 31/38.7

Q15323 Keratin, type I KRT31 1167 48693 45/40.6

P13645 Keratin, type I KRT10 1157 59703 29/32.9

P78386 Keratin, type II KRT85 1150 57306 29/29.0

O76009 Keratin, type I KRT33A 1117 47166 41/36.4

P35908 Keratin, type II KRT2 1001 66110 20/30.9

Q14525 Keratin, type I KRT33B 990 47325 31/35.1

O76011 Keratin, type I KRT34 955 50818 21/39.2

P35527 Keratin, type I KRT9 735 62320 19/36.8

P47929 Galectin-7 228 15123 5/36.8

Q3LI72 Keratin-associated protein

19-5

122 7847 1/30.6

P27482 Calmodulin-like protein 3 102 16937 1/11.4

P10599 Thioredoxin 88 12015 1/12.4

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CHAPTER 3

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GAMMA KERATOSE MAINTAINS CELL VIABILITY IN VITRO AFTER THERMAL

STRESS BY REGULATING THE EXPRESSION OF CELL DEATH PATHWAY

SPECIFIC GENES

Poranki D, Van Dyke M

Wake Forest Institute for Regenerative Medicine

Wake Forest School of Medicine, Medical Center Boulevard, Winston Salem, NC 27157

The following manuscript has not yet been submitted for publication. Deepika Poranki prepared

the manuscript. Dr. Mark Van Dyke acted in an advisory and editorial capacity.

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Abstract:

When skin is thermally burned, transfer of heat energy into the skin results in the destruction of

cells. Some of these skin cells are damaged but may be capable of self-repair and survival,

thereby contributing to spontaneous healing of the wound. This phenomenon can be replicated in

cell cultures through models that utilize mild heating, where cells are thermally stressed and their

response to injury studied in the presence and absence of therapeutic treatments. Keratin protein-

based biomaterials have been suggested as potential treatments for burn injury. Isolation of

cortical proteins from hair fibers results in an acid soluble fraction of keratin proteins referred to

as “gamma” keratose. In the present study, treatment with this fraction dissolved in media was

able to maintain cell viability after thermal stress in an in vitro model using primary mouse

dermal fibroblasts. PCR array analysis identified that at 12hr compared to the pre-treatment

control (6hr), gamma keratose treated cells has a significant (p<0.01) up regulation of Ulk1

(autophagy) and Casp3 (apoptosis and autophagy). Whereas in the growth media treated cells

although there is relatively high expression (fold change) of the genes, significant difference was

not observed. At 18hr, necrotic (Parp2) and autophagic (Atg16l2) genes were highly expressed in

growth media treated cells with statistically significant (p<0.01) difference compared to the pre-

treatment control (6hr), whereas none of the cell death related genes were highly expressed in the

gamma keratose treated cells. Moreover, gamma keratose treatment significantly (p<0.01) down

regulated autophagic (Esr1) and necrotic (Parp2) genes compared to the pre-treatment control

(6hr). By 24 hours, there was similar expression in both the treatment groups. These results

suggest that gamma keratose treatment may assist in the survival and salvage of thermally

stressed cells, maintaining their viability by regulating cell death pathway related genes. Gamma

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keratose may be a promising material for burn treatment that aids in spontaneous wound healing

from viable tissue surrounding the burn.

Keywords: gamma keratose, thermal stress, PCR array, apoptosis, necrosis, autophagy

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1. Introduction

When skin is thermally burned, transfer of the heat into the tissue results in cell

destruction. Some damaged cells may be capable of self-repair and survival, if properly treated,

thereby contributing to spontaneous healing of the wound. Complete tissue necrosis is often

observed in the region where the injury occurs, beginning with the surface of the skin and

radiating downward into the tissue. The tissue surrounding this necrotic zone can be termed a

“thermally stressed zone”, which has received the energy as an initiating factor but is susceptible

to several further insults that result in cell death [1]. This phenomenon can be replicated in cell

culture by models that utilize mild heating (i.e. 42-48oC), resulting in a heterogeneous population

of cells, some of which die as a direct result of the thermal injury and others that die secondary to

the thermal insult due to the inflammatory cytokines released into the media. There are also some

cells that survive both insults and are capable of self repair. Heating cells has been shown to

induce various cell death pathways [2-8]. Depending on the intensity and duration of the

exposure, apoptosis, necrosis or autophagy pathways are triggered. Apoptosis is a genetically

programmed cell death mechanism that is associated with the activation of cysteine-dependent

aspartate-specific proteases called caspases and is involved in the development of homeostasis

[9]. Necrosis is morphologically characterized by cellular swelling, swelling of organelles,

plasma membrane rupture and loss of intracellular contents [10]. Until recently, it was

considered as non-programmed and unregulated cell death, but recent evidence indicates that

programmed necrosis occurs through the activation of death receptors in a caspase independent

manner termed “necroptosis”[11]. Autophagy is a catabolic process involving degradation of the

cell’s own components and is usually activated by oxidative, nutritional or toxic stresses [12]. In

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contrast to other cell death pathways, autophagy reflects a cell survival mechanism by

maintaining normal cellular function during nutrient deficient conditions or by removing

damaged organelles and aggregated proteins [8, 13].

Numerous studies have been published in which in vitro models have been used to

investigate thermally-induced cell death. In one study the log-phase cultures of mouse

mastocytoma cells exposed to heat from 42ºC - 44ºC showed an increase in apoptosis; at 45ºC

both necrosis and apoptosis were observed, and at 46º and 47ºC only necrosis was observed [2].

In another study, prolonged energy deprivation followed by thermal stress (43ºC for 10 min) of

murine P3 O1 myeloma and Ehrlich ascites carcinoma cells induced high expression levels of

Hsp70 that protected the cells from necrosis [4]. A third study used osteoblasts exposed to 48ºC

for 10 min and showed a 15-20% increase in necrotic cells within minutes after heating. After a

12 hour recovery at 37ºC, there was a 10% increase in apoptotic cells compared to control cells

maintained at 37ºC, indicating that thermal stress induced necrosis in the early phase and

apoptosis in the late phase [5]. These studies have shown that mild heating of cells in culture can

induce both necrosis and apoptosis at different stages, thus serving as a basis for further

investigation of these pathways at the cell and molecular level.

More extensive studies have been published in which pathway analysis at the gene level

has been conducted. For example, exposure of NIH3T3 cells to 45ºC for 15 min induced the

activation of Akt mediated by PI3Kinase. Phosphotidylinositol 3 kinase (PI3 kinase) activates

Akt (a serine threonine kinase) downstream in the signaling pathway of growth factors and

cytokines [14-16]. Akt is also activated by PI3Kinase in cell stress conditions such as hydrogen

peroxide (H2O2) and heat shock [17]. Phosphorylated Akt gradually decreased to basal levels in

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9 hours after insult and a high rate of cell proliferation was observed within 24 hours. Whereas

when the cells were exposed to prolonged heating at 45ºC for 60min, apoptosis was increased by

about 70% compared to untreated cells at 37ºC. This treatment failed to activate Akt, indicating

that cells might be irreversibly damaged when exposed to high temperatures for prolonged

periods[6]. In another study, heat treatment of Statens Seruminstitut Rabbit Cornea (SIRC) cells

at 42ºC for 30 min triggered a mitochondrial apoptotic pathway indicated by a change in

Bax/Bcl-2 ratios, resulting in caspase-9 activity [7]. Thermal stress to cells was also shown to

trigger death receptor apoptotic pathways by a change in Fas ligand expression, which in turn

resulted in the activation of caspase 8. Addition of an antioxidant decreased heat mediated

apoptosis, suggesting that ROS plays a critical role in the mitochondrial and death receptor

pathways of these cells [7].

The aforementioned studies shed further light on the time/temperature dependence on cell

death pathways, and begin to elucidate some of the molecular pathways involved in necrosis and

apoptosis. However, autophagy has been less widely studied. There are published in vitro studies

showing that in spite of apoptosis being completely inhibited, cell death still occurs and is

usually independent of caspases, following a specific signaling pathway where the cell

morphology resembles necrotic cell death [18-22]. If anti-apoptotic proteins block the caspases,

then necrosis occurs through the caspase independent pathway [20]. Embryonic fibroblasts from

double knock out Bax/Bak-/-

mice are resistant to apoptotic inducers. Bax and Bak regulate the

intrinsic pathway of apoptosis by controlling the activation of caspases [23]. Bax/Bak-/-

double

knockouts are used in research to study the different mechanisms of cell death (autophagy and

necrosis) that occur when apoptosis is completely inhibited. When treated with etoposide, the

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cells failed to undergo apoptosis and instead manifested a massive autophagy response followed

by cell death, but knocking down the autophagy related gene (Atg) products beclin-1 and Atg5,

reduced etoposide induced cell death [24]. Using other apoptotic inhibitors did not trigger the

autophagic cell death pathway, indicating that Bax and Bak absence played an important role in

autophagy activation [24]. In other cell types like L929 mouse fibrosarcoma, human Jurkat T

lymphoma, and U937 monocytoid cells, inhibition of cystein proteases (caspases) was shown to

induce autophagy [25, 26]. In stress conditions like nutrition depletion or loss of growth factors,

autophagy gets activated by the inhibition of apoptosis and promotes cell survival [27, 28]. If

autophagy is inhibited in cell stress conditions, the cells usually die through apoptosis. This cell

death can be postponed by inhibiting Bax or Bak or caspases [29]. In vitro and in vivo

experiments have shown induced apoptotic cell death by the inactivation of Atg genes Atg 5 or

7, and beclin-1[30-33]. Autophagy may develop as a primary response to stress stimuli, which

then triggers either apoptosis or necrosis [34, 35].

Previous studies (Chapter 2) have shown that gamma keratose was able to maintain cell

viability post thermal stress compared to the other treatments in an in vitro model using primary

mouse fibroblasts. It was evident in these studies that a large number of cells died from the initial

heat treatment, additional cells died within hours after heating, and surviving cells re-established

the culture. Thus, we concluded that all three pathways of cell death may be present in our cell

culture model, and that keratin treatment may affect one or more of these pathways. In the

present study, we hypothesize that treating primary dermal fibroblast cells with gamma keratose

six hours post thermal stress will induce differential expression of genes related to the cell death

pathways. The RT2 Profiler

TM PCR array was used to investigate cell death pathway related gene

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expression in thermally stressed cells treated with gamma keratose dissolved in the media

compared to cells treated with fibroblast growth media only.

2. Methods

2.1. Fibroblast cell Isolation: Primary mouse fibroblasts were isolated from adult CD1

mouse ear pads. The ear pads were incubated in Dispace II overnight and then the

dermis was peeled off using forceps and the remaining tissue minced before incubating

in 0.35% type I collagenase reconstituted in DMEM high glucose for 30 min at 37˚C on

a shaker. The suspension was filtered using a 100 micron meshed filter and the cells

were pelleted by centrifugation. The cell pellet was washed once with Hank’s balanced

salt solution (HBSS) to remove the residual collagenase. Finally, the fibroblast cells

were seeded in a 150 mm tissue culture plastic (TCP) dish at 2.5 x 106 cells/mL in

fibroblast growth media [DMEM high glucose plus 10% Fetal Bovine Serum (Hyclone®

Laboratories,Inc. Logan,Utah)] and 1% Antibiotic/Antimycotic solution (Hyclone®

Laboratories,Inc. Logan,Utah)).

2.2. Gamma Keratose Extraction: Keratin biomaterial was extracted from human hair

using the oxidative protocol specified by de Guzman et al. [36]. The extracted keratin

biomaterial was termed crude keratose. The gamma keratose fraction was further

separated from crude keratose by isoelectric precipitation. Concentrated hydrochloric

acid was added dropwise to the crude keratose solution with stirring until a pH 4.2 is

reached. At this pH, the alpha keratose precipitates leaving the acid-soluble gamma

fraction in solution. The insoluble alpha keratose was separated by fixed angle

centrifugation at 1500 rpm (252 RCF) for 15min at 4ºC. After neutralizing the gamma-

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containing supernatant to pH 8.4, it was dialyzed against endotoxin free water using a

5kDa spiral wound cartridge (Millipore, Billerica, MA) for 5 volume washes. Finally,

the solution was concentrated to minimal volume, pH adjusted to 7.4, lyophilized, and

sterilized by gamma irradiation at 1 MRad prior to use.

2.3. In vitro thermal stress model: For this experiment, primary mouse fibroblasts between

passages 3 to 6 were seeded at approximately 8x105cells/mL in 100mm TCP dishes. The

cells were allowed to grow in fibroblast growth media until they were 95% confluent.

Media changes were performed every 3 days. For a thermal stress experimental run, a

media change was performed a day prior to heating the cells. On the day of the

experiment, cultures were moved from a 37˚C, 5% CO2 incubator to a 44˚C, 5% CO2

incubator and heated for 150 minutes. After the heat treatment, culture dishes were

moved back to a 37˚C, 5% CO2 incubator to allow re-attachment of cells and the media

temperature to equilibrate. Six hours post heat treatment, cultures were randomized and

treatment was applied in fresh media. Treatments included 0.1mg/mL gamma keratose

supplemented to fibroblast growth media and fibroblast growth media alone (control

containing 10% FBS). The former will be termed gamma keratose treatment and the

latter growth media treatment in the rest of the chapter.

2.4. Trypan Blue Assay: Cell counting with a hemocytometer was performed using 0.4%

trypan blue reagent (Invitrogen, Gibco, Grand Island, NY), which stains cells with a

disrupted plasma membrane. Cells were counted at 6hr, 12hr, 18hr and 24 hr post heat

treatment. Out of two separate in vitro experiments performed, three samples showing

the highest percent cell viability after gamma keratose treatment and three samples

showing the lowest percent cell viability with growth media treatment were selected for

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further analysis using PCR arrays. Cell viability data from these samples was presented

as mean ± SEM (standard error of the mean).

2.5. RNA extraction & quantification: RNA extraction was performed using 5 prime

perfect pure RNA cultured cell kit (5 Prime Inc., Gaithersburg, MD 20878). After

trypsinization and cell counting, the cell pellet obtained after centrifugation was lysed

with a solution provided in the kit. The suspension was loaded onto a column with a

filter and centrifuged, followed by two washes with wash solution and RNA elution

from the column filter was performed using an elution buffer. Extracted RNA was

quantified using a Thermoscientific Nanodrop 2000 spectrophotometer. Out of the two

separate thermal stress experiments that were performed, RNA was extracted from three

individual (10cm) tissue culture plates for each treatment that showed the highest

percent cell viability after gamma keratose treatment and the least percent cell viability

after growth media treatment.

2.6. PCR arrays: cDNA synthesis was performed with the RT2

First Strand Kit

(SABiosciences) using an AB Applied Biosystems Veit 96 well thermocycler (850

Lincoln Center Drive, Foster City, CA) following the protocol specified by the

manufacturer. After the cDNA synthesis was performed, RT2 Profiler

TM PCR

microarray mouse cell death pathway finder (SABiosciences) was used to determine the

expression of genes that are involved in cell death. RT2 Sybr green ROX qPCR master

mix (SABiosciences) was used to perform the gene array experiment using an AB

Applied Biosystems 7300 Real time PCR system (850 Lincoln center drive, Foster City,

CA) following the protocol specified by the manufacturer. RT2 Profiler

TM PCR array

mouse cell death pathway finder analyzes the real-time expression of 86 genes

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(Supplementary Table 1) related to cell death pathways, which includes apoptosis,

necrosis and autophagy.

3. Data Analysis: Data was analyzed using the Auto Ct settings provided by the 7300 Real

Time PCR system software. Further data analysis was done using RT2

profiler PCR array

web based software provided by the manufacturer that follows ∆∆Ct method of calculation

[37]. This provided a comparison of post-treatment (12hr, 18hr, and 24hr) to the pre-

treatment control (6hr). To compare the relative up or down regulation between the gamma

keratose and fibroblast growth media treatment groups, the ratio of gene expression (fold

change) for gamma keratose treatment to growth media treatment was calculated. Using this

calculation, if the ratio is less than 1, it indicates that the gene expression is higher in growth

media treated cells, if it is equal to 1, the expression is the same in both the treatments, and if

it is greater than 1, the expression is greater in the gamma keratose treated cells.

4. Results:

4.1. In vitro thermal stress model: Six hours after heating, the average percent cell viability

was 53.7%±4.35. At 12hr, there is a significant difference (p<0.05) in the percent cell

viability between gamma keratose treated cells and growth media only treated cells. The

average percent cell viability for gamma keratose treated cells was 37.8%±0.85, whereas

growth media treated cells was 24.41%±2.20. At 18 hr, gamma keratose treated cells

were able to maintain cell viability at 39.83%±5.54, whereas in growth media treated

cells, additional cell death was noted with an average percent cell viability of only

20.18%±4.58. This difference was statistically significant (p<0.01). At 24 hr, the average

percent cell viability for gamma keratose treated cells was 36.9%±3.74 compared to

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growth media treated cells at 24.83%±0.69. This difference was not statistically

significant (p>0.05) (Figure 1).

4.2. RNA quantification: The total amount of RNA extracted from all the 24 samples is

listed in Table 2. The A260:280 and A260:230 ratios were approximately 2.1 and 2.0

indicating the purity of extracted RNA (Table 1).

4.3. PCR array data analysis: Gene expression of gamma keratose and growth media

treated cells at 12hr, 18hr and 24hr was compared to that of post thermal stress control

(i.e. cells at 6 hours post-heating, but immediately prior to treatment with fresh growth

media or gamma keratose supplemented media).

At 12hr compared to the pre-treatment control (6hr), the growth media treated

cells have no genes that were statistically significant even though their gene expression

(fold change) was high. In the gamma keratose treated cells, 29 genes were significantly

(p<0.05) up-regulated compared to the pre-treatment control. Among these 29 genes, 19

genes were down-regulated, six genes were up-regulated in gamma keratose treated cells

and four genes had a similar expression when compared to growth media treated cells.

The 19 genes that were down-regulated were involved in necrosis ( 9430015G10Rik,

Bmf, Txnl4b, Parp2, Pvr, Jph3), autophagy (Atg7, Atg5, Atg12, Map1lc3a), both

apoptosis and autophagy (Akt1, Bcl2, Bax) and apoptosis (anti-apoptotic [Traf2, Xiap]

and pro-apoptotic [Dffa, Fasl, Apaf1, Birc2]).The six up-regulated genes were involved

in autophagy (Htt, Ulk1, Atg16l1, Irgm1), both apoptosis and autophagy (Casp3) and

apoptosis (anti-apoptotic [Mcl1]). The genes that had similar expression between both

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growth media and gamma keratose treated cells were involved in autophagy (App,

Mapk8, Rps6kb1) and apoptosis (pro-apoptotic [Cflar]) (Table 2).

At 18hr, compared to the pre-treatment control (6hr), 34 genes in the growth

media treated cells were statistically significant (p<0.05). Of them seven genes were

down-regulated and 27 genes were up-regulated in the growth media treated cells. In the

gamma keratose treated cells, 18 genes had a gene expression that was statistically

significant (p<0.05) compared to pre-treatment control (6hr). Of these, six genes were

down-regulated and 12 genes were up-regulated in the gamma keratose treated cells.

When the 34 genes that were significantly expressed in growth media treated cells were

compared to the gamma keratose treated cells, six genes involved in necrosis (Galbt5,

Foxi1, Olfr1404), autophagy (Ins2, Esr1) and apoptosis (anti-apoptotic [Tnfrsf11b]) were

down-regulated and six genes involved in necrosis (Parp2, Rab25, Bmf, S100a7a),

autophagy (Atg16l1) and apoptosis (pro-apoptotic [Fasl]) were up-regulated in growth

media treated cells. Twenty two genes involved in necrosis (Commd4, Txnl4b,

9430015g10Rik), autophagy (Htt, Atg7, Pik3c3, Atg5, Ulk1, Mapk8, Map1lc3a),

apoptosis (anti-apoptotic [Xiap, Birc3] and pro-apoptotic [Dffa, Birc2, Casp1, Apaf1,

Gadd45a]) and both apoptosis and autophagy (Casp3, Bcl2l1, Bcl2, Bax, Akt1) had a

similar expression in both the groups. Comparison of the 18 genes that were expressed in

gamma keratose treated cells to growth media have shown that three genes involved in

necrosis (Rab25, Parp2) and autophagy (Atg16l1) were down-regulated and three genes

involved in necrosis (Galnt5), autophagy (Esr1) and anti-apoptosis (Sqstm1) were up-

regulated in gamma keratose treated cells, The 12 remaining genes involved in necrosis

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(Txnl4b), autophagy (Htt, Atg7, Atg5, Ulk1, Mapk8, Map1lca), apoptosis (anti-apoptotic

[Xiap, Birc3] and pro-apoptotic [Birc2]) and both apoptosis and autophagy (Casp3, Bax)

had a similar expression between both the groups (Table 3 & 4).

At 24hr compared to the pre-treatment control (6hr), 21 genes were expressed in

growth media treated cells with statistical significance (p<0.05). Of them, 10 genes were

down-regulated and 11 genes were up-regulated in the growth media treated cells.

Similar gene expression was noticed in the gamma keratose treated cells. Twenty one

genes in gamma keratose treated cells had significant difference (p<0.05) compared to

pre-treatment control. Of them, 10 genes were down-regulated and 11 genes were up-

regulated in the gamma keratose treated cells. When the 21 genes that were significantly

expressed in growth media treated cells were compared to gamma keratose treated cells,

seven genes involved in necrosis (Galnt5, Bmf, Txnl4b), autophagy (Esr1), apotosis (pro-

apoptotic [Gadd45a] and anti-apoptotic [Tnfrsf11b]), apoptosis and necrosis (Sycp2)

were up-regulated. The remaining 14 genes involved in necrosis (Olfr1404, Commd4,

S100a7a, Parp2), autophagy (Ifng, Ctsb, Atg16l1, Gaa), apoptosis (anti-apoptotic [Xiap]

and pro-apoptotic [Birc2, Nol3, Casp1]) and both apoptosis and autophagy (Bcl2l1, Bax)

were expressed similarly between the two groups. Comparison of the 21 genes that were

significantly expressed in gamma keratose treated cells to the growth media treated cells

have shown that six genes involved in necrosis (Galnt5), autophagy (Esr1, Ins2),

apoptosis (anti-apoptotic [Tnfrsf11b] and pro-apoptotic [Gadd45a]) and both apoptosis

and necrosis (Sycp2) were down-regulated and one gene involved in apoptosis (pro-

apoptotic [Abl1]) were up-regulated in gamma keratose treated cells. The remaining 14

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92

genes involved in necrosis (Rab25, Olfr1404, Commd4, S100a7a, Parp2), autophagy

(Ifng, Ctsb, Atg16l1, Gaa), apoptosis (anti-apoptotic [Xiap] and pro-apoptotic [Birc2,

Nol3, Casp1]) and both apoptosis and autophagy (Bcl2l1) were similarly expressed

between the groups (Table 5 & 6).

5. Discussion

Thermal burns typically display an injury pattern dictated by the transfer of the thermal

energy into the skin and underlying tissues. According to Jackson’s burn model, the zone of

stasis contains viable cells initially but due to the inflammatory cytokines released by the

surrounding necrotic tissue, more cell death occurs [38]. Necrosis is the major mechanism of cell

death that occurs as thermally damaged tissue converts to dead tissue and the wound progresses

from second degree to third degree. This process has sometimes been described as burn wound

progression [39]. This concept was shown by Clark et al. using a validated porcine hot comb

model in which high mobility group box 1 (HMGB-1) protein was used as a marker for cellular

necrosis and activated cleaved caspase-3 as a marker for apoptosis. The authors showed that

cellular necrosis in the zone of stasis occurred between one and four hours and apoptosis was

responsible for cell death only after 24 hr. They also suggested that an early intervention within

the first four hours is necessary to limit the irreversible cellular changes that account for burn

injury progression. However, there have been relatively few therapies that specifically target

cessation of burn injury progression by treating the thermally stressed tissue immediately

following injury. In one rare example, c-jun amino terminal kinase, a member of the mitogen-

activated protein kinase family involved in both apoptosis and necrotic pathways was

investigated as a potential target [40, 41]. In a mouse thermal injury burn model, a peptide

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93

inhibitor of c-jun was shown to promote re-epithelialization faster and also reduced cell death

surrounding the wounded region by 24 hr post injury [42]. Clearly, treatments that can arrest the

death of potentially viable cells are feasible and represent a novel therapy that may provide tissue

sparring treatment, but surprisingly little research has been conducted in this field.

Previous studies (Chapter 2) showed that gamma keratose was able to reduce the TBSA

in in vivo studies and maintain cell viability in an in vitro thermal stress model. Using the same

thermal stress model, we attempted to understand whether gamma keratose induces differential

expression of cell death pathway genes as a possible mechanism of its apparent tissue-sparing

activity. There are four main findings from the present experiments:

1. As expected, heat treatment of mouse dermal fibroblasts up regulates cell death

pathway related genes.

2. Gene expression was more consistent (i.e. smaller p values) in cells treated

with gamma keratose compared to cells treated with fibroblast growth media at

the early time point post thermal stress (12hr).

3. A single gamma keratose treatment at 0.1mg/mL appeared to influence gene

expression at 12 and 18 hr post-thermal stress (6 and 12 hr post-treatment,

respectively), but this effect was diminished by 24 hr.

4. In general, treating these thermally stressed cells with gamma keratose

substantially diminishes the gene up regulation compared to treatment with

fresh fibroblast growth media.

To further explain the first point, real time PCR array data analysis showed that there was

an overall up regulation of the genes involved in cell death pathways (necrosis, autophagy,

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94

apoptosis) six hours post thermal stress, and the cell viability data from the in vitro thermal stress

model correlates with this point. After the 6hr post thermal stress interval, there was a drop in the

cell viability from 100% to 53.7%±4.35 (Figure 1).

To further explain the second point, at 12hr post thermal stress, gamma keratose treated

cells showed statistically significant (p<0.05) alterations in the expression of 29 genes compared

to the pre-treatment control (6hr). Whereas in the growth media treated cells although there was

high expression (fold change) of the genes compared to the pre-treatment control, no statistical

significance was observed (p>0.05) due to the variability in the sample replicates for this

treatment group (Table 2).

As mentioned in point three, treating the thermally stressed cells once with 0.1mg/mL

gamma keratose was able to maintain cell viability by regulating the cell death related genes at

12hr and 18hr. At 12hr, 29 genes had a significant (p<0.05) gene expression in gamma keratose

treated cells compared to the pre-treatment control (6hr). Of these, 19 genes were highly

expressed by the growth media treated cells. By 18hr, only 18 genes were significantly (p<0.05)

expressed in gamma keratose treated cells, whereas 34 genes were significantly (p<0.05)

expressed in growth media treated cells compared to the pre-treatment control (6hr). The

regulation of cell death related genes by gamma keratose strongly correlates with the in vitro

thermal stress model cell viability results. In the in vitro model at 12hr post thermal stress,

gamma keratose treated cells were able to maintain cell viability at 37.8%±0.85 compared to

growth media treated cells at 24.41%±2.20 and the difference was statistically significant

(p<0.05). At 18 hr, gamma keratose treated cells were able to maintain cell viability at

39.83%±5.54, whereas in growth media treated cells, additional cell death was observed with an

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95

average percent cell viability of only 20.18%±4.58. This difference was statistically significant

(p<0.01). By 24hr, there was no significant difference in the cell viability and the gene

expression was similar between the two treatment groups (Figure 1 and Table 7 respectively).

To further explain the fourth point, at 12hr, 10 genes in gamma keratose treated cells

showed a statistically significant difference (p<0.01) compared to the pre-treatment control.

Among these 10 genes, two genes involved in autophagy (Ulk1) and both apoptosis and

autophagy (Casp 3) were up-regulated, 8 genes involved in necrosis (Parp2, Pvr), autophagy

(Atg5, Map1lc3a, Atg7), apoptosis (pro-apoptotic[Fas1]) and both apoptosis and autophagy

(Bax, Akt1) were down-regulated in the gamma keratose treated group compared to the growth

media treated cells (Table 2).

Tozhe et al. screened embryonic kidney 293 cells by kinase specific si-RNA library using

autophagy assay and identified Ulk1 as a novel candidate for autophagy [43]. In mammalian

cells, Ulk1/2 forms a complex with Atg13, Atg101 and FIP200 that was positively regulated by

AMPK (AMP-activated protein kinase) and negatively regulated by mTOR (mammalian target

of rapamycin) [44, 45]. AMPK is an evolutionarily conserved energy sensing kinase that is

activated by metabolic stress [45]. mTOR regulates growth, proliferation and protein synthesis of

cells in response to growth inducing stimuli [46]. Caspase 3 (Casp 3) is involved in the cleavage

of Atg4D (regulates autophagosome formation) and the truncated Atg4D is highly toxic and

couples both apoptosis and autophagy [47]. Fasl is a member of tumor necrosis family (TNF) of

type II transmembrane proteins. It is prototypic death factor that induces apoptosis when it binds

to its receptor Fas [48]. Atg5 and Atg7are involved in the formation of autophagosome [49].

Starved mouse embryonic fibroblasts lacking the autophagy gene Atg7 failed to undergo cell

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96

cycle arrest. Whereas prolonged metabolic stress resulted in augmented DNA damage and

increased p-53 mediated apoptotic cell death [50].

The gene expression data suggests that gamma keratose treatment was able to control the

up regulation of most of the cell death genes at 12hr compared to the growth media. The genes

that were up regulated in the gamma keratose treatment are involved in the autophagy pathway,

suggesting that the cell death observed in the gamma keratose treatment from 6 to 12 hrs post

thermal stress was due to autophagy. In vitro studies have shown that in stressed conditions like

starvation and energy deprivation, cell death occurs through autophagy when other cell death

pathways (like apoptosis) were inhibited [24-26]. At 18hr compared to the pre-treatment control,

gamma keratose treated cells had a significant (p<0.01) expression of 4 genes. Of the four genes,

two genes involved in autophagy (Esr1) and necrosis (Parp2) were down-regulated compared to

growth media treated cells (Table 3). The remaining two genes had a similar expression between

both the groups. Even though down regulation of Esr1 was observed in our thermal stress model,

no studies were published that suggest that Esr1 expression regulates autophagy when the cells

are exposed to stress conditions. Parp2 was shown to be involved in programmed necrosis (also

called necroptosis). Hitomi et al. treated L929 cells with TNF-α (tumor necrosis receptor family

involved in apoptosis by the activation of caspase 3) and zVAD.fmk (caspase inhibitor) and

performed a genome wide siRNA screen to identify the regulators of necroptosis. They identified

Parp-2 as a key regulator of necroptosis and showed that knockdown of Parp-2 inhibits

necroptosis [51]. This suggests that gamma keratose treated cells were able to maintain their cell

viability (39.83%±5.54) at 18hr when compared to 12hr (20.18%±4.58) by controlling the down

regulation of genes involved in autophagy and necrosis. By 24 hrs, the gene expression between

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97

the two treatment groups was similar, suggesting that the effect of the gamma keratose treatment

had diminished (Table 5 & 6). Although the mechanism of interaction of the gamma keratose

with the cell was not investigated in this study, the metabolic demand of thermally stressed cells

is known to be pathologically high [52-55], so it would not be surprising if the keratose were

consumed rapidly and its effects short lived.

6. Conclusion:

This study has shown that a single treatment of gamma keratose at 0.1mg/mL after

thermal stress can mediate survival of mouse dermal fibroblasts and faster recovery of the

culture. At 12hr compared to pre-treatment control (6hr), there was approximately 15% decrease

in cell viability and an increase in expression of autophagy related genes in the gamma keratose

treated cells. However, by 18hr gamma keratose was able to maintain cell viability similar to that

observed at 12 hrs and also down regulated the genes related to autophagy. This study suggests

that along with apoptosis and necrosis, autophagy plays an important role in the viability of

thermally stressed cells. Importantly, gamma keratose treatment was able to maintain cell

viability by down regulating the expression of the genes involved in apoptosis, necrosis and

autophagy. Gamma keratose, as a component in a keratin biomaterial hydrogel, may therefore

have been beneficial as a burn treatment as shown in earlier studies due to this mechanism.

Further detailed studies are necessary to determine if this is indeed the case, as the processes of

cell death are complex and any potential role of gamma keratose would need to be examined in

more detail, particularly with regard to how the cell interacts with the gamma keratose

molecules.

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98

Conflict of interest statement

Mark Van Dyke, Ph.D. holds stock and is an officer in the company, KeraNetics LLC, which has

provided partial funding for this research. Wake Forest Health Sciences has a potential financial

interest in KeraNetics, LLC through licensing agreements.

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99

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Figures & Tables:

Figure 1: In vitro Heat shock Model

Gamma keratose treated cells were able to maintain cell viability at 12hr, 18hr post-thermal

stress compared to the fibroblast growth media treated cells with statistical significance. P<0.01-

**, P<0.05*

* **

P<0.05 *

P<0.01 **

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Table 1: RNA Quantification

The 260/280 and 260/230 ratios were close to 2.1 and 2.0 indicating the purity of the extracted

RNA

# Sample ID

Nucleic Acid

Conc.(ng/µl) A260 A280 260/280 260/230

1 0h/N1 2210.9 55.3 26.1 2.1 2.2

2 0h/N2 3974.9 99.4 47.2 2.1 2.2

3 0h/N3 1973.9 49.3 23.1 2.1 2.2

4 6h/N1 835.6 20.9 9.8 2.1 2.2

5 6h/N2 1742.7 43.6 20.5 2.1 2.2

6 6h/N3 1270.6 31.8 14.8 2.2 2.2

7 12h/N1 486.7 12.2 5.6 2.2 2.2

8 12h/N2 477.9 11.9 5.5 2.2 2.1

9 12h/N3 574.5 14.4 6.6 2.2 2.2

10 12h/N1G 682.8 17.1 7.9 2.2 2.2

11 12h/N2G 385 9.6 4.6 2.1 1.9

12 12h/N3G 383.7 9.6 4.6 2.1 2.2

13 18h/N1 203.8 5.1 2.4 2.1 2.2

14 18h/N2 285.1 7.1 3.4 2.1 1.9

15 18h/N3 279.8 7.0 3.3 2.1 2.0

16 18h/N1G 285.4 7.1 3.4 2.1 1.8

17 18h/N2G 334.1 8.4 4.0 2.1 2.2

18 18h/N3G 732.9 18.3 8.7 2.1 2.2

19 24h/N1 114.9 2.9 1.4 2.1 2.2

20 24h/N2 185.6 4.6 2.2 2.1 2.1

21 24h/N3 226.1 5.7 2.7 2.1 2.1

22 24h/N1G 241.3 6.0 2.9 2.1 2.2

23 24h/N2G 324.8 8.1 3.9 2.1 2.1

24 24h/N3G 180.7 4.5 2.1 2.1 2.2

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107

Table 2: Gene expression of gamma keratose treated cells at 12hr

These are list of genes that were having significant p-value <0.05 for the gamma keratose treated

cells at 12hrs compared to the post heat shock pre-treated cells. Red color represents the up

regulated genes and blue color represents down regulated genes.

Gene

Symbol

Gamma

keratose

treated

cells p-

value

<0.05

Growth

media

treated

cells

fold

change

Gamma

keratos

e

treated

cells

fold

change

Gamma

keratose

treated cells

fold

change/Growt

h media

treated cells

fold change Pathway Involved

Traf2 0.02 20.63 2.14 0.1 Anti-apoptotic

Xiap 0.02 5.41 3.85 0.71 Anti-apoptotic

Mcl1 0.04 1.28 2.46 1.93 Anti-apoptotic

Akt1 0.01 21.75 2.86 0.13 Apoptosis and Autophagy

Bcl2 0.04 10.64 2.68 0.25 Apoptosis and Autophagy

Bax 0 4.65 2.84 0.61 Apoptosis and Autophagy

Casp3 0.01 0.49 3.03 6.17 Apoptosis and Autophagy

Atg7 0.01 41.19 2.63 0.06 Autophagy

Atg5 0 15.49 2.44 0.16 Autophagy

Atg12 0.02 9.09 3 0.33 Autophagy

Map1lc3a 0 6.8 2.91 0.43 Autophagy

App 0.03 2.65 2.31 0.87 Autophagy

Mapk8 0.05 2.32 2.1 0.91 Autophagy

Rps6kb1 0.04 2.68 2.64 0.98 Autophagy

Htt 0.02 2.87 5.06 1.77 Autophagy

Ulk1 0.01 3.76 8.02 2.13 Autophagy

Atg16l1 0.02 0.85 4.62 5.43 Autophagy

Irgm1 0.05 0.39 2.15 5.46 Autophagy

9430015G1

0Rik 0.02 70.88 3.39 0.05 Necrosis

Bmf 0.03 44.63 7.14 0.16 Necrosis

Txnl4b 0.04 15.37 3.61 0.24 Necrosis

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108

Parp2 0 9.51 2.62 0.28 Necrosis

Pvr 0 5.87 2.14 0.36 Necrosis

Jph3 0.02 13.64 9.91 0.73 Necrosis

Dffa 0.01 15.23 2.67 0.18 Pro-Apoptotic

Fasl 0 38.16 8.48 0.22 Pro-Apoptotic

Apaf1 0.01 43.33 9.91 0.23 Pro-Apoptotic

Birc2 0.05 15.78 3.81 0.24 Pro-Apoptotic

Cflar 0.02 4.55 3.92 0.86 Pro-Apoptotic

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109

Table 3: Gene expression of gamma keratose treated cells at 18hr

These are list of genes that were having significant p-value <0.05 for the gamma keratose treated

cells at 18hrs compared to the post heat shock pre-treated cells. Red color represents the up

regulated genes and blue color represents down regulated genes.

Gene

Symbol

Gamma

keratose

treated

cells p-

value

<0.05

Growth

media

treated

cells

fold

change

Gamma

keratose

treated

cells

fold

change

Gamma

keratose treated

cells fold

change/Growth

media treated

cells fold change Pathway Involved

Xiap 0.01 5.54 4.16 0.75 Anti-apoptotic

Birc3 0.03 2.3 2.03 0.88 Anti-apoptotic

Sqstm1 0.02 1.71 2.2 1.28 Anti-apoptotic

Casp3 0.03 3.13 2.54 0.81 Apoptosis and Autophagy

Bax 0.01 2.96 2.62 0.88 Apoptosis and Autophagy

Atg16l1 0.02 5.89 4.4 0.75 Autophagy

Htt 0.05 5.41 4.57 0.84 Autophagy

Atg7 0.02 3.05 2.64 0.87 Autophagy

Atg5 0.02 2.41 2.2 0.91 Autophagy

Ulk1 0.03 7.42 7.03 0.95 Autophagy

Mapk8 0.03 2.05 2.06 1.01 Autophagy

Map1lc3a 0.02 2.14 2.54 1.19 Autophagy

Esr1 0 0.02 0.08 3.42 Autophagy

Rab25 0.05 7.91 4.74 0.6 Necrosis

Parp2 0.01 3.27 2.33 0.71 Necrosis

Txnl4b 0.01 3.36 4.03 1.2 Necrosis

Galnt5 0.01 0.26 0.34 1.29 Necrosis

Birc2 0.04 4.92 4.06 0.82 Pro-Apoptotic

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Table 4: Gene expression of growth media treated cells at 18hr

These are list of genes that were having significant p-value <0.05 for the growth media treated

cells at 18hrs compared to the post heat shock pre-treated cells. Red color represents the up

regulated genes and blue color represents down regulated genes.

Gene Symbol

Growt

h

media

treated

cells p-

value

<0.05

Fibroblas

t media

treated

cells fold

change

Gamma

keratos

e

treated

cells

fold

change

Gamma keratose

treated cells fold

change/Fibroblas

t Media treated

cells fold change Pathway Involved

Xiap 0.00 5.54 4.16 0.75 Anti-apoptotic

Birc3 0.00 2.30 2.03 0.88 Anti-apoptotic

Tnfrsf11b 0.01 0.40 0.51 1.26 Anti-apoptotic

Casp3 0.01 3.13 2.54 0.81

Apoptosis and

Autophagy

Bcl2l1 0.00 3.11 2.55 0.82

Apoptosis and

Autophagy

Bcl2 0.03 2.59 2.17 0.84

Apoptosis and

Autophagy

Bax 0.00 2.96 2.62 0.88

Apoptosis and

Autophagy

Akt1 0.02 2.30 2.32 1.01

Apoptosis and

Autophagy

Atg16l1 0.00 5.89 4.40 0.75 Autophagy

Htt 0.04 5.41 4.57 0.84 Autophagy

Atg7 0.00 3.05 2.64 0.87 Autophagy

Pik3c3 0.02 2.10 1.85 0.88 Autophagy

Atg5 0.00 2.41 2.20 0.91 Autophagy

Ulk1 0.00 7.42 7.03 0.95 Autophagy

Mapk8 0.03 2.05 2.06 1.00 Autophagy

Map1lc3a 0.03 2.14 2.54 1.19 Autophagy

Ins2 0.05 0.23 0.37 1.60 Autophagy

Esr1 0.00 0.02 0.08 3.42 Autophagy

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Rab25 0.02 7.91 4.74 0.60 Necrosis

Bmf 0.01 7.75 4.73 0.61 Necrosis

S100a7a 0.02 9.00 6.27 0.70 Necrosis

Parp2 0.00 3.27 2.33 0.71 Necrosis

Commd4 0.01 2.01 1.60 0.80 Necrosis

9430015G10Ri

k 0.05 3.40 3.04 0.90 Necrosis

Txnl4b 0.02 3.35 4.03 1.20 Necrosis

Galnt5 0.00 0.26 0.34 1.29 Necrosis

Foxi1 0.05 0.23 0.37 1.60 Necrosis

Olfr1404 0.05 0.23 0.37 1.60 Necrosis

Fasl 0.04 7.88 5.65 0.72 Pro-Apoptotic

Dffa 0.00 2.87 2.26 0.79 Pro-Apoptotic

Birc2 0.00 4.92 4.06 0.82 Pro-Apoptotic

Casp1 0.01 2.61 2.22 0.85 Pro-Apoptotic

Apaf1 0.02 6.15 5.25 0.85 Pro-Apoptotic

Gadd45a 0.00 0.43 0.47 1.10 Pro-Apoptotic

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Table 5: Gene expression of gamma keratose treated cells at 24hr

These are list of genes that were having significant p-value <0.05 for the gamma keratose treated

cells at 24hrs compared to the post heat shock pre-treated cells. Red color represents the up

regulated genes and blue color represents down regulated genes.

Gene

Symbol

Gamma

keratose

treated

cells p-

value

<0.05

Growth

media

treated

cells

fold

change

Gamma

keratose

treated

cells

fold

change

Gamma keratose

treated cells fold

change/Growth

media treated cells

fold change Pathway Involved

Tnfrsf11b 0.00 0.34 0.24 0.73 Anti Apoptotic

Xiap 0.02 3.71 3.59 0.97 Anti Apoptotic

Sycp2 0.03 0.23 0.13 0.58 Apoptosis and Necrosis

Bcl2l1 0.00 2.93 3.09 1.06 Apotosis and Autophagy

Esr1 0.00 0.10 0.04 0.41 Autophagy

Ins2 0.03 0.21 0.13 0.64 Autophagy

Ifng 0.03 0.14 0.13 0.95 Autophagy

Ctsb 0.00 0.46 0.45 0.96 Autophagy

Atg16l1 0.04 3.74 3.84 1.03 Autophagy

Gaa 0.03 0.47 0.57 1.20 Autophagy

Galnt5 0.00 0.24 0.13 0.51 Necrosis

Rab25 0.01 6.96 5.51 0.79 Necrosis

Olfr1404 0.03 0.14 0.13 0.95 Necrosis

Commd4 0.01 2.18 2.21 1.01 Necrosis

S100a7a 0.01 7.87 8.18 1.04 Necrosis

Parp2 0.01 2.04 2.50 1.23 Necrosis

Gadd45a 0.00 0.46 0.33 0.71 Pro Apoptotic

Birc2 0.00 4.22 3.21 0.76 Pro Apoptotic

Nol3 0.04 0.43 0.44 1.02 Pro Apoptotic

Casp1 0.01 2.44 2.55 1.05 Pro Apoptotic

Abl1 0.01 3.76 5.23 1.39 Pro Apoptotic

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Table 6: Gene expression of growth media treated cells at 24 hr

These are list of genes that were having significant p-value <0.05 for the growth media treated

cells at 24hrs compared to the post heat shock pre-treated cells. Red color represents the up

regulated genes and blue color represents down regulated genes.

Gene

Symbol

Growth

media

treated

cells p-

value

<0.05

Growth

media

treated

cells

fold

change

Gamma

keratose

treated

cells fold

change

Gamma keratose

treated cells fold

change/Growth

media treated

cells fold change Pathway Involved

Tnfrsf11b 0.00 0.34 0.24 0.73 Anti Apoptotic

Xiap 0.01 3.71 3.59 0.97 Anti Apoptotic

Bcl2l1 0.00 2.93 3.09 1.06

Apoptosis and

Autophagy

Bax 0.03 2.14 2.29 1.07

Apoptosis and

Autophagy

Sycp2 0.04 0.23 0.13 0.58 Apoptosis and Necrosis

Esr1 0.00 0.10 0.04 0.41 Autophagy

Ifng 0.03 0.14 0.13 0.95 Autophagy

Ctsb 0.00 0.46 0.45 0.96 Autophagy

Atg16l1 0.01 3.74 3.84 1.03 Autophagy

Gaa 0.01 0.47 0.57 1.20 Autophagy

Galnt5 0.01 0.24 0.13 0.51 Necrosis

Bmf 0.03 3.55 2.30 0.65 Necrosis

Txnl4b 0.02 2.91 2.14 0.73 Necrosis

Olfr1404 0.03 0.14 0.13 0.95 Necrosis

Commd4 0.04 2.18 2.21 1.01 Necrosis

S100a7a 0.02 7.87 8.18 1.04 Necrosis

Parp2 0.05 2.04 2.50 1.23 Necrosis

Gadd45a 0.01 0.46 0.33 0.71 Pro Apoptotic

Birc2 0.01 4.22 3.21 0.76 Pro Apoptotic

Nol3 0.03 0.43 0.44 1.02 Pro Apoptotic

Casp1 0.00 2.44 2.55 1.05 Pro Apoptotic

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Table 7: Total number of genes significantly expressed in gamma keratose treated cells and

growth media treated cells with p<0.05

The third column in the table indicates the total number of genes showing significant differential

expression in each of the treatments at that particular time point. The columns 4, 5 and 6 are

breakdown numbers of column 3. Columns 4 and 6 show how many of the total differentially

expressed genes were expressed higher in gamma keratose treatment and in growth media

treatment respectively. Column 5 represents the number of genes that have similar expression in

both the treatments.

Treatment Treatment

hours

Total number of

genes showing

significant

differential

expression

Number

of genes

expressed

higher in

gamma

keratose

treatment

Number of

genes

showing

similar

expression in

both the

treatments

Number of

genes

expressed

higher in

growth media

treatment

Gamma

Keratose

12hrs 29 6 4 19

18hrs 18 3 12 3

24hrs 21 1 14 6

Fibroblast 12hrs 0 0 0 0

18hrs 34 6 22 6

24hrs 21 0 14 7

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Supplementary Table 1: Genes included in the PCR array

Gene Symbol Gene Name

Cell death

Pathway

Associated

9430015G10Rik RIKEN cDNA 9430015G10 gene Necrosis

Abl1 C-abl oncogene 1, non-receptor tyrosine kinase Pro-Apoptotic

Akt1 Thymoma viral proto-oncogene 1 Apoptosis and

Autophagy

Apaf1 Apoptotic peptidase activating factor 1 Pro-Apoptotic

App Amyloid beta (A4) precursor protein Autophagy

Atg12 Autophagy-related 12 (yeast) Autophagy

Atg16l1 Autophagy-related 16-like 1 (yeast) Autophagy

Atg3 Autophagy-related 3 (yeast) Autophagy

Atg5 Autophagy-related 5 (yeast) Autophagy

Atg7 Autophagy-related 7 (yeast) Autophagy

Atp6v1g2

ATPase, H+ transporting, lysosomal V1 subunit

G2

Apoptosis and

Necrosis

Bax Bcl2-associated X protein Apoptosis and

Autophagy

Bcl2 B-cell leukemia/lymphoma 2 Apoptosis and

Autophagy

Bcl2a1a B-cell leukemia/lymphoma 2 related protein A1a Anti-apoptotic

Bcl2l1 Bcl2-like 1 Apoptosis and

Autophagy

Bcl2l11 BCL2-like 11 (apoptosis facilitator) Pro-Apoptotic

Becn1 Beclin 1, autophagy related Autophagy

Birc2 Baculoviral IAP repeat-containing 2 Pro-apoptotic

Birc3 Baculoviral IAP repeat-containing 3 Anti-apoptotic

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Bmf Bcl2 modifying factor Necrosis

Casp1 Caspase 1 Pro-apoptotic

Casp2 Caspase 2 Anti-apoptotic

Casp3 Caspase 3 Apoptosis and

Autophagy

Casp6 Caspase 6 Pro-apoptotic

Casp7 Caspase 7 Pro-apoptotic

Casp9 Caspase 9 Pro-apoptotic

Ccdc103 Coiled-coil domain containing 103 Necrosis

Cd40 CD40 antigen Pro-apoptotic

Cd40lg CD40 ligand Pro-apoptotic

Cflar CASP8 and FADD-like apoptosis regulator Pro-apoptotic

Commd4 COMM domain containing 4 Necrosis

Ctsb Cathepsin B Autophagy

Ctss Cathepsin S Autophagy

Cyld Cylindromatosis (turban tumor syndrome) Apoptosis and

Necrosis

Defb1 Defensin beta 1 Necrosis

Dennd4a DENN/MADD domain containing 4A Necrosis

Dffa DNA fragmentation factor, alpha subunit Pro-apoptotic

Dpysl4 Dihydropyrimidinase-like 4 Necrosis

Eif5b Eukaryotic translation initiation factor 5B Necrosis

Esr1 Estrogen receptor 1 (alpha) Autophagy

Fas Fas (TNF receptor superfamily member 6) Apoptosis and

Autophagy

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Fasl Fas ligand (TNF superfamily, member 6) Pro-apoptotic

Foxi1 Forkhead box I1 Necrosis

Gaa Glucosidase, alpha, acid Autophagy

Gadd45a

Growth arrest and DNA-damage-inducible 45

alpha

Pro-apoptotic

Galnt5 UDP-N-acetyl-alpha-D-galactosamine:polypeptide

N-acetylgalactosaminyltransferase 5

Necrosis

Grb2 Growth factor receptor bound protein 2 Necrosis

Hspbap1 Hspb associated protein 1 Necrosis

Htt Huntingtin Autophagy

Ifng Interferon gamma Autophagy

Igf1 Insulin-like growth factor 1 Autophagy

Igf1r Insulin-like growth factor I receptor Anti-apoptotic

Ins2 Insulin II Autophagy

Irgm1 Immunity-related GTPase family M member 1 Autophagy

Jph3 Junctophilin 3 Necrosis

Kcnip1 Kv channel-interacting protein 1 Necrosis

Mag Myelin-associated glycoprotein Necrosis

Map1lc3a Microtubule-associated protein 1 light chain 3

alpha

Autophagy

Mapk8 Mitogen-activated protein kinase 8 Autophagy

Mcl1 Myeloid cell leukemia sequence 1 Anti-apoptotic

Nfkb1 Nuclear factor of kappa light polypeptide gene

enhancer in B-cells 1, p105

Autophagy

Nol3 Nucleolar protein 3 (apoptosis repressor with

CARD domain)

Pro-apoptotic

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Olfr1404 Olfactory receptor 1404 Necrosis

Parp1 Poly (ADP-ribose) polymerase family, member 1 Necrosis

Parp2 Poly (ADP-ribose) polymerase family, member 2 Necrosis

Pik3c3 Phosphoinositide-3-kinase, class 3 Autophagy

Pvr Poliovirus receptor Necrosis

Rab25 RAB25, member RAS oncogene family Necrosis

Rps6kb1 Ribosomal protein S6 kinase, polypeptide 1 Autophagy

S100a7a S100 calcium binding protein A7A Necrosis

Snca Synuclein, alpha Autophagy

Spata2 Spermatogenesis associated 2 Apoptosis and

Necrosis

Sqstm1 Sequestosome 1 Autophagy

Sycp2 Synaptonemal complex protein 2 Apoptosis and

Necrosis

Tmem57 Transmembrane protein 57 Necrosis

Tnf

Tumor necrosis factor

Apoptosis and

Autophagy

Tnfrsf10b Tumor necrosis factor receptor superfamily,

member 10b

Pro-apoptotic

Tnfrsf11b Tumor necrosis factor receptor superfamily,

member 11b (osteoprotegerin)

Anti-apoptotic

Tnfrsf1a Tumor necrosis factor receptor superfamily,

member 1a

Apoptosis and

Necrosis

Traf2

Tnf receptor-associated factor 2

Anti-apoptotic

Trp53 Transformation related protein 53 Apoptosis and

Autophagy

Txnl4b Thioredoxin-like 4B Necrosis

Ulk1 Unc-51 like kinase 1 (C. elegans) Autophagy

Xiap X-linked inhibitor of apoptosis Anti-apoptotic

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CHAPTER 4

_____________________________________________________________________________________

DEVELOPMENT OF A PORCINE DEEP PARTIAL THICKNESS BURN MODEL

Carmen Gaines*a, Deepika Poranki*

a, Wei Du

b, Richard A. F. Clark

c, Mark Van Dyke

a

a Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC

27157, USA

b Department of Pathology-Tumor Biology, Wake Forest School of Medicine, Winston-Salem,

NC 27157, USA

c Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY 11794,

USA

* Both Authors contributed equally to this work

This manuscript was submitted for publication to Burns Journal. Stylistic variations are due to

requirements of the journal. Deepika Poranki and Carmen Gaines prepared the manuscript. Mark

Van Dyke acted in an advisory and editorial capacity.

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120

Abstract:

Swine are the preferred animal models to study the effects of burns on dermal wound healing.

Various studies have been published in which little emphasis was placed on minimizing burn

variability and inconsistency. We developed a novel method to create deep partial thickness

burns that are highly consistent.

A custom‐made burn device was fabricated to control the pressure applied on the swine skin

during burn creation. Cylindrical brass blocks, measuring 3 cm in diameter, are used to create the

burns. A stainless steel post extends from the block for insertion into the device holder. In this

study, burns were created in four female Yorkshire swine. Heating of the brass blocks was

conducted using a boiling azeotropic solution of 80% polyethylene glycol (PEG) and 20% water

and boiling water alone. Contact times ranging from 12 to 20s were used. At 24h and 7d

post‐injury, two swine were euthanized and tissues collected for histological assessment using

Gomori trichrome staining. Histological analysis showed that burns created using boiling water

were superficial compared to those created using the boiling PEG:H2O solution. With a burn

contact time of 20s, 48.5% ± 5.7 tissue damage was demonstrated at 24h when the PEG:H2O

solution was used, whereas only 11.9% ± 1.3 was observed with boiling water.

Keywords: contact burn, polyethylene glycol, azeotropic mixture, deep partial thickness burn,

swine, skin

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121

1. Introduction

The structural similarities of swine and human skin render swine as preferred subjects for in vivo

burn therapy research [1, 2]. Additionally, the large bodies of swine allow for ample surface area

for multiple wound creation and clinical evaluation [3]. Of the many contact injury models

published [4-6], few studies have considered the incidence of burn wound variability and

inconsistency as a function of burn technique. The use of models with such inherent sources of

error may give an inaccurate picture of the treatment being employed in studies where

differences between treatments is being quantified and statistically compared.

Kempf et al. [7] reported the occurrence of variable deep partial thickness scald burn wounds

upon creation, despite full adherence to a consistent burn procedure. Consequently, reddened,

superficially-burned regions within the wounds healed faster than the surrounding eschar-

covered regions by 14 days post-injury (Figure 1). Papp et al. [8] reported similar difficulties in

burn wound creation, displaying obvious signs of inconsistency in a representative photo (Figure

2). In the aforementioned studies, a single operator created all burn wounds with only the weight

of gravity on the heat source serving as applied pressure. Singer et al. [9] have shown that the

amount of pressure applied on the animal has a direct effect on the intensity and depth of burn.

As such, maintaining a constant pressure every time is important in creating consistent burns.

The observed incidence of burn wound variability suggests that better control over the pressure

applied by the heat source on the skin combined with a more effective heat transfer technique

may produce wound uniformity. A large portion of the published literature describes the use of

boiling water to heat the brass blocks employed in wound creation [8, 10-12]. Boiling water

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122

produces air bubbles that adhere to the brass blocks and can create non-uniform heating (i.e.

“cold spots”). Uniform thermal energy transfer is then compromised when the brass block is in

contact with the skin of the animal. The cold spots can produce superficially-injured skin regions

that, over time, develop into regenerated ‘islands’ of new skin within the wound at earlier time

points than the rest of the burn. As migration of healthy cells from the periphery of normal skin

to the wounded area is part of the wound healing process, the wounds that have these islands of

less damaged, faster regenerating skin heal faster compared to wounds without islands [13-15].

Our lab has developed a model in which a custom-made, spring-loaded burn device was

fabricated to standardize the pressure applied on the swine skin during burn creation,

independent of the operator. Cylindrical brass blocks served as our heat source for wound

creation. Preliminary evaluations of the brass block temperature upon immersion in boiling water

indicated that the blocks did not thermally equilibrate in the water bath and reach an optimal

temperature. Use of a boiling azeotropic mixture of deionized water and polyethylene glycol

raised the equilibration temperature of the brass blocks and also appeared to eliminate the

formation of cold spots. This allowed for the creation of highly consistent burn wounds and

facilitated the determination of an optimal contact time to achieve the desired burn depth.

2. Materials and methods

0.1. Animal model: This swine study was approved by the Wake Forest University Animal

Care and Use Committee. Six female, Yorkshire swine (Baux-Mountain, Winston-

Salem, NC) weighing 20-25 kg at the time of surgery were used in this study. The

animals were housed in individual pens upon their arrival and allowed to become

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123

acclimated for at least 7 days. The animals were washed and shaved one day prior to

surgery, at which time they were pretreated with a transdermal fentanyl patch (50mcg/h)

for pain management. While on study, the animals were fasted each night prior to the

administration of anesthesia.

0.2. General anesthesia and surgical monitoring: Anesthesia was induced intramuscularly

with ketamine (10mg/kg) and dexmedetomidine (0.05mg/kg), and was later continued

with isoflurane during surgery. The previously applied fentanyl patch was removed and

a new patch (75mcg/h) applied during surgery, which was replaced every 3 days. Blood

pressure, heart rate and body temperature were monitored during surgery for any

complications. Ten burn wounds were to be created with 3 cm diameter cylindrical brass

blocks, 5 on each side of the mid-dorsal line between the shoulder and the hip.

0.3. Wound creation

2.3.1 Pressure-controlled burn device: Cylindrical brass blocks (360 grade), measuring 3 cm

in diameter and 5 cm in height, were used to create the burn wounds (Figure 3). The

blocks were heated in a boiling solution until used. A stainless steel post protruded from

the top of each block to allow for easy insertion into the burn device holder, made of

insulative Delrin®. An adjustable spring was housed within the otherwise hollow body of

the holder to enable a consistent force response when the block was pressed onto the

animal’s body (Figure 4). During burn creation, the operator positioned the device holder

over the brass block in solution, dislodging a pin connector to allow for insertion of the

steel post into the holder. Release of the pin connector ensured secure locking of the

block within the holder. The holder was then used to remove the block from solution,

residual liquid was quickly removed with a towel, and the block placed onto the animal’s

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124

skin in a randomly assigned location. Enough force was applied so that the bottom of the

device holder was flush with the brass block, allowing for a consistent block pressure

controlled by the spring. Prior to use on animal subjects, the burn device was tested for

operational consistency by two separate researchers using pressure-sensitive film (Sensor

Products Inc., Madison, NJ). This was further verified under experimental conditions

through visual observation within the current study. A total of 3 identical brass blocks

were rotated for use during the burn surgery to assure temperature equilibration and

minimize the amount of time needed to maintain the animal under general anesthesia.

2.3.2 Metal block surface temperature validation: Initial studies conducted by our lab have

shown that the brass blocks reached a temperature range of only 88‐92°C when

equilibrated in boiling water. A potential solution was to raise the temperature of the

water through the addition of PEG. An 80:20 ratio of PEG:H2O produced a negative

azeotropic mixture that raised the boiling temperature to 115-125°C. Temperature of the

bottom surface of the brass blocks after equilibration in the boiling liquids was tested

using temperature sensing strips (Omega Engineering, Stamford, CT). An unanticipated

effect of the use of this azeotropic liquid was its wetting characteristics and the potential

to reduce the prevalence and adherence of vapor bubbles on the under side of the brass

blocks. As the azeotropic solution contained mostly PEG, we evaluated the difference in

surface tension compared to water alone. A contact angle meter (KSV Cam100

Instruments Ltd, Helsinki, Finland) was used to determine the average contact angle

measurements of water and the azeotropic PEG:H2O mixture on polished brass metal

coupons. The coupons were of the same composition and surface finish as the brass

blocks used with the burn device. Contact angles were measured in 5 places along the

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125

diameter of the metal coupons and the difference between average values was evaluated

using a Student’s T-test with p<0.05 considered significant.

2.3.3 Burn wound creation: Two Yorkshire swine were used for each boiling solution - water

alone and the 80:20 PEG:H2O mixture. Ten burn wounds were created on each animal

using the custom-made device. Brass block contact times from 12-20s, in 2s increments,

were used in wound creation. Two replicate burns were created for each contact time and

were randomly assigned locations on the animal. Sixty minutes after burning, the wounds

were covered with saline soaked non-adhesive gauze (Telfa, Tyco Healthcare, Mansfield,

MA) followed by an occlusive adhesive dressing (IobanTM

2, 3M, St.Paul, MN), a

protective plastic shield and a nylon jacket to keep the dressings in place. Atipamezole

(0.05mg/kg) was administered intramuscularly to reverse the effect of the

dexmedetomidine post-surgery. Two animals were euthanized after 24h and the wounds

were harvested for further histological analysis. The other two animals were euthanized

after 7days. Dressing changes were performed every 3 days for the 7 day animals.

2.4 Data acquisition

2.4.1 Digital wound capture: Digital pictures were taken at 0, 1, 3 and 7 days post-surgery

(Nikon-D90, Nikon Inc., Melville, NY) to document the presence or absence of islands of

normal tissue. A ruler and color wheel were located within each photograph for later

processing of the image.

2.4.2 Histology: The entire wound was excised from the animal at the designated time point

and half of the tissue was fixed in 10% neutral buffered formalin for 2 days. After

fixation, a tissue cross section closest to the center of the wound was cut and processed in

70% isopropyl alcohol (IPA), 80% IPA, 95% IPA, 100% IPA and xylene (Leica

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126

ASP300S Tissue processor, Buffalo Grove, IL) overnight. The tissue was embedded in

paraffin and cut in 5 �m sections using a microtome (Leica CM 1850, Leica

Microsystems Inc., Buffalo Grove, IL) and stained with Gomori trichrome.

Representative sections of each wound were evaluated by an experimental pathologist

blinded to the protocol. At pre-determined distance intervals from the edge of the wound,

tissue morphology was scored according to the following scale:

Epidermis

0 Normal

1 Flattening of the dermal-epidermal (DE) junction

2 Flattening and disruption of the DE junction

3 Absence of epidermis

Dermis

0 Normal

1 Loss of collagen architecture (papillary dermis)

2 Loss of collagen architecture and dye intensity color change

3 Loss of collagen architecture in reticular dermis (mid dermis)

4 Loss of collagen (full thickness)

Digital images of stained tissue sections were captured (Axio Imager M1 Microscope,

Carl Zeiss USA, Thornwood, NY) and the burn areas (i.e. demonstrating infiltration of

inflammatory cells, edema, and damaged collagen) and total area of the skin from the

epidermis down to the dermis-fat junction were measured (ImageJ, National Institutes of

Health). Total percent tissue loss was determined by calculating the area of burn (as

indicated by a change from green to red color and denaturation of the collagen bundles

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127

[16]) divided by the total area of the skin. Average percent tissue loss was calculated

from two replicate wounds and reported as mean ± standard deviation.

3 Results

3.1 Brass block temperature validation and surface chemistry: The brass blocks heated in

boiling azeotropic mixture of 80:20 PEG:H2O reached a temperature range of 99‐103°C

(Figure 5B), whereas the blocks heated with water only reach 88-92oC (Figure 5A). The

contact angle analysis showed a relatively lower average contact angle for the PEG:H2O

mixture compared to water alone (Figure 6). During heating, it was observed that vapor

bubbles did not adhere to the surface of the brass blocks.

3.2 Visual differences due to heating method: When water alone was used, burn wounds can

be uneven as shown in other studies. Islands of superficially-burned skin are evident,

causing an unwanted increase in burn edges that would be expected to affect the rate of

healing. These islands presumably arise from the presence of vapor bubbles that adhere to

the bottom of the brass blocks, thereby insulating the surface and creating cold spots. The

PEG:H2O azeotropic mixture has low surface tension compared to water alone, creating a

continuous liquid interface along the bottom of the block surface. In addition, the PEG:H2O

mixture does not boil as vigorously as water alone, resulting in noticeably fewer vapor

bubbles. The combination of fewer bubbles and better wetting mitigated the formation of

cold spots and created uniform heating when the PEG:H2O azeotrope was used. This

difference can be seen visually with digital pictures of wounds taken on days 0, 3 and 7

post-surgery (Figure 7). Wounds created by two separate operators and using the PEG:H2O

azeotropic mixture showed greater consistency (Figure 8), as verified by an initial

experiment using pressure-sensitive film (data not shown).

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128

3.3 Histological analysis

3.3.1 Wound variability evaluation: Wound scoring data for day 1 is shown graphically in

Figure 9. Each bar represents the mean value for contact time with the burning device,

and the error bars provide a measure of the variance seen between the two replicate burns

(i.e. large relative error bars indicate larger variance). As can be seen in the graphs, the

damage created by the water heated blocks (Figure 9A) was relatively less severe than

those created by the PEG:H2O heated blocks (Figure 9B) in the epidermis. In addition,

the PEG:H2O burns were more consistent as suggested by the small error bars. Evaluation

of the dermis showed similar results (Figures 9C and 9D) in that the PEG:H2O burns

were relatively more severe and more consistent across replicates and contact times. By

day 7, the epidermis in both groups was necrotic and had been completely removed by

debridement during dressing changes. In the dermis, the extent of damage in the

PEG:H2O burns was extensive as these wounds had progressed to nearly full thickness in

most cases and were not scored, while the water burns showed only modest damage and

continued variability (scoring data not shown).

3.3.2 Percent dermal tissue damage: Gomori trichrome staining was used to measure the

burn depth. When water alone was used for wound creation, the burns were more

superficial, whereas the PEG:H2O heating system created a more severe partial thickness

burn (Figure 10). Importantly, there was no correlation of burn depth with the contact

time as has been reported in earlier studies [8]. When the PEG:H2O mixture was used, the

burns were more consistent and deep and there was a measurable correlation between the

contact time and intensity of the burn. For a 20s contact time, 48.5% ± 5.7 of tissue loss

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was observed with the PEG:H2O boiling solution but only 11.9% ± 1.3 tissue loss was

observed with boiling water alone (Figure 11).

4. Discussion

The creation of highly-reproducible burn wounds is essential in the assessment of healing

progression in therapeutic models. Researchers have recognized the need to create consistent

burn wounds, but have cited the change in underlying tissue structure of the animal as a

hindrance. While we acknowledge the inherent variability encountered in working with animal

subjects, the present model offers control over variability through two simple means, a burn

device that creates operator-independent pressure, and a heating system that ensures consistent

and uniform heating of the brass blocks. Several other groups have limited their attempts at

controlling variability to designating a single operator to create burn wounds during surgery.

While this method has led to slightly better control, this study demonstrates that a higher level of

control can be achieved. Through the customized design of our spring-loaded device, any

operator can create the burn wounds without concern for uneven pressure when applied

appropriately. Pressure applied from the operator is only transferred along the periphery of the

device holder, where the device holder hits the animal’s skin along its insulated bottom surface.

This portion of the holder does not directly encounter the hot bath and does not achieve a

temperature as high as the actual brass block. In addition, the device and mode of operation is

potentially less hazardous to users than some other methods such as scalding [7].

The use of the PEG:H2O azeotrope improved the heat transfer efficiency within our

system. Moreover, the azeotropic mixture appears to maintain a contiguous coating along the

contact surface of the brass blocks, even in the presence of boiling fluid. Water vapor bubbles

adhere more strongly to the brass surface and are more difficult to dislodge through wetting

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because of the hydrophobicity of the brass block. The lower surface tension of the PEG:H2O-

brass interface minimizes the insulating effects of adhered vapor bubbles and thus the formation

of cold spots. If such uneven heating is present, a more superficial burn is created in this area,

accounting for the ‘islands’ that many researchers have encountered. The use of a non-toxic,

biocompatible polymer such as PEG for thermal burn models not only increases overall

maximum temperature, but provides an added benefit of ensuring more even distribution of heat

to allow for more consistent burning and the elimination of lateral and depth temperature

gradients. PEG is used in a variety of applications such as coating the surfaces of nanoparticles

to increase the amount of surface active reactive groups [17], and in many cosmetic products

[18]. It has also been used to neutralize the residual phenol left in a chemical burn [19-21].

Therefore it is unlikely that residual PEG on the brass block surface would induce a chemical

burn or toxicity tissue reaction.

Histological analysis with Gomori trichrome staining showed that using PEG:H2O as a

heating solution created more consistent and deep burns compared to boiling water alone. For a

20s burn, more tissue loss was observed with PEG:H2O compared to water after 24 hours. One of

the possible reasons might be that the brass blocks are not being heated to 100oC with water

alone, as shown by other investigators [8]. Also, given the dimensions of the brass blocks used in

this study, they did not provide an adequate heat sink for 20s when the temperature reached

using water alone was only 88-92oC. This is demonstrated by the fact that we expected to see

more superficial burns with a contact time of 12s compared to 20s. However, when boiling water

was used the burns were more variable overall and the 20s burns were more superficial

compared to the 12s burns.

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There are many variables that need to be considered when creating a burn wound, and

controlling those variables will help in creating a consistent burn. Several prior studies have

discussed the use of animals of similar size/age, controlling contact time, and using the same

operator. Randomization and the use of a large number of replicate wounds is also advocated.

The need for randomization is evident in the data for the 18s contact time in the water only group

where the percent burn was unusually high. Despite randomly locating all wounds, these two

burns ended up over the abdomen, which likely contributed to a high value for this test condition.

Heating brass blocks to a consistently high temperature and avoiding the formation of

cold spots are also important considerations in developing a burn model with consistent, efficient

thermal energy transfer. This study demonstrates that these aspects can be addressed through the

application of simple surface chemistry and thermodynamic principles.

Conflict of interest statement

Mark Van Dyke, Ph.D. holds stock and is an officer in the company, KeraNetics LLC, which has

provided partial funding for this research. Wake Forest Health Sciences has a potential financial

interest in KeraNetics, LLC through licensing agreements.

Acknowledgements

This work is supported by the Armed Forces Institute for Regenerative Medicine (DoD Contract

# W81XWH-08-2-0032) and KeraNetics LLC.

The authors would like to thank Christina Ross and Mary Ellenburg (WFIRM) for their

assistance with the burn surgeries.

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M, Provinciali M, Guerrieri M, Di Primio R, Giacometti A, Offidani A. Tigecycline

accelerates staphylococcal-infected burn wound healing through matrix metalloproteinase-

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nanoparticles through dual solvent exchange. Nano Lett. 2011 Sep 14;11(9):3720-6. Epub

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19. Horch R, Spilker G, Stark GB. Phenol burns and intoxications. Burns. 1994 Feb;20(1):45-

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20. Brown VK, Box VL, Simpson BJ. Decontamination procedures for skin exposed to

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21. Monteiro-Riviere NA, Inman AO, Jackson H, Dunn B, Dimond S. Efficacy of topical

phenol decontamination strategies on severity of acute phenol chemical burns and dermal

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104.

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Figure 1. Differences in healing of deep partial thickness scald burns due to inconsistent

burning. Consistent, deep partial thickness burn wound (A) compared to a wound containing red,

superficially-burned areas (inside dashed lines; B) will heal more uniformly (C) compared to the

superficially-burned areas which were healed by day 14 (inside dashed lines; D). Used with

permission [7].

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Figure 2. Burns with non-uniform severity created with brass block heated in boiling water

along the right flank of the animal. A burn wound created with a contact time of 12s is indicated

by the arrow. Used with permission [8].

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Figure 3. 360 grade, 3 cm diameter brass block used for burn wound creation. Notched stainless

steel post extends from block for easy insertion into the device holder.

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Figure 4. Burn device operation. The brass block is held within the device holder by engaging

a pin connector at the notched end. Upon depression of the fully-extended assembly, the spring

contracts exerting an even force onto the block with no extraneous forces from the operator.

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Figure 5. Heated brass blocks with temperature sensors. Equilibrium temperatures of the

block contact surfaces upon heating in boiling water alone (A; up to 88-92oC; arrow) and the

PEG:H2O azeotropic mixture (B; 99-103oC; arrow).

H2O Only PEG:H2O

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Figure 6. Contact angle analysis using water and PEG:H2O azeotropic mixture to determine

surface wetting. The addition of PEG caused a significant drop in surface tension and

consequently, static contact angle (n=5, p<0.05, Student’s T-test).

Water Only 80:20 PEG:H2O0

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Figure 7. Healing progression of wounds with and without islands. The presence of

superficially burned skin in the lower left of the water heated block would be expected to heal

more non-uniformly. The PEG:H2O azeotropic mixture avoided the island formation and created

a more uniform burn.

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Figure 8. Visually consistent burn wounds created by using boiling PEG:H2O azeotropic

mixture. Each row of wounds was created by a different operator.

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Water Only

PEG:H2O

Figure 9. Histological scoring data from day 1 wounds. Wounds created using water heating

caused less severe damage to the epidermis and were more variable (A) compared to wounds

created using PEG:H2O (B). This trend continued in the dermis (C and D, respectively).

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Figure 10. Tissue sections stained with Gomori trichrome stain showing a variation in the

burn depth for a 20s contact burn after 24h when PEG:H2O azeotropic mixture and boiling water

alone were used – Boiling water alone has created superficial burn compared to PEG:H2O

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Figure 11. Percent dermal tissue damage. For a 20s contact time, 48.5% ± 5.7 tissue loss was

observed using PEG:H2O boiling solution for heating whereas only 11.9% ± 1.3 tissue loss was

observed with boiling water after 24h.

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CHAPTER 5

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ASSESSMENT OF DEEP PARTIAL THICKNESS BURN TREATMENT WITH

KERATIN BIOMATERIAL HYDROGELS IN A SWINE MODEL

Poranki Da, Gaines C

a, Van Dyke M

a

aWake Forest School of Medicine, Wake Forest Institute for Regenerative Medicine

Wake Forest School of Medicine, Medical Center Boulevard, Winston Salem, NC 27157

The following manuscript has not yet been submitted for publication. Deepika Poranki prepared

the manuscript. Dr. Mark Van Dyke acted in an advisory and editorial capacity.

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Abstract:

Partial thickness burns can advance to full thickness burns due to inadequate tissue

perfusion and increased production of inflammatory cytokines, even with proper hospitalization

and treatment. This advancement of wound severity in the first several days after injury has been

referred to as burn wound progression and is the result of a cascade of events that results in

further tissue loss in the periphery of the original burn. There are currently no commercially

available treatments that can control burn wound progression. In our previous work (Chapters 2

and 3), we demonstrated that a keratin biomaterial hydrogel placed over a burn wound appeared

to reduce progression and that this may be due to a particular fraction of keratin, gamma

keratose, that showed the ability to regulate cell death pathway genes.

In the present thermal burn study, we tested the hypothesis that a modified formulation of

keratin, hereafter referred to as modified keratose hydrogel or MKH, which contained a reduced

amount of the gamma keratose fraction would heal wounds faster compared to the original

formulation of crude keratose. Crude keratose was used in the original pilot studies and was

included here as a control, as well as a collagen hydrogel (Coloplast®) and silver sulfadiazine

cream (SSD; clinical standard of care).

Fourteen female Yorkshire swine weighing 20-25 kg were used in the study.

Standardized burn wounds were created and treated within 60 minutes. Two animals were

euthanized on days 1, 3, 6, 9, 12, 15 and 30; their tissues collected. Digital images of the wounds

immediately prior to sacrifice were captured and analyzed using ImageJ software. The tissue

sections were stained and analyzed for burn depth, granulation of tissue thickness and re-

epithelialization.

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Wound assessment from digital images showed a significant difference between the

MKH and SSD on days 9 (p<0.05), 12 (p<0.001) and 15(p<0.05). Significant differences were

also found between crude keratose and SSD on days 12 (p<0.001) and 15 (p<0.05), and between

Coloplast and SSD on days 9 and 12(p<0.01). Rates of re-epithelialization at early time points

showed a 220% increase for the MKH compared to SSD. A Linear regression model predicted

that MKH and crude keratose had a time to wound closure of 19 days while Coloplast and SSD

treatments took 24 days. However, these data were not statistically significant due to high

variability in the SSD treated wounds. Measurements of burn depth and granulation tissue

thickness showed that by day 9, vertical burn progression had occurred in all wounds. Moreover,

crude keratose treatment appeared to result in a relatively smaller increase in burn depth (5.56%)

compared to other treatments (SSD - 9.81%, MKH - 20.01%, Coloplast - 22.5%). SSD treatment

appeared to show a higher difference (1.115cm) in the thickness of granulation tissue from days

9 to 15 compared to other treatments, while crude keratose appeared to show the least difference

(0.746cm). Again, no statistically significant differences between treatment groups were noted.

These results did demonstrate a smaller wound area with treatment using the MKH at mid-time

points during healing, and appeared to show a faster healing rate for this biomaterial compared to

the standard of care, SSD. However, the inconsistency of healing in the SSD treated wounds

contributed to a loss of statistical significance in several outcome measures. Additional studies

using keratin biomaterials that more specifically monitor burn wound progression, preferably in

a non-spontaneously healing burn injury model, are warranted.

Keywords: keratin biomaterial, deep partial thickness burns, hydrogel, crude keratose, alpha

keratose, gamma keratose, collagen, silver sulfadiazine, re-epithelialization, granulation tissue.

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1. Introduction:

Burns are one of the most catastrophic injuries to treat. Often, partial thickness burns

convert to full thickness burns due to inadequate tissue perfusion and increased production of

inflammatory cytokines leading to protein denaturation and necrosis [1]. The phenomenon of

burn injury progression occurs when events downstream of the original damage cause further

tissue loss. As a result, wounds increase in surface area and deepen, progressing from second

degree to third degree burns and increasing overall total body surface area (TBSA) burned,

thereby increasing risk to the patient, healing time, and treatment expense. In 1953, Jackson

described a model of burn injury in terms of three zones of tissue damage: 1) the inner zone of

coagulation, which is characterized by necrotic tissue, 2) an intermediate zone of stasis, which is

characterized by damaged cells that may live or die depending on treatment and other factors,

and 3) an outer zone of hyperemia where cells are stressed but will likely survive if the wound

does not get infected and standard treatment is given [2].

Superficial and partial thickness burns heal from the remaining epithelial structures in the

dermis [3], so the zone of stasis represents an opportunity for treatment as these cells are capable

of survival if an appropriate therapy can be developed that increases their survival, yet limited

clinical research has been directed in this area. In vivo studies however, have shown that

treatments like cerium nitrate, curcumin, activated protein C (APC), recombinant tissue type

plasminogen (r-TPA), TAK-044 (nonselective endothelin receptor antagonist), simvastatin, and

beraprost sodium (prostaglandin I2 analogue) were able to control burn injury progression in rats

by preventing tissue necrosis in the zone of stasis [4-11]. In general these studies have made use

of drug compounds that require intravenous or enteric administration, thus complicating their

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clinical development. Topical application of an efficacious therapeutic agent represents a

simplified, local treatment modality.

Previous studies (Chapter 2) in our lab have shown that crude keratose, which is a

heterogeneous mixture composed of alpha keratose, gamma keratose and keratin associated

proteins (KAPs) fractions increased tissue sparing and enhanced wound healing in both mice

chemical and swine thermal burn studies. Another study in an in vitro thermal injury model using

primary mouse dermal fibroblasts showed that the gamma keratose fraction was able to maintain

increased cell viability compared to the other treatments post thermal stress (Chapter 2). Finally,

gamma keratose was shown to salvage the cells by down regulating many genes involved in cell

death pathways (Chapter 3). Mass spectrometry analysis identified the major constituents of the

gamma fraction as keratins 81, 83, 85, 86, 31, 33A, 33B, 34 with lower than expected molecular

weights, suggesting that these were degraded alpha keratin proteins (Chapter 2). As such, they

represent peptides with biological function different than that of their parent molecules. In the

present study, we hypothesized that the peptides found in the gamma keratose fraction would

provide a therapeutic benefit when used in a topical keratin biomaterial hydrogel formulation.

This postulate was tested by comparing a “crude” keratose hydrogel with a re-formulated

hydrogel, modified keratose hydrogel or MKH, that contained a reduced amount of gamma

keratose, in a swine burn injury model. The amount of gamma keratose was reduced to reflect

the concentration at which gamma keratose was effective in the heat stress cell culture model. A

collagen hydrogel, Coloplast®, and the clinical standard of care, silver sulfadiazine cream (SSD)

were used as controls.

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2. Methods:

2.1. Keratin Protein Extraction: Keratin protein was extracted from human hair using the

oxidative protocol specified by de Guzman et al. [12]. The extracted keratin is referred

to as crude keratose.

2.2. Separation of Alpha and Gamma Keratose Fractions: Crude keratose was further

separated into alpha and gamma keratose fractions by isoelectric precipitation.

Concentrated hydrochloric acid was added dropwise to the crude keratose solution with

stirring until a pH 4.2 was reached. At this pH, the alpha keratose precipitates leaving

the acid-soluble gamma fraction in solution. The insoluble alpha keratose was separated

by fixed angle centrifugation at 1500 rpm (252 RCF) for 15min at 4ºC. After

neutralizing the gamma-containing supernatant to pH 8.4, it was dialyzed against

endotoxin free water using a 5kDa spiral wound cartridge (Millipore, Billerica, MA) for

5 volume washes. Finally, the solution was concentrated to minimal volume, pH

adjusted to 7.4, lyophilized, and sterilized by gamma irradiation at 1 MRad prior to use.

The precipitated alpha keratose was re-dissolved in 0.1M sodium hydroxide (NaOH)

solution and dialyzed against endotoxin free water using a 5kDa spiral wound cartridge

(Millipore) for 5 volume washes. Finally, the solution was concentrated to minimal

volume, pH adjusted to 7.4, lyophilized, and sterilized by gamma irradiation at 1 MRad

prior to use.

2.3. Hydrogel Preparation: The MKH was prepared using a proprietary mixture of alpha

and gamma keratose powder and reconstituted with phosphate buffered saline (PBS) to

form a hydrogel. Crude keratose powder was similarly reconstituted with PBS to form a

15% w/v hydrogel. The gels were made under sterile conditions, centrifuged to remove

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air bubbles and incubated in warm room (37ºC) overnight on a shaker. Gels were loaded

into syringes for easy application onto the burn wounds. Collagen hydrogel

(Woun’Dress® Collagen Hydrogel, Coloplast) and SSD (Watson Pharmaceuticals) were

obtained from commercial sources and used as received.

2.4. Swine burn study: The swine study was approved by the Wake Forest University

Animal Care and Use Committee (ACUC). Fourteen female, Yorkshire swine (Baux-

Mountain, Winston-Salem, NC) weighing 20-25 kg at the time of surgery were used in

this study. The animals were housed in individual pens upon their arrival and allowed to

become acclimated for at least 7 days.

2.5. General Anesthesia and Monitoring: The animals were washed and shaved one day

prior to the surgery, at which time they were pretreated with a transdermal fentanyl patch

(50mcg/h) for pain management. While on study, the animals were fasted each night

prior to the administration of anesthesia. Anesthesia was induced intramuscularly with

ketamine (10mg/kg) and dexmedetomidine (0.05mg/kg). Isoflurane was used to maintain

the animal in an anesthetic state during the surgery and during dressing changes. The

previously applied fentanyl patch was removed and a new patch (75mcg/h) was applied

during surgery, which was replaced every 3 days. Blood pressure, heart rate and body

temperature were monitored during surgery for any complications.

2.6. Burn Wound Creation: Wounds were created using a pressure controlled burn device

(described in Chapter 4). This spring loaded device was made of insulative Delrin®.

Cylindrical brass blocks (360 grade), measuring 3 cm in diameter and 5 cm in height,

were used to create the burn wounds. The blocks were heated in a boiling solution of

polyethylene glycol (PEG; Mn 400; Sigma-Aldrich) and deionized (DI) water. A

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stainless steel post protruded from the top of each block to allow for easy insertion into

the burn device holder. A total of three identical brass blocks were rotated for use during

the burn surgery to assure temperature equilibration and minimize the amount of time

needed to maintain the animal under general anesthesia. As described previously in

Chapter 4, twelve burns were created on each swine, six on either side of the mid dorsal

line between shoulder and the hip. For each time point, twenty four wounds (on two

swine) were randomly assigned to four treatment groups and treatments were

administered 60 minutes after creating burns. Treatments included Coloplast (Collagen

Hydrogel), Crude Keratose, MKH, and SSD. Each treatment had six replicates.

Following the treatments, the wounds were covered with saline soaked non-adhesive

gauze (Telfa, Tyco Healthcare, Mansfield, MA), followed by an occlusive adhesive

dressing (IobanTM 2, 3M, St.Paul, MN), a fabric stocking, a protective plastic shield,

and a nylon jacket to keep the dressings in place and prevent the animal from rubbing

directly on the wounds. Atipamezole (0.05mg/kg) was administered intramuscularly to

reverse the effect of the dexmedetomidine after surgery. Two animals were euthanized at

1, 3, 6, 9, 12, 15 and 30 days after the surgery and tissues harvested for histological

analysis after euthanasia on those respective days. Dressing changes were performed

every three days.

2.7. Digital wound capture: Digital pictures were captured on 0, 1, 3, 6, 9, 12, 15, 21 and 30

days post-surgery during the dressing changes, immediately after cleaning and debriding

the wounds (Nikon-D90, Nikon Inc., Melville, NY). All the images were taken with the

camera placed exactly at the same distance and at the same angle (90º, perpendicular) to

the wound. A ruler and grey scale were located within each photograph for later

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processing of the image. Wound area was measured using ImageJ software (National

Institutes of Health, Bethesda, MD). These data were represented as mean ± standard

deviation.

2.8. Histology: The entire wound was excised from the animal at the designated time point

and half of the tissue was fixed in 10% neutral buffered formalin for 2 days. After

fixation, a tissue cross section closest to the center of the wound was cut and processed

in 70% isopropyl alcohol (IPA), 80% IPA, 95% IPA, 100% IPA and xylene (Leica

ASP300S Tissue processor, Buffalo Grove, IL) overnight. The tissue was embedded in

paraffin, cut in 5 µm sections using a microtome (Leica CM 1850, Leica Microsystems

Inc., Buffalo Grove, IL), and stained with hemotoxylin and eosin (H&E). Representative

sections of each wound were evaluated histomorphometrically by a blinded reviewer.

Burn depth measurement: Digital images of stained tissue sections (1.5cm in length)

were captured (Axio Imager M1 Microscope, Carl Zeiss USA, Thornwood, NY). The

thickness of the normal skin section from the epidermis down to the dermis-fat junction

and the residual dermis in the burned region were measured. An average of three

measurements was taken, one at the burned-normal dermis junction, one in the center of

the burned region, and another on the opposite end of the burned region. Burn depth was

determined by calculating the difference of the thickness of the residual dermis in the

burned region from the thickness of the normal skin section.

Re-epithelialization measurement: The burned region without epithelial cell coverage

(defined as at least one layer of cells staining dark pink) was measured from the stained

tissue sections. The percent re-epithelialization was determined by subtracting the burned

region from the measured radius of the wound (as determined from the digital image for

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the same wound at that time point). Re-epithelialization data was analyzed in two phases:

Early time points that included day 3 and 6, and later time points that included day 9, 12

and 15. This was done because the re-epithelialization curves demonstrated two phase

healing behavior, suggesting that wound healing rates were different between these two

time ranges and that distinct phases of healing (tissue salvage and cell survival followed

by matrix deposition and tissue remodeling) were present.

Granulation tissue thickness measurement: The thickness of the new granulation tissue

(characterized by connective tissue and blood vessels stained pink with H&E) was

determined by taking an average of three measurements obtained throughout the length of

the stained tissue sections as previously described.

3. Data Collection and Statistical Analysis: For visual wound assessment digital images,

wound size values for each treatment group were averaged (e.g. n=36 on day 3, n=30 on day

6, n=24 on day 9, etc.) and reported as mean ± standard deviation (SD). For burn depth, re-

epithelialization and granulation tissue assessment, values for each treatment group from six

replicates were averaged and represented as mean ± standard deviation (SD). Two-way

ANOVA and Bonferroni analyses were performed using Graph pad Prism at 95% confidence

intervals for all the data analyses.

4. Results:

4.1. Visual wound assessment: The eschar (dead skin) typically sloughs off completely

within 9-12 days, so re-epithelialization was observed most distinctly in the digital

images of the wounds from 9 to 30 days. To this end, image analysis data showed a

significant difference between MKH and SSD on days 9 (p<0.05), 12 (p<0.001) and

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15(p<0.05), and crude keratose and SSD on days 12 (p<0.001) and 15 (p<0.05). Also, a

significant difference (p<0.01) was noted between Coloplast and SSD on days 9 and 12.

(Figure 1)

4.2. Histology:

Percent burn depth: On day 3, the average burn depth was minimal in Coloplast

treatment group (26.6%) compared to the other treatment groups (MKH - 30.9%, Crude

KOS – 34.51%, SSD – 34.98%). By day 9, vertical progression was evident in all the

treatments. Crude keratose treatment (5.56%) had a minimal increase in burn depth

compared to the other treatments (Coloplast - 22.5%, MKH - 20.01%, SSD - 9.81%)

(Figure 2 & Table 1). There was no statistically significant difference in burn depth

between the treatments.

Re-epithelialization: Burns treated with crude keratose and MKH demonstrated an

upward trend in re-epithelialization at early time points (days 3and 6) and later time

points (days 9, 12 and 15) (Figure 3&4). The rate of re-epithelialization was relatively

higher in the MKH group (4.1 +/- 0.96) compared to other treatments at early time points

(Coloplast - 3.4 +/- 1.1, crude keratose - 3.4 +/- 1.2, SSD - 1.3 +/- 1.7; Table 2). This

observation corresponded with previous findings that SSD can impede re-

epithelialization [13, 14]. In addition, healing was generally inconsistent across the

replicates in the SSD treatment group as indicated by the higher standard deviations.

At later time points (days 9, 12 and 15), the rate of re-epithelialization was

relatively higher in the MKH treatment group (7.6 +/- 1.2) compared to other treatments

(Coloplast - 6.9 +/- 1.1, crude keratose - 7.3 +/- 1.4, SSD- 5.9 +/- 1.2; Table 3), although

not as markedly as at the early time points. Healing was generally more consistent across

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replicates as indicated by the relatively smaller standard deviations compared to early

stages of healing. When the data was extrapolated to determine a predicted value for the

number of days taken to complete wound closure, crude and MKH treatments required 19

days, whereas both Coloplast and SSD treatments required 24 days (Table 4).

Thickness of granulation tissue: Compared to the other treatments, SSD had a larger

difference (1.115cm) in the thickness of granulation tissue from days 9 to 15 and crude

keratose had the least difference (0.746cm). Differences in all the treatment groups were

not statistically significant (Figure 5).

5. Discussion:

Due to tissue necrosis and burn wound progression, deep partial thickness burns can

convert to full thickness burns, thereby increasing risk to the patient, length of hospital stay, and

the costs associated with treatment. At the early stages after burn injury, the processes of healing

and wound progression are in opposition to each other, and most often progression prevails due

to a lack of specific treatments that can address tissue loss at the level of cellular pathways.

Currently there are no approved treatments that can control burn injury progression by regulating

death signals within the cell. The standard of care is generally focused on avoiding infection

while allowing the burn to take its natural course, followed by excision of dead tissue after some

short time period to facilitate healing from the healthy periphery of the burn. However, the

standard of care, SSD, did not perform as well as the other treatments in this study. One of the

more striking findings was the variability in healing observed in this treatment group. Despite its

beneficial antimicrobial properties [15, 16], the use of SSD as a standard of care has been

questioned due to its reported interference with re-epithelialization [13, 14, 17, 18]. Tian et al.

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suggested that the use of silver nano-particles as a more effective alternative to SSD [17].

Crystalline silver has also been shown to have beneficial wound healing properties [19], but to

our knowledge no comparative testing of these different forms of silver in the same burn model

have been published.

Assessment of digital images of wound areas did show that MKH had a significantly

greater effect on minimizing wound size at days 9, 12, and 15 compared to SSD, but not so

compared to crude keratose and Coloplast. Even though significant difference between MKH

and SSD was observed in the visual wound assessment, histological analyses have not shown

any significant difference at the time points 9, 12 and 15. This might be due to the parameters

considered when measuring a wound visually from digital images. Visually a wound is

considered to be re-epithelialized if it is covered with a thin pink or white opalescent non glossy

covering, but there is a chance that such a coating is present even in the absence of neo-

epidermis [20]. Additionally there is a higher chance for the fragile neo-epidermis to be

accidentally removed during the processing and sectioning of the tissue samples. In the present

study, percent re-epithelialization as determined using stained tissue sections showed that crude

keratose and MKH treated wounds were able to re-epithelialize faster compared to SSD,

especially at early time points where the rate for MKH was 220% higher than SSD. However,

these data were not statistically significant, largely due to the high standard deviation in the SSD

treatment group (the percent relative standard deviation was 131% for SSD versus 23% for

MKH; the R squared for the least squares linear regression of these data was 0.03 for SSD versus

0.54 for MKH). Later stages of re-epithelialization appeared to be similar in all treatment groups.

As expected, the injury model did demonstrate burn injury progression in all treatment

groups, but it was the crude keratose that showed the smallest increase in burn depth compared

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to the other treatments. This finding may indirectly support our hypothesis as the crude keratose

formulation contained the highest level of gamma keratose, which may be the reason that wound

progression was relatively lower. However, this outcome measure also failed to demonstrate

statistical significance.

Granulation tissue depth, a measure of later stages of healing, was highest in the SSD

treatment group and minimal in the crude keratose treatment. However, this may have been due

to the fact that this treatment group experienced the most wound progression, and consequently

had the most extensive tissue loss. Higher amounts of granulation tissue may have been the

normal response to tissue loss and not necessarily indicative of better healing. Regardless, the

differences in this outcome measure were again found to not be statistically significant when

comparing the different treatments.

In order to better understand the apparent lack of statistical power in our experimental

design, power calculations were performed based on the re-epithelialization data. This showed

that two more replicates would be required to show a significant (p<0.05) difference in re-

epithelialization rate between MKH and SSD, and more than 100 replicates would be required to

show a significant difference between MKH and crude keratose or Coloplast. These numbers

point toward improvement in arguably the most important outcome in burn treatment, re-

epithelialization, but they do not necessarily support the conclusion that the amount of gamma

keratose was optimal, and/or that the route of administration was ideal in this study. As was

shown in Chapter 2, the gamma keratose fraction contains large peptide molecules, and delivery

of these compounds through the skin and especially through a thick layer of eschar, would be

expected to be difficult and inefficient. Interestingly, Nunez et al. have shown that soluble

keratose has vasodilation properties when administered intravenously or topically [21].

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Considering that the zone of stasis is characterized by reduced blood circulation due to the

clogging of blood vessels after burn injury [2], perhaps part of the mechanism of tissue sparing

may be due to keratin’s ability to dilate blood vessels in the tissue surrounding the burn.

Moreover, a different route of keratin administration such as intravenous injection may show

even greater efficacy, as has been the case with other compounds tested in burn models.

However, as discussed previously, local delivery is often preferred so a higher dose of gamma

keratose, such as was demonstrated using crude keratose, may represent a viable solution.

Regardless, a better understanding of the mechanism of action, optimal dose, and efficient

administration of the most efficacious compounds will require more research.

6. Conclusion:

In this study, we tried to understand if a particular formulation of keratin hydrogel

containing a reduced concentration of gamma keratose, MKH, could have a beneficial effect on

burn wound healing. We compared MKH to the crude keratose that was previously shown to

promote burn wound healing (Chapter 2) and that contained a fraction of protein, gamma

keratose, which rescued thermally stressed cells (Chapter 2 and 3). The amount of gamma

keratose was reduced in MKH to reflect the concentration that demonstrated efficacy in a cell

culture model of thermal stress. A statistically significant difference in wound size was observed,

but this did not translate to the histological measurements. The rate of early re-epithelialization

appeared faster in the MKH treatment group, particularly at early time points where it was

expected to have its greatest effect, but statistical significance was lost due to variance in the

SSD treatment group. Regardless, the outcome measures in this study were not able to

differentiate MKH (i.e. low gamma keratose concentration) from crude keratose (high keratose

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concentration). More research to investigate the mechanism of keratins in burn wound healing,

and more specifically in burn injury progression, are needed.

Conflict of interest statement

Mark Van Dyke, Ph.D. holds stock and is an officer in the company, KeraNetics LLC, which has

provided partial funding for this research. Wake Forest Health Sciences has a potential financial

interest in KeraNetics, LLC through licensing agreements.

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References:

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Plast Surg. 2007;59:109-15.

[2] Jackson DM. [The diagnosis of the depth of burning]. Br J Surg. 1953;40:588-96.

[3] Wasiak J, Cleland H, Campbell F. Dressings for superficial and partial thickness burns.

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[4] Goldman RD, Grin B, Mendez MG, Kuczmarski ER. Intermediate filaments: versatile

building blocks of cell structure. Curr Opin Cell Biol. 2008;20:28-34.

[5] Rogers MA, Langbein L, Praetzel-Wunder S, Winter H, Schweizer J. Human hair keratin-

associated proteins (KAPs). Int Rev Cytol. 2006;251:209-63.

[6] Popescu C, Hocker H. Hair--the most sophisticated biological composite material. Chem Soc

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[7] Carney DH, Redin W, McCroskey L. Role of high-affinity thrombin receptors in postclotting

cellular effects of thrombin. Semin Thromb Hemost. 1992;18:91-103.

[8] Freedberg IM, Tomic-Canic M, Komine M, Blumenberg M. Keratins and the keratinocyte

activation cycle. J Invest Dermatol. 2001;116:633-40.

[9] Norfleet AM, Bergmann JS, Carney DH. Thrombin peptide, TP508, stimulates angiogenic

responses in animal models of dermal wound healing, in chick chorioallantoic membranes, and

in cultured human aortic and microvascular endothelial cells. Gen Pharmacol. 2000;35:249-54.

[10] Freyberg S, Song YH, Muehlberg F, Alt E. Thrombin peptide (TP508) promotes adipose

tissue-derived stem cell proliferation via PI3 kinase/Akt pathway. J Vasc Res. 2009;46:98-102.

[11] Olszewska-Pazdrak B, Hart-Vantassell A, Carney DH. Thrombin peptide TP508 stimulates

rapid nitric oxide production in human endothelial cells. J Vasc Res. 2010;47:203-13.

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[12] de Guzman RC, Merrill MR, Richter JR, Hamzi RI, Greengauz-Roberts OK, Van Dyke ME.

Mechanical and biological properties of keratose biomaterials. Biomaterials. 2011;32:8205-17.

[13] Hussain S, Ferguson C. Best evidence topic report. Silver sulphadiazine cream in burns.

Emerg Med J. 2006;23:929-32.

[14] Lansdown AB. A review of the use of silver in wound care: facts and fallacies. Br J Nurs.

2004;13:S6-19.

[15] Atiyeh BS, Costagliola M, Hayek SN, Dibo SA. Effect of silver on burn wound infection

control and healing: review of the literature. Burns. 2007;33:139-48.

[16] Modak SM, Fox CL, Jr. Binding of silver sulfadiazine to the cellular components of

Pseudomonas aeruginosa. Biochem Pharmacol. 1973;22:2391-404.

[17] Tian J, Wong KK, Ho CM, Lok CN, Yu WY, Che CM, et al. Topical delivery of silver

nanoparticles promotes wound healing. ChemMedChem. 2007;2:129-36.

[18] Demling RH, Leslie DeSanti MD. The rate of re-epithelialization across meshed skin grafts

is increased with exposure to silver. Burns. 2002;28:264-6.

[19] Wright JB, Lam K, Buret AG, Olson ME, Burrell RE. Early healing events in a porcine

model of contaminated wounds: effects of nanocrystalline silver on matrix metalloproteinases,

cell apoptosis, and healing. Wound Repair Regen. 2002;10:141-51.

[20] Singer AJ, Hirth D, McClain SA, Clark RA. Lack of agreement between gross visual and

histological assessment of burn reepithelialization in a porcine burn model. J Burn Care Res.

2012;33:286-90.

[21] Nunez F, Trach S, Burnett L, Handa R, Van Dyke M, Callahan M, et al. Vasoactive

properties of keratin-derived compounds. Microcirculation. 2011;18:663-9.

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Figures and Tables:

Figure 1: Visual wound assessment

MKH has a significant difference compared to silver sulfadiazine (SSD) at days 9 (p<0.05), 12

(p<0.001) and 15(p<0.05). Crude keratose has a significant difference compared to SSD on days

12 (p<0.001) and 15 (p<0.05) and coloplast has a significant difference to SSD days 9 (p<0.01)

and 12 (p<0.01).

0 10 20 30 400.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 Crude Keratose

MKH

Coloplast

Silver Sulfadiazine

Days

Rela

tive W

ound S

ize

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Figure 2: Burn depth

Vertical progression in burn depth was observed for all the treatments by day 6. Error bars

indicate the variance in the data. Significant difference was not observed between the treatments.

Day

3

Day

6

-20

0

20

40

60

80Coloplast

Crude KOS

MKH

SSD

Time Points

Burn

depth

thic

kness

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Figure 3: Early time points rates of re-epithelialization

Burns treated with keratose (KOS) treatments demonstrated a general upward trend in re-

epithelialization at early stages of treatment, whereas Coloplast and SSD generally trended

downward. There was no statistically significant difference between treatment groups (n=6,

mean +/- standard deviation, p>0.05)

Coloplast

0 3 60

20

40

60

Day

% Re-Epithelialization

Crude KOS

3 60

20

40

60

Day

% Re-Epithelialization

SSD

3 6

-20

0

20

40

60

Day

% Re-Epithelialization

KeraHeal

0 3 60

20

40

60

Day

% Re-Epithelialization

MKH

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Figure 4. Rates of re-epithelialization during later stages of wound healing

Compared to the other treatments, keratose (MKH and CrudeKOS) treatments promote faster re-

epithelialization. There was no statistically significant difference between treatment groups (n=6,

mean +/- standard deviation, p>0.05)

Coloplast

9 12 150

20

40

60

80

100

Day

% Re-Epithelialization

Crude KOS

9 12 150

20

40

60

80

100

Day

% Re-Epithelialization

SSD

9 12 150

20

40

60

80

100

Day

% Re-Epithelialization

KeraHeal

9 12 150

20

40

60

80

100

Day

% Re-Epithelialization

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Figure 5: Thickness of granulation tissue

Silver sulfadiazine has a higher difference in the thickness of granulation tissue from day 9 to 15

compared to other treatments. No significant difference was observed between treatments.

Day

9

Day

12

Day

15

0.0

0.5

1.0

1.5

2.0

2.5Coloplast

Crude KOS

MKH

SSD

Time Points

Gra

nula

tion tis

sue thic

kness

Table 1: Percent increase in burn depth from day 3 to 6

Crude KOS showed the minimal increase in burn depth (5.57%) and coloplast showed the

maximum increase in burn depth (22.59%). No significant difference was observed between the

treatments (p>0.05).

Treatments

Percent increase in burn

depth from day 3 to 6

MKH 20.016%

Crude KOS 5.570%

Coloplast 22.590%

SSD 9.812%

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Table 2. Rates of re-epithelialization at day 3 and day 6.

Compared to silver sulfadiazine, MKH has the highest difference in the rate of re-

epithelialization in the early stages of wound healing (days 3 and 6).

Treatment

Rate (% Re-

Epithelialization/Day) R Squared % Difference vs. SSD

Coloplast 3.4 +/- 1.1 0.40 +160%

Crude KOS 3.4 +/- 1.2 0.35 +160%

MKH 4.1 +/- 0.96 0.54 +220%

SSD 1.3 +/- 1.7 0.03 --

Table 3. Rates of re-epithelialization between days 9 and 15.

Compared to silver sulfadiazine, MKH has the highest difference in the rate of re-

epithelialization in the later stages of wound healing (days 9 to 15).

Treatment

Rate (% Re-

Epithelialization/Day) R Squared % Difference vs. SSD

Coloplast 6.9 +/- 1.1 0.72 17%

Crude KOS 7.3 +/- 1.4 0.64 24%

MKH 7.6 +/- 1.2 0.72 29%

SSD 5.9 +/- 1.2 0.62 --

Table 4. Days to wound closure.

Keratose (Crude KOS and MKH) treatments have a faster wound closure compared to coloplast

and silver sulfadiazine.

Treatment Mean Days to Wound Closure

Coloplast 24.2

Crude KOS 19.5

MKH 19.2

SSD 23.4

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CHAPTER – 6

_____________________________________________________________________________________

DELAYED TREATMENT OF PORCINE DEEP PARTIAL THICKNESS BURNS

SUBJECT TO THERAPEUTIC KERATIN BIOMATERIALS

Authors, Affiliation:

Gaines Ca, Poranki D

a, Van Dyke M

a

aWake Forest School of Medicine, Wake Forest Institute for Regenerative Medicine

Wake Forest School of Medicine, Medical Center Boulevard, Winston Salem, NC 27157

This manuscript was not yet submitted for publication. Deepika Poranki assisted in the design

and execution of the animal study and data analysis. Dr. Mark Van Dyke acted in an advisory

and editorial capacity.

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Abstract

Burn wound healing outcomes rely heavily upon the timely administration of first aid treatments

and can affect the quality of later healing. The previously-reported wound healing capabilities of

keratin biomaterials makes them a viable treatment option as they may be capable of influencing

cell survival in the peri-burn region. In this study, the healing efficacy of deep partial thickness

burns subject to keratin biomaterial treatments 10 hrs after injury was evaluated. While burn

wound digital image examination suggested that keratin biomaterials enhance wound closure

rates at 12 days post-injury, histological evaluation suggested that the standard of care silver

sulfadiazine (SSD) encouraged faster reepithelialization rates for the first 15 days of the 30-day

study. By Day 30, the extent of reepithelialization due to keratin biomaterial treatments was

significantly greater in comparison to SSD (p≤0.001). Measurement and evaluations of

additional endpoints during the second half of the study are needed to determine the true

downstream healing efficacy of keratin biomaterials for the delayed treatment of deep partial

thickness burn wounds.

Keywords: deep partial thickness burns, keratin biomaterials, delayed treatment, swine model,

reepithelialization

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1. Introduction

Burn wound healing outcomes rely heavily upon the timely administration of first aid

treatments. Arguably the most precarious burn wounds to care for, deep partial thickness burns,

are those in which damage extends through the epidermis and well into the reticular dermis.

Unmediated treatment of these wounds is likely to result in burn wound progression, in which

tissue damage moves radially from a centralized region and evolves into a full thickness burn

condition. Within this centralized “zone of coagulation,” damage to blood vessels and dermal

appendages result in an ischemic state that starts within 48 hrs of injury [1-3] and lasts up to 3-7

days post-injury [4, 5]. Fibroblasts, keratinocytes and affiliated dermal cells within the adjacent

“zone of stasis” will succumb to either cell death effectively extending the zone of coagulation

and increasing the size and severity of the wound or revitalization and proliferation based upon

decided treatment modalities. Proper assessment of burn severity dictates the optimal course of

treatment for challenging conditions encountered downstream such as contracture, hypertrophic

scarring and microbial infection that often accompany full thickness burns.

Timely access to first aid burn treatments is often limited by physical proximity to

appropriate medical personnel or facilities specializing in burn care. Burn victims are

responsible for 45,000 hospitalizations in the US, 55% of who were transferred to specialized

“burn units [6].” At the time of injury, the victim may be located hundreds of miles away from

assistance, as often seen in rural communities nationwide or with members of the armed forces

serving overseas needing specialized care. With a total of 128 self-reported burn centers (51

American Burn Association-verified) dispersed throughout the US, Canada and Australia [7],

easy access by the multitudes of severely burned victims may not be easily attainable. While

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Klein [7] reported that approximately 80% of the US lives within 1-2 hrs of ground or air

transport of a verified burn center, many cover large geographic areas in which initial treatment

is transferred to a closer local hospital for initial assessment. As such, actual transfer to a burn

center may result in delays, ultimately leading to longer hospital stays, increased complications

and possible mortality. Sheridan [8] reported that a 5-day delay in transporting pediatric burn

victims with severe burns at >20% total body surface area (TBSA) burned to a designated burn

center resulted in significantly greater incidences of bacterial infection, wound sepsis and longer

wound closure times. MacKenzie [9] found that the in-hospital and one year mortality rates for

patients seen at a trauma center vs. non-trauma center was significantly lower. A study

conducted at the University of Washington Burn Center reported an average transport time post-

injury of 7.2 hrs; initial assessments described wounds with a 17% TBSA [10]. In this study, the

estimated burn size by non-trauma care personnel was overestimated, as TBSA of 15% is the

threshold for fluid resuscitation needed.

According to the United States Army Institute of Surgical Research (USAISR) Burn Center,

hand and facial burns were the most prevalent seen in military personnel, with approximately 73-

76% of combat victims experiencing this type of injury within a 2-year timeframe [11-13]. As

perhaps the most difficult regions of the body to treat, inadequate healing may lead to impaired

functionality and in some cases removal from previous military duties [14]. From 2003 to 2007,

transport time for injured soldiers from Iraq and Afghanistan to the USAISR was up to 4 days

from time of injury to admission to the burn center [12]. While 5.6% of the victims died from

their injuries at the USAISR, the ventilation support provided to over one-third of the victims in-

flight likely attributed to the relatively low casualty rate. These studies suggest that preventative

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measures taken to stabilize burns prior to more extensive treatments are crucial to limiting

negative outcomes.

Spontaneous wound healing largely depends on the following conditions: a) systemic

hydration via fluid resuscitation, b) local hydration of the wound bed to encourage

reepithelialization and c) wound infection control. Microbial control may have the most critical

local effects on progressive wound healing. Stander [15] suggests that microbial control within

the wound bed be maintained if delayed transport to a burn center is over 12 hrs. D’Avignon

[16] suggested the application of silver sulfadiazine (SSD) within 8 hrs of injury in combat-

related burns. Topical application of SSD cream has long been considered the ‘gold standard’

for microbial control [17, 18]. In deep partial thickness burns, sweat glands and hair follicles

may still be present in deeper layers of the dermis. If left untreated, gram-positive bacteria will

migrate from these appendages to colonize the wound within 48 hrs post-injury [19, 20]. By 7-

days post-injury, gram-positive and gram-negative microbes migrate from other regions in the

body to inhabit the burn wound eschar, creating a ripe environment for infection [21-23].

Additionally, researchers suggest that the application of SSD along with gentle debridement is

most effective in containing wound progression if patients cannot be transported for at least 72

hrs [17, 24]. While infection control is important, SSD alone is inadequate to keep the wound on

the path of healing. In fact, some studies have reported that SSD has a deleterious effect on

healing through exhibiting cytotoxic effects that delays wound closure. A study conducted by

Fraser [25] suggested that keratinocytes grown in culture exhibited a significant reduction in cell

number when exposed to SSD for 72 hrs.

An optimal first-line treatment for burns would be to encourage cell proliferation within the

wound bed without causing further infection to the wound. Burns have been characterized as

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having an exaggerated inflammatory phase [19]. Growth factor (i.e. transforming growth factor

beta or TGF-β) infiltration into the wound bed accounts for the initial influx of fibroblasts during

early wound healing, and is often responsible for scarring and contracture [26].

The first line of defense in burn care is the use of topical treatments. From a

physiological standpoint, the primary goals of such treatments are to prevent further tissue loss

and infection, and to promote spontaneous healing. In order for this to occur, revascularization

of the wound must happen within a few days post-injury. One such benefit of topicals is their

convenience and ease of use. This is especially important for victims to be able to treat

themselves at home or on the battlefield. In lieu of SSD, biologic dressings that exhibit some

bioactivity have been proven to enhance wound healing [27-31]. Common themes among these

treatments are non-immunogenicity, biocompatibility, non-toxicity and biodegradability.

A potential wound healing treatment that embodies the aforementioned characteristics, as

well as the added benefit of prompting no spontaneous protolytic activity within the body, are

keratin biomaterials. Keratin proteins derived from human hair have shown promise in a variety

of regenerative medicine applications, and is comprehensively outlined in Rouse et al. [32].

Within cell culture models, keratin hydrogels encouraged cell adhesion and proliferation on par

with collagen hydrogels [33]. Keratin dressings have also been shown to accelerate wound

healing and closure through the stimulation of keratinocyte migration and proliferation along the

wound margin within a porcine partial thickness model [34]. Previous studies conducted by our

lab demonstrate enhanced wound healing rates in a swine contact burn model treated with a

crude extract of keratin hydrogel (Chapter 2). Additionally, keratin hydrogel-treated chemical

burns within a rodent model showed containment of the burn wound (Chapter 2). The present

study was designed to test the healing efficacy of delayed treatment of deep partial thickness

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burns with a crude keratose hydrogel and a modified keratose hydrogel (MKH) within a tightly

controlled swine contact burn model. This delayed treatment scheme was conducted to

compliment a standard treatment paradigm evaluated in our lab (Chapter 5) and more closely

represents cases encountered by first responders in an attempt to provide initial care for burn

wounds and prevent the need for more invasive surgical treatments.

2. Materials & Methods

2.1. Animal study logistics: This study was approved by the Wake Forest Animal Care and

Use Committee. Fourteen female, Yorkshire swine (Baux Mountain, Winston-Salem,

NC) weighing 20-25 kg at the time of surgery were used. The animals were housed in

individual pens and allowed to acclimate to their surroundings for at least 7 days prior to

surgery. Deep partial thickness burns were created on pairs of swine treated and

monitored for 1, 3, 6, 9, 12, 15 or 30 days relative to the date of injury. The order in

which these designated endpoints occurred were randomly selected. One day prior to

surgery, the swine were cleaned and shaved along the dorsum. At this time, a fentanyl

patch (50 mcg/h) was adhered to the shoulder or hip of each animal as an analgesic in

preparation for the upcoming surgery. The animals were fasted the night before the

administration of anesthetization for each procedure conducted throughout the study.

2.2. Burn surgery protocol: Details of the burn surgery protocol used were reported

previously [35]. Briefly, the animals were anesthetized intramuscularly with a

combination of ketamine (10 mg/kg) and dexmedetomidine (0.05 mg/kg), intubated and

sustained on isofluorane during the surgical procedure. Vitals, such as blood pressure

and body temperature, were closely monitored during surgery to ensure animal safety.

Three cm diameter wounds were created using an innovative burn system in which a

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custom-made, pressure-controlled burn device housed a detachable 360 grade brass

block. As the designated heat source, the brass block was immersed in a boiling

negative azeotropic mixture of 80:20 polyethylene glycol (Mn = 400; Sigma-Aldrich, St.

Louis, MO):deionized water to ensure proper heat transfer from the source to the animal.

For each wound, the brass block made contact with the animal’s skin for 20s. Six

wounds were created on each side of the dorsal mid-line, between the shoulder and hip.

A total of 24 wounds were created, 12 per animal (Figure 1). The wounds were allowed

to set for 1 hr prior to being covered with saline-soaked gauze and bandaged as

described previously [35]. For the Day 30 endpoint, black dots were tattooed in the 4

corners surrounding each wound as a means to monitor contracture over time. Post-

surgical care consisted of the intramuscular administration of atipamezole (0.05 mg/kg)

to reverse the sedative effects of the dexmedetomidine and careful monitoring during

recovery.

2.3. Bandage changes:A typical bandage change occurred approximately every 3 days.

Under general anesthesia, all bandaging was removed and the wounds were lightly

debrided with saline-soaked gauze. Digital images of each wound were captured

(Nikon D60, Nikon, Melville, NY); a gray scale (Kodak, Rochester, NY) was included

in the field of view for image optimization along with a ruler to ensure proper scaling

during analysis. After treatment application, the animals were re-bandaged and

monitored during recovery. Saline was administered intravenously every other bandage

change to ensure proper systemic hydration. A new fentanyl patch (75 mcg/h) was

placed on each animal for pain management.

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2.4. Delayed treatment: Test and control treatments were randomly applied to wounds over

the swine pair for each endpoint. Test treatments consisted of Crude and modified

keratose formulations of keratin-based hydrogels, which will be referred to as Crude

KOS and MKH throughout the paper. Keratin proteins were extracted from a

commercial source of human hair via oxidative methods as described in de Guzman

[36]. Crude keratose was further separated into alpha and gamma keratose fractions by

isoelectric precipitation. Concentrated hydrochloric acid was added dropwise to the

crude keratose solution with stirring until a pH 4.2 was reached. At this pH, the alpha

keratose precipitates leaving the acid-soluble gamma fraction in solution. The insoluble

alpha keratose was separated by fixed angle centrifugation at 1500 rpm for 15min at 4ºC.

After neutralizing the gamma-containing supernatant to pH 8.4, it was dialyzed against

endotoxin free water using a 5kDa spiral wound cartridge (Millipore, Billerica, MA) for

5 volume washes. Finally, the solution was concentrated to minimal volume, pH

adjusted to 7.4, lyophilized, and sterilized by gamma irradiation at 1 MRad prior to use.

The precipitated alpha keratose was re-dissolved in 0.1M sodium hydroxide (NaOH)

solution and dialyzed against endotoxin free water using a 5kDa spiral wound cartridge

(Millipore) for 5 volume washes. Finally, the solution was concentrated to minimal

volume, pH adjusted to 7.4, lyophilized, and sterilized by gamma irradiation at 1 MRad

prior to use. Crude KOS and MKH formulations were sterilized via gamma irradiation (2

MRad). Under sterile conditions, powdered formulations were reconstituted in saline

and agitated overnight at 37°C to form 15 weight% hydrogels. Control treatments

consisted of a collagen-based hydrogel wound dressing indicated for the treatment of

superficial and partial thickness burns (Woun’Dres™ Collagen Hydrogel, Coloplast,

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Minneapolis, MN) and 1 wt% silver sulfadiazine (SSD) cream (Watson Pharmaceuticals,

Parsippany, NJ), long considered the standard of care for burn injuries [18]. All topical

treatments were transferred to 10 cc syringes for controlled application, with

approximately 2 cc applied to each wound. Treatment was delayed for approximately 10

hrs post-surgery and administered under general anesthesia. The animals were then

bandaged and allowed to recover as previously stated. All hydrogel treatments were

applied for 15 days, and SSD for 9 days, and was replaced by saline-soaked gauze for

protection during additional healing.

3. Data analysis

3.1. Digital Image Capture: Digital images of the burn wounds at progressive stages of

healing were captured on surgery day and at every subsequent bandage change and end

point. The margin of each wound was traced using Image J software (NIH, Bethesda,

MD); the area of the open wound was calculated. For each series of wounds

corresponding to an endpoint, the average area value was normalized to the surgery day

area value. The data set was analyzed via 2-way ANOVA and Bonferroni post hoc

analyses using GraphPad Prism (GraphPad Software, La Jolla, CA). Additionally, the

series of Day 30 wounds were evaluated for contracture where the circumference of the

“boxed” area around each of the wounds was measured by connecting the dots using

Image J. The data set was analyzed using 2-way ANOVA and Bonferroni post hoc

analyses using GraphPad Prism.

3.2. Histological analysis: At each specified endpoint, a pair of swine was euthanized and

each of the wounds was harvested in full as reported previously [35]. Briefly, the

wounds were cut into quadrants and fixed in 10% neutral buffered formalin for 48 hrs.

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A 2-cm thick slice from the innermost portion of the quadrant of each wound was

sampled and paraffin processed as previously reported. The slice represented the region

of the wound with the greatest degree of damage. The tissue samples were embedded in

paraffin and sectioned into 5 µm thick slices. One tissue section was evaluated per

wound, representing a sample size of 6 for each treatment. Sections stained with

hexatoxylin and eosin (H&E) were used to qualitatively access burn depth and the

relative density of granulation tissue. The extent of reepithelialization was calculated by

subtracting the length of the non-epithelialized burn region of the tissue sample from the

measured radius of the corresponding surgery day digital image of the wound to reveal

the length of the reepithelialized region. The length of the reepithelialized region was

normalized to the surgery day wound radius to determine the % reepithelialization. All

measurements were taken with Image J. Each data set was analyzed via linear

regression up to Day 15. At Day 30, the data was analyzed via 1-way ANOVA and

Bonferroni post hoc analysis. All statistical evaluations were done using GraphPad

Prism.

4. Results

4.1. Digital Image Capture: An approximate 10% increase in measured wound size

for up to 6 days post-injury was observed for all treatments, followed by a steady

decrease to <25% of the original wound size by Day 30 (Figure 2). The fastest rates of

size reduction were seen between Days 9 and 12. The healing patterns among treatments

were not significantly different (p>0.05) over the course of 30 days, except for significant

differences in the extent of healing in wounds treated with the keratin hydrogels vs. SSD

(p<0.001) and Coloplast vs. SSD (p<0.05) at Day 12. In measuring the progressive

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occurrence of wound contracture, the mean distance surrounding each wound expanded

in all treatment groups within the first 6 days of injury (Figure 3). Thereafter, these

“borders” steadily contracted to near baseline with some variation at the later time points.

There was no significant difference in the degree of wound contracture among treatment

groups at any specified time point (p>0.05). On average, MKH and Coloplast exhibited

the least amount of contracture over 30 days of healing, while Crude KOS and SSD

exhibited slightly higher amounts.

4.2. Histological analysis:

Burn Depth: Representative images of the degree of tissue damage incurred as a result

of a deep partial thickness burn wounds at Day 1 post-injury are depicted in Figure 4. In

all cases, the epidermis had been completely destroyed, leaving a gradient of dermal

damage relative to the proximity of the heat source throughout the depth of the tissue.

The upper to mid dermal regions revealed fully coagulated tissue, as defined by the

damaged appendages (e.g., hair follicles, sweat glands) and the absence of distinct

collagen bundles and blood vessels. In the lower dermal regions, next to the adipose

layer of skin, was dermis that incurred a slightly lesser degree of damage than the upper

layers. While distinct collagen bundles were present, they were loosely packed and show

evidence of potentially early coagulation. The depth of burn damage remained relatively

constant over all treatment groups.

Inflammatory Cell Infiltration: At Day 6 post-injury, inflammatory cells infiltrated the

wound margin as evidenced by the purple bands of nuclei depicted in Figure 5. For SSD-

treated wounds, evidence of this band is barely visible and appears to be hidden within

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the adipose layer. The deposition of collagen and new blood vessel formation within

interstitial spaces along and below the lower dermis encouraged cells from fatty layer to

disperse and spread throughout the entire dermal region. Coagulated tissue initially

found toward the top of the dermal layer eventually sloughed off, leaving behind

extensive granulation tissue by Day 12 (Figure 6). Lower densities of granulation tissue

were observed in the Crude KOS and SSD treated wounds relative to the other

treatments.

Re-epithelialization: Minimal amounts of regenerated epithelium were present up to

Day 6 post-injury. Within the first 6 days of treatment, rates of re-epithelialization

among treatment groups showed a general downward trend hovering at 20% or lower

(Figure 7). The MKH treatment showed the greatest stability after Day 3 at

approximately 10% wound closure. Re-epithelialization has progressed to the greatest

degree in wounds treated with SSD as indicated by a sharp initial slope to Day 3 and at

least a 40% greater re-epithelialization rate as compared to that of the keratin treatments

and 28% compared to Coloplast (Table 1). Although healing is generally inconsistent

across replicates as indicated by the large error bars, the tightest control is seen at Day 3

with the SSD treatment. Overall, no statistically significant difference in healing rate was

observed between treatment groups (p>0.05).

Re-epithelialization rates during the later stages of healing indicated an upward trend

among all treatment groups, with the most linear progression demonstrated by MKH

(Figure 8). SSD maintains the fastest re-epithelialization rate from Days 9 to 15, which is

40% greater than that of the MKH and Coloplast treatments and 15% greater than Crude

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KOS (Table 2). Healing was more consistent across replicates in comparison to the

earlier stages of wound healing as evidenced by the smaller error bars, although the

tightest control in this case was demonstrated by the Crude KOS treatment.

Qualitatively, well-organized, multilayered epithelium was present with all treatments at

Day 12 (Figure 9). Distinct rete ridges were seen in the Coloplast and SSD treatments.

In the keratin treatments, the papillary dermis appears to be loosely packed in comparison

to other treatments and may be indicative of mechanical weakness. No statistically

significant difference in healing rate observed between treatment groups was reported

(p>0.05). The degree of re-epithelialization shifts dramatically at Day 30 post-injury as

the hydrogel treatments exhibit significantly greater wound closure than SSD at p≤0.001

(Figure 10).

5. Discussion

Delays in the administration of adequate first aid treatments for burn wounds may have a

deleterious effect on downstream wound healing processes. In our study, keratin biomaterial

treatments were applied to deep partial thickness burns at 10 hrs post-injury in order to evaluate a

more realistic scenario during which potential complications that may arise from delayed burn

care. The 10-hr threshold was chosen based on discussions with burn care clinicians, logistical

constraints for conducting the study within our animal care facility and the desire to inflict

minimal stresses onto the animals during the study.

Quantitative analyses of the burn wound digital images showed significant decreases in

wound size over time in all treatment groups (Figure 2). Wound closure along the burn surface

is a function of the migration and proliferation of keratinocytes originating from healthy dermal

tissue along the wound margin and appendages. The keratin biomaterial treatments showed the

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greatest reduction in wound size at Day 12 post-injury in comparison to SSD. From a

histological standpoint, there was no significant difference in the degree of re-epithelialization

over time between treatment groups (Figure 7). Interestingly, the data exposes a slightly greater

re-epithelialization rate in SSD between Days 9 and 12 in comparison to all other treatment

groups (Table 2). While in direct conflict with recent reports on the stagnated closure of burn

wounds when treated with SSD as mentioned previously, its potential usefulness in healing

cannot be ignored [37, 38]. In the case of the present study, the latter concept was evident at

Days 9-15. To address the apparent discrepancy between the statistically significant wound size

evaluation and non-significant histological evaluations, attention must be brought to the integrity

of the reepithelialized layer [39]. While the hydrogel materials may have encouraged a greater

degree of re-epithelialization at Day 12 as demonstrated by the wound digital images, perhaps

portions of the re-epithelialized layers as defined by migrating keratinocytes were lost during

histological processing [40]. Mechanical stresses incurred during gross tissue preparation or

paraffin block sectioning may be contributing factors. The multilayered appearance of the

emerging epithelium and presence of distinct rete ridges and density of the papillary dermis may

be considered in determining epithelial integrity. The representative H&E images of the

Coloplast- and SSD-treated burns at Day 12 (Figure 9) showed a more robust papillary dermis to

support epithelial growth as compared to the other treatment groups. Interestingly, wound

closure as exhibited by the hydrogel treatments was significantly greater than that of SSD by

Day 30 (Figure 10). This suggests that perhaps the keratin and Coloplast treatments showed the

greatest activity at time points between Days 15 and 30, and that accurate re-epithelialization

rates cannot be determined without more definitive data from this timeframe.

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Beneath the open wound, granulation tissue at various stages of remodeling was present

at Day 12 (Figure 6). An abundance of cells from the fatty layer was dispersed within the

granulation tissue with the Coloplast and MKH treatments. Evidence of densely-packed

collagen deposits with relatively smaller regions of cells from fatty layer dispersed was seen with

the Crude KOS and SSD treatments. The latter condition suggests that these treatments may be

further along in the wound healing process, creating a dermal foundation of greater integrity to

support closure of the open wound.

Up to Day 6 post-injury, the influence of all treatments appears to be minimal. After a

slight increase in the degree of re-epithelialization at Day 3, all treatment groups trended

downward upon reaching Day 6. This was likely due to the formation of wound eschar requiring

cleaning at each bandage change prior to treatment application. Although careful and deliberate

debriding techniques were used, it is possible that growing epithelium at the interface between

the eschar and healthy wound margin was removed as well. Overall, the lack of significant

differences between treatment groups raises the question of how well, or if, penetration of the

topical treatments occur through the eschar to more centralized regions of the burns [41].

Additionally, H&E images indicate relatively consistent initial burn depths across treatments at

Day 1 and a similarly-emerging presence of an inflammatory cell band to begin the processes of

removing damaged tissue and debris from the wounds and collagen deposition.

In contrast to a similar burn study conducted at our lab in which treatments were applied

1 hr post-injury, the treatment of deep partial thickness burns after 10 hrs with keratin

biomaterials for enhanced wound healing prior to Day 15 cannot be validated based on the

outcomes of this study. In the former case, the mean rates of re-epithelialization during the

earlier (up to Day 6) and later (Days 9-15) endpoints were 220% and 29% greater than those for

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SSD, respectively (Chapter 5). The current study demonstrated outcomes in favor of SSD up to

Day 15, with a striking end result of significant wound closure using Crude KOS, MKH and

Coloplast by Day 30. While our surgical protocol was fully qualified to produce consistent deep

partial thickness burns and the wounds were randomly treated as indicated in Figure 1, the

thickness and microstructure of the underlying tissue along the dorsum of the swine may have

also introduced variation that was exposed in our results. Additional endpoints between Days 15

and 30 post-injury needed to provide greater insight into the re-epithelialization activity during

the second half of the study.

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187

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Figures and Tables:

Figure 1. Schematic of randomized placement of treatments. The dorsal of a pair of swine is

depicted, with burn wounds color-coded to represent the type of treatment applied. Replicates

are numbered from 1 to 6 to represent a sample size of 6 for each treatment group.

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Figure 2. Extent of burn wound size reduction over time. At Day 12, wounds treated with

Crude KOS and MKH exhibited significantly greater healing than those treated with SSD

(p<0.001). Additionally, wounds treated with Coloplast exhibited significantly greater healing

than those treated with SSD (p<0.05) at this time point. Otherwise, there was no statistically

significant difference between treatment groups (n=6, mean +/- standard deviation, p>0.05)

0 3 6 9 12 15 18 21 24 27 300.00

0.25

0.50

0.75

1.00

1.25Crude KOS

MKH

Coloplast

SSD

Days Post-Burn

Norm

alized W

ound S

ize

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Figure 3. Degree of wound contracture as a function of time. There was no statistically

significant difference between treatment groups (n=6, mean +/- standard deviation, p>0.05). On

average, MKH and Coloplast exhibited the least amount of contracture over 30 days of healing,

while Crude KOS and SSD exhibited slightly higher amounts.

0 3 6 9 12 15 18 21 24 27 30

0.90

0.95

1.00

1.05

1.10

1.15Crude KOS

MKH

Coloplast

SSD

Days Post-Injury

Norm

alized W

ound C

ontr

actu

re

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Figure 4. H&E images of deep partial thickness burn depth at Day 1. Representative

images of burned dermal tissue subject to A) Coloplast, B) Crude KOS, C) MKH D) SSD

treatments at Day 1. Fully coagulated tissue is labeled in the upper to mid dermal region, while

gradients of thermal damage is depicted by arrows within the lower dermal regions. The extent

of dermal damage remains consistent at this end point.

Figure 5. H&E images of deep partial thickness burns at Day 6. Representative images of

burned dermal tissue subject to A) Coloplast, B) Crude KOS, C) MKH D) SSD treatments at

Day 6. Emerging band of inflammatory cells is depicted by arrows within the mid to lower

dermal regions. Cell infiltration in the SSD-treated wounds is barely present in the adipose

region.

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Figure 6. H&E images of deep partial thickness burns at Day 12. Representative images of

granulation tissue subject to A) Coloplast, B) Crude KOS, C) MKH D) SSD treatments at Day

12. Fatty tissue that has been dispersed through the dermal region by the deposition of collagen

via fibroblastic activity is indicated, with scant regions present in the Crude KOS- and SSD-

treated wounds.

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Figure 7. Initial rates of re-epithelialization in deep partial thickness burns. All wounds

treated at 10 hrs post-burn demonstrated a general downward trend in reepithelialization at early

stages of treatment, with the greatest stability observed after Day 3 using MKH. Healing was

generally inconsistent across replicates as indicated by the large error bars, with the greatest

consistency observed at Day 3 using the SSD treatment. There was no statistically significant

difference between treatment groups (n=6, mean +/- standard deviation, p>0.05).

Coloplast

0 3 60

20

40

60

Days Post-Burn

% Reepithelialization

Crude KOS

0 3 60

20

40

60

Days Post-Burn

% Reepithelialization

MKH

0 3 60

20

40

60

Days Post-Burn

% Reepithelialization

SSD

0 3 60

20

40

60

Days Post-Burn

% Reepithelialization

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Figure 8. Rates of re-epithelialization in second degree burns during late stages of healing.

All treatments demonstrated an upward trend in healing. Healing was generally more consistent

across replicates as indicated by the relatively smaller error bars compared to early stages of

healing (see Figure 7). There was no statistically significant difference between treatment

groups (n=6, mean +/- standard deviation, p>0.05).

Coloplast

9 12 150

20

40

60

80

100

Days Post-Burn

% Reepithelialization

Crude KOS

9 12 150

20

40

60

80

100

Days Post-Burn

% Reepithelialization

MKH

9 12 150

20

40

60

80

100

Days Post-Burn

% Reepithelialization

SSD

9 12 150

20

40

60

80

100

Days Post-Burn

% Reepithelialization

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Figure 9. H&E images of deep partial thickness epithelium at Day 12. Representative

images of emerging epithelium subject to A) Coloplast, B) Crude KOS, C) MKH D) SSD

treatments at Day 12. All images show relatively well-organized, multilayered epithelium. Well

defined rete ridges are seen in the Coloplast and SSD treatments. The papillary dermis (as

indicated by arrows) for all treatments except Coloplast and SSD appear to be loosely packed

due to potential mechanical stress or the density of adipose tissue.

Figure 10. Degree of reepithelialization at Day 30 post-burn. Wounds treated with Coloplast,

Crude KOS and MKH KOS exhibited significantly greater healing than those treated with SSD

by Day 30 (n=6, mean +/- standard deviation, p≤0.001).

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Table 1. Initial rates of reepithelialization between Days 0 and 6. SSD exhibited a rate of 1.9

± 1.4 %re-epithelialization/day within the first 6 days of injury, at rates of 28% greater than

Coloplast, 42% greater than Crude KOS and 48% MKH.

Treatment

Rate

(% Reepithelialization/Day) R2 % Difference vs. SSD

Coloplast 2.6 +/- 1.7 0.12 -28%

Crude KOS 2.1 +/- 1.2 0.17 -42%

MKH 1.9 +/- 1.4 0.10 -48%

SSD 3.6 +/- 1.0 0.44 --

Table 2. Rates of reepithelialization between Days 9 and 15. SSD exhibited a rate of 5.7 ±

0.78 %re-epithelialization/day between Days 9 and 15, at rates of 43% greater than Coloplast,

15% greater than Crude KOS and 41% MKH.

Treatment

Rate

(% Reepithelialization/Day) R2 % Difference vs. SSD

Coloplast 3.2 +/- 1.1 0.37 -43%

Crude KOS 4.8 +/- 1.0 0.58 -15%

MKH 3.4 +/- 1.1 0.36 -41%

SSD 5.7 +/- 0.78 0.77 --

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CHAPTER 7

_____________________________________________________________________________________

SUMMARY, CONCLUSIONS AND PROSPECTIVE RESEARCH

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SUMMARY

The overall goal of this dissertation work was to test the wound healing properties of

human hair derived keratin biomaterials. Keratins represent a class of biomaterials that have been

used in a variety of biomedical applications [1-9], but the understanding of how keratin proteins

function in these applications is only at its nascent stages. Keratin biomaterial hydrogels possess

all the physical attributes of a burn wound dressing like providing a moist environment, acting as

a protective barrier and absorbing excess wound fluid. As part of this work, pilot in vivo studies

in mice and swine showed that an oxidized form of keratin biomaterial termed “keratose

hydrogel” was able to promote faster wound healing by preventing an initial increase in wound

area that often accompanies a burn (known a burn wound progression). This keratose hydrogel

was provided as a heterogeneous mixture of proteins representing “alpha” and “gamma”

fractions of keratin. These different fractions have disparate properties including molecular

weights, amino acid content, and solubility characteristics and can be separated by techniques

such as dialysis or isoelectric precipitation. Burn progression is mainly due to tissue necrosis and

therefore, we hypothesized that the interaction of keratin with burned tissue would promote cell

survival and reduce the total body surface area burned. The preceding chapters provide insight

into the differential wound healing activity associated with keratin fractions (alpha and gamma)

and the molecular mechanism by which the oxidized form of keratin biomaterial promotes cell

survival after thermal injury.

The first research aim, as presented in Chapter II, identified the keratose fraction that

displayed differential activity in salvaging thermally stressed cells. An in vitro thermal stress

model similar to Jackson’s burn model was developed in which a large number of cells died from

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the initial heat treatment, additional cells died within hours after heating, and surviving cells re-

established the culture. Using this in vitro thermal stress model, the efficacy of different keratose

preparations (alpha, gamma and crude keratose) in cell survival was tested. Our results showed

that gamma keratose was better able to maintain cell viability compared to crude keratose, alpha

keratose, and fibroblast growth media. As this is the first study to show that this keratose fraction

has particular efficacy for cell salvage after thermal stress, mass spectrometry analysis was

performed to understand the composition of proteins present in the gamma keratose fraction.

Mass spectrometry analysis results suggested that gamma keratose was a degraded protein

product of alpha keratose.

Chapter III provided an in depth analysis of the potential cell survival mechanism(s)

when gamma keratose treatment was given. Using the same thermal stress model (developed in

Chapter II), we attempted to understand whether gamma keratose induces differential expression

of cell death pathway genes as a possible mechanism of its apparent cell salvage activity. RT2

ProfilerTM

PCR array (specific to cell death pathways: apoptosis, necrosis and autophagy) was

used to investigate gene expression in thermally stressed cells treated with gamma keratose

dissolved in the media compared to cells treated with fibroblast growth media only. Our results

showed that gene expression was more significant (i.e. smaller p values) in cells treated with

gamma keratose compared to cells treated with fibroblast growth media at the early time point

post thermal stress. Additionally, we observed that treating the thermally stressed cells once with

gamma keratose was able to maintain cell viability by down regulating the genes involved in

apoptosis, necrosis and autophagy.

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The creation of highly-reproducible burn wounds is essential in the assessment of healing

progression in therapeutic models. Chapter IV explains in detail the development of a porcine

deep partial thickness burn model. This model offered control over variability through two

simple means, a burn device that creates operator-independent pressure, and a heating system

that ensures consistent and uniform heating of the brass blocks. Heating the brass blocks with an

azeotropic solution of polyethyleneglycol (PEG) and water created deeper burns compared to

boiling water only. Using the same in vivo burn model, a modified keratose hydrogel (MKH)

that contained a reduced amount of gamma keratose was tested for its wound healing properties

along with crude keratose, collagen hydrogel and silver sulfadiazine. Results showed that MKH

promoted faster re-epithelialization and wound closure compared to the other treatments. To

demonstrate potential clinical relevance and real-world practicality, a delayed treatment study

was performed with the same treatment groups wherein treatment was delayed 10 hours, thereby

representing transportation and admission to a burn unit. Results showed that MKH was still able

to promote faster re-epithelialization compared to the other treatments, but not with the same

efficacy as more immediate treatment.

LIMITATIONS AND PROSPECTIVE RESEARCH

Further investigation of the composition of the gamma keratose fraction by gel

electrophoresis and mass spectrometry identified degraded alpha keratose proteins with different

molecular weights. As gamma keratose showed differential activity in cell salvage after thermal

stress, further work needs to be done to elucidate whether the differential activity is associated

with a single excised band or multiple alpha keratose peptides. Additionally, the interaction of

gamma keratose with cells needs to be further investigated to better understand the mechanism of

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cell survival (for example, how do(does) the alpha keratose peptide(s) “communicate” with the

cell?). Ultimately, this knowledge will help to develop second generation keratin biomaterials for

wound healing.

Gamma keratose showed an overall down regulation of the genes involved in cell death

pathways (apoptosis, necrosis and autophagy). Autophagy, unlike apoptosis and necrosis

involves cell survival mechanism by maintaining normal cellular function during nutrient

deficient conditions or by removing damaged organelles and aggregated proteins [10, 11].

Therefore, additional molecular studies need to be performed to understand whether autophagy

helps in survival of thermally stressed cells when treated with gamma keratose. Also, in vitro

studies have shown that in stressed conditions like starvation and energy deprivation, cell death

occurs through autophagy when other cell death pathways (like apoptosis) are inhibited [12-14].

Further work needs to be done to understand if the down regulation of apoptotic and necrotic

genes is triggering the activation of the autophagic pathway. In addition, the effect of gamma

keratose on other skin cell types (e.g. endothelial cells, keratinocytes) should be investigated to

gain insight into the total wound healing mechanism(s) of gamma keratose.

In the in vivo studies we performed, there were some limitations that need to be

addressed in future work. Although an in vivo burn model using a burn device and an azeotropic

mixture solution successfully created highly consistent deep partial thickness burns, 3 cm burns

were not critical size wounds that convert to full thickness if proper treatment was not provided.

To study whether keratose treatment is able to prevent the conversion of the zone of stasis region

to necrotic tissue and further limit burn progression, using the contact burn comb model would

have provided more definite information. Moreover, a non-spontaneously healing model could

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have been used in which full wound closure would only happen if autografting is performed. In

adition, the experimental design for the in vivo studies used 24 hours as the earliest time point

for euthanasia, but according to Clark et al. cellular necrosis in the zone of stasis occurs between

one and four hours and apoptosis is responsible for cell death only after 24 hours. Therefore,

including early time points within hours after creation of the burn would provide more insight

into the tissue salvage mechanism of gamma keratose. Another interesting facet of this research

that should be investigated relates to the fact that the zone of stasis is characterized by reduced

blood circulation due to the clogging of blood vessels after burn injury [15]. Nunez et al. have

shown that alpha keratose and KAPs (referred to as crude keratose in this work) had vasodilation

properties when administered intravenously or topically [1]. Future investigations utilizing

keratin as an IV administered fluid may therefore be worth pursuing. In addition, performing

immunohistochemical staining specific to blood vessel may provide more insight into the role of

blood vessel plugging (early time points), or even new blood vessel formation (later time points)

when keratin treatment is given. Like many other compounds, there is likely a limit to the

beneficial dose of gamma keratose. In vitro studies in the lab have shown that very high

concentrations of gamma keratose (i.e. >10mg/mL) can be detrimental for thermally stressed

cells. Determining an optimum ratio of alpha and gamma keratose fractions in pilot studies and

then using that ratio to study burn wound healing may have provided more statistical difference

between treatment groups. Finally, determining the optimum time in which keratose treatment

can promote tissue salvage would be important information for clinical application as most

changes at the cell and molecular level are irreversable.

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CONCLUSIONS

In summary, this dissertation work outlines the importance of keratose biomaterial in the

treatment of burns. In vitro studies demonstrated that the gamma keratose fraction was able to

promote survival of thermally stressed cells by down regulating the genes involved in cell death

pathways. In vivo studies showed that keratose biomaterial was able to promote faster re-

epithelialization and wound closure in deep partial thickness burns. Although further work is

needed to understand the specific mechanism(s) of wound healing in both in vitro and in vivo

studies, particularly those related to burn wound progression, the results so far have indicated the

potential benefit of using keratin biomaterial in burn therapy.

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References

[1] Nunez F, Trach S, Burnett L, Handa R, Van Dyke M, Callahan M, et al. Vasoactive

properties of keratin-derived compounds. Microcirculation. 2011;18:663-9.

[2] Saul JM, Ellenburg MD, de Guzman RC, Van Dyke M. Keratin hydrogels support the

sustained release of bioactive ciprofloxacin. J Biomed Mater Res A. 2011;98:544-53.

[3] Dias GJ, Mahoney P, Swain M, Kelly RJ, Smith RA, Ali MA. Keratin-hydroxyapatite

composites: biocompatibility, osseointegration, and physical properties in an ovine model. J

Biomed Mater Res A. 2010;95:1084-95.

[4] Lu G, Ling K, Zhao P, Xu Z, Deng C, Zheng H, et al. A novel in situ-formed hydrogel

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2010;18:70-9.

[5] Pechter PM, Gil J, Valdes J, Tomic-Canic M, Pastar I, Stojadinovic O, et al. Keratin

dressings speed epithelialization of deep partial-thickness wounds. Wound Repair Regen. 2012.

[6] Richter JR, de Guzman RC, Van Dyke ME. Mechanisms of hepatocyte attachment to keratin

biomaterials. Biomaterials. 2011;32:7555-61.

[7] Shen D, Wang X, Zhang L, Zhao X, Li J, Cheng K, et al. The amelioration of cardiac

dysfunction after myocardial infarction by the injection of keratin biomaterials derived from

human hair. Biomaterials. 2011;32:9290-9.

[8] Sierpinski P, Garrett J, Ma J, Apel P, Klorig D, Smith T, et al. The use of keratin biomaterials

derived from human hair for the promotion of rapid regeneration of peripheral nerves.

Biomaterials. 2008;29:118-28.

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[9] Tachibana A, Nishikawa Y, Nishino M, Kaneko S, Tanabe T, Yamauchi K. Modified keratin

sponge: binding of bone morphogenetic protein-2 and osteoblast differentiation. J Biosci Bioeng.

2006;102:425-9.

[10] Nishida K, Kyoi S, Yamaguchi O, Sadoshima J, Otsu K. The role of autophagy in the heart.

Cell Death Differ. 2009;16:31-8.

[11] Zhang Y, Calderwood SK. Autophagy, protein aggregation and hyperthermia: a mini-

review. Int J Hyperthermia. 2011;27:409-14.

[12] Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, et

al. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on

autophagy genes. Nat Cell Biol. 2004;6:1221-8.

[13] Madden DT, Egger L, Bredesen DE. A calpain-like protease inhibits autophagic cell death.

Autophagy. 2007;3:519-22.

[14] Xu Y, Kim SO, Li Y, Han J. Autophagy contributes to caspase-independent macrophage

cell death. J Biol Chem. 2006;281:19179-87.

[15] Jackson DM. [The diagnosis of the depth of burning]. Br J Surg. 1953;40:588-96.

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SCHOLASTIC VITA

DEEPIKA PORANKI_______________________________________________

Wake Forest Institute for Regenerative Medicine

Wake Forest School of Medicine

Medical Center Boulevard

Winston Salem, NC-27157

EDUCATION______________________________________________________

Aug 2012 Ph.D. in Molecular Genetics

Wake Forest University

May 2005 M.S. in Molecular Genetics

Andhra University, INDIA

May 2003 B.S. in Biotechnology

Andhra University, INDIA

PROFESSIONAL EXPERIENCE_____________________________________

Jun 2012 – Aug 2012 Business Development Intern

Aerial Biopharma, Cary, NC

Aug 2007 – Aug 2012 Graduate Research Assistant

Institute for Regenerative Medicine, WFU

Aug 2011 – Apr 2012 Technology Transfer Intern

Office of Technology Asset Management, WFU

May 2011 – Oct 2011 Undergraduate Student Mentor

Institute for Regenerative Medicine, WFU

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210

Nov 2004 – Mar 2005 Research Intern

Central Tobacco Research Institute, INDIA

Aug 2003- Sep 2004 Graduate Teaching Assistant in Biochemistry

Andhra University, INDIA

HONORS AND AWARDS___________________________________________

Aug 2007 – Aug 2012 Graduate Student Fellowship

Wake Forest University Graduate School

Apr 2012 NC Biotech Center Summer Internship Award

North Carolina Biotech Center

Jan 2012 NABS Research Scholarship

North American Burn Society

Jan 2012 NABS Student Travel Award

North American Burn Society

Jan 2012 Alumni Student Travel Award

Wake Forest University

Nov 2011 Graduate Student Travel Award

Institute for Regenerative Medicine, WFU

Oct 2011 Grand Prize – Poster completion

10th

Annual Charlotte Biotechnology Conference

Jun 2011 AFIRM Fellow’s Career Enrichment Award

Armed Forces Institute for Regenerative Medicine

Jun 2008 Finalist – National Student Scholarship

Paul Foundation, INDIA

May 2005 University Second by GPA – Molecular Genetics

Andhra University, INDIA

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211

PUBLICATIONS___________________________________________________

Poranki, D *., Gaines, C

*., Du, W., Clark, R.A.F., Van Dyke, M.E. Development of deep partial

thickness burn model. (accepted in Burns Journal)

Poranki, D., Whitener, W., Howse S., Mesen, T., Howse, B., Burnell, J., Greengauz-Roberts,

O., Molnar. J., Van Dyke. M.E. A keratin biomaterial promotes skin regeneration after burns in

vivo and rescue cells after thermal stress in vitro. (manuscript in preparation)

Poranki, D., Van Dyke, M.E. Gamma keratose maintains cell viability in vitro after thermal

stress by regulating the expression of cell death pathway specific genes. (manuscript in

preparation)

Poranki, D., Gaines, C., Van Dyke, M.E. Assesment of deep partial thickness burn treatment

with keratin biomaterial hydrogels in a swine model. (manuscript in preparation)

Gaines, C., Poranki, D., Van Dyke, M.E. Delayed treatment of porcine deep partial thickness

burns subject to therapeutic keratin biomaterials. (manuscript in preparation)

ABSTRACTS – PODIUM PRESENTATIONS__________________________

Poranki ,D., Gaines, C., Van Dyke, M.E., Dec 2011. Development of a pivotal pre-clinical

porcine deep partial thickness burn model and testing of a keratin biomaterial hydrogel product.

Tissue Engineering and Regenerative Medicine North America (TERMIS-NA). Houston, TX

Poranki ,D., Gaines, C., Van Dyke, M.E., Jan 2012. A pre-clinical study testing the

effectiveness of keratin biomaterial hydrogel in porcine deep partial thickness burns. North

American Burn Society. Utah., CO

ABSTRACTS – POSTER PRESENTATIONS__________________________

Poranki, D., Gaines, C., Van Dyke, M.E., Mar 2012. Keratin biomaterial in the treatment of

burns. Wake Forest Institute for Regenerative Medicine Annual Retreat.

Poranki, D., Gaines, C., Van Dyke, M.E., Mar 2012. Keratin biomaterial in the treatment of

burns. Wake Forest University Graduate Student and post-doctoral Research Day.

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Gaines C, Poranki D, Van Dyke ME., Dec 2011. Optimized deep partial thickness swine burn

model for the evaluation of keratin hydrogel treatments. Materials Research Science.

Poranki, D., Gaines, C., Van Dyke, M.E., Nov 2011. Keratin biomaterial helps in the survival of

thermally stressed fibroblasts in vitro and promotes wound healing in vivo. Division of Surgical

Sciences 19th Annual Resident’s and Fellow’s Research Day.

Gaines, C., Poranki, D., Van Dyke, M.E., Nov 2011 Keratin Mediated healing of deep partial

thickness burns. Division of Surgical Sciences 19th Annual Resident’s and Fellow’s Research

Day.

Poranki, D., Gaines, C., Van Dyke, M.E., Nov 2011. Development of a porcine deep partial

(2⁰) thickness burn model. North Carolina Tissue Engineering & Regenerative Medicine

(NCTERM).

Poranki, D., Gaines, C., Van Dyke, M.E., Oct 2011. Keratin Biomaterial for Burn Wound

Healing. 10th Annual Charlotte Biotechnology Conference.

Gaines C, Poranki D, Van Dyke ME., Oct 2011. Optimized Wound Creation in a Deep Partial

Thickness Porcine Burn Model. Biomedical Engineering Society Annual Meeting (BMES).

Poranki, D., Gaines, C., Van Dyke, M.E., Jan 2011. Development of a porcine deep partial (2⁰)

thickness burn model. AFIRM All Hands Meeting.

Poranki, D., Gaines, C., Van Dyke, M.E., Dec 2010. Keratin Biomaterials Augment Burn

Wound Healing. Tissue Engineering & Regenerative Medicine North America (TERMIS-NA).

Poranki, D., Gaines, C., Van Dyke, M.E., Aug 2010. Keratin Biomaterials Promote Cell and

Tissue Survival in Burns. ATACC Meeting.

Poranki, D., Gaines, C., Van Dyke, M.E., Mar 2012. Heat shock response of Keratin

Biomaterial on Primary Mouse Fibroblasts – An in vitro Study. Wake Forest University

Graduate Student and Post Doctoral Research Day.

Poranki, D., Gaines, C., Van Dyke, M.E., Jan 2010 Effect of Keratin Biomaterials on Thermally

Injured Skin cells. AFIRM All Hands Meeting.

Poranki, D., Vinukonda, N., Katakam, A. Data Mining Colorectal Cancer CGH data to

investigate gender disparities. Association of Genetic Technologists Annual Meeting.

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Poranki, D. Are we losing hope for a cure to Down’s syndrome? Annual Meeting of American

College of Clinical Genetics.

Poranki, D., Sarala, K., Aug 2005 An RAPD based genetic diversity study to determine the

genetic similarity among advanced tobacco breeding lines. Central Tobacco Research Institute

Research Day.