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EX VIVO INVESTIGATION OF NOVEL WOUND HEALING THERAPIES AND DEVELOPMENT OF A 3-D HUMAN SKIN EQUIVALENT WOUND MODEL Yan Xie Bachelor of Medicine, Ningxia Medical University 2004 Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Faculty of Life Sciences Queensland University of Technology November 2008

INVESTIGATION OF NOVEL WOUND HEALING THERAPIES AND ...eprints.qut.edu.au/26541/1/Yan_Xie_Thesis.pdf · chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the

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EX VIVO INVESTIGATION OF NOVEL WOUND

HEALING THERAPIES AND DEVELOPMENT OF A

3-D HUMAN SKIN EQUIVALENT WOUND MODEL

Yan Xie Bachelor of Medicine, Ningxia Medical University 2004

Submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Faculty of Life Sciences

Queensland University of Technology

November 2008

Page i

Keywords

chronic

differentiation

fibroblasts

growth factors

healing

hyaluronic acid

keratinocytes

migration

proliferation

skin

skin equivalent model

synthetic fibrin-like gel

vitronectin

wound healing

Page ii

Abstract

It has previously been found that complexes comprised of vitronectin and growth factors

(VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these

complexes have been shown to significantly enhance the migration of dermal

keratinocytes derived from human skin. In view of this, it was thought that these

complexes may hold potential as a novel therapy for healing chronic wounds. However,

there was no evidence indicating that the VN:GF complexes would retain their effect on

keratinocytes in the presence of chronic wound fluid. The studies in this thesis

demonstrate for the first time that the VN:GF complexes not only stimulate proliferation

and migration of keratinocytes, but also these effects are maintained in the presence of

chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture

system provided insights into how the cells might respond to the VN:GF complexes, this

investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this,

a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo

environment, was used to test the VN:GF complexes on epidermopoiesis. These studies

revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and

differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified

epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the

DED in the presence of the VN:GF complexes and hyaluronic acid, another important

biological factor in the wound healing cascade. This HSE model was then further

developed to enable studies examining the potential of the VN:GF complexes in

epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound

model was created in fully-defined media and monitored as it healed. In this situation, the

VN:GF complexes were shown to significantly enhance keratinocyte migration and

Page iii

proliferation, as well as differentiation. This model was also subsequently utilized to

assess the wound healing potential of a synthetic fibrin-like gel that had previously been

demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown

to be markedly improved in the presence of this 3-D matrix, highlighting its future

potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this

synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created

in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as

revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix

was found to be dependent upon the presence of the fibroblasts. Taken together, these

data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo

tool to assess potential wound healing therapies. Moreover, the models will decrease our

reliance on animals for scientific experimentation. Additionally, it is clear that these

models will significantly assist in the development of novel treatments, such as the

VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately

facilitating their clinical trial in the treatment of chronic wounds.

Page iv

Table of Contents

CHAPTER 1: LITERATURE REVIEW ...............................................................1

1.1 Introduction.........................................................................................................1

1.2 Skin and wound healing......................................................................................3 1.2.1 Skin and Wounds........................................................................................3 1.2.2 Skin Wound Healing...................................................................................5 1.2.3 Keratinocyte Function and Normal Wound Healing Responses ...............7 1.2.4 Fibroblast Function and Normal Wound Healing Responses ...................9 1.2.5 Chronic Wounds.......................................................................................11 1.2.6 Keratinocyte and Fibroblast Behaviours in Chronic Wounds.................13

1.3 Growth factors and ECM for wound healing....................................................15 1.3.1 Growth Factors and ECM .......................................................................15 1.3.2 Vitronectin: Growth Factors Complex ....................................................17 1.3.3 Hyaluronic Acid.......................................................................................18 1.3.4 Hyaluronic Acid and Its role in Skin Wound Healing .............................19 1.3.5 Fibrin .......................................................................................................21 1.3.6 Synthetic Fibrin-like Gel..........................................................................22

1.4 In vivo and in vitro models ...............................................................................24 1.4.1 Animal Studies .........................................................................................24 1.4.2 2-Dimensional Monolayer Cell Culture System ......................................25 1.4.3 Tissue-Engineered Skin............................................................................26

1.5 Methods for wounding......................................................................................30

1.6 Project hypothesis and aims..............................................................................31

CHAPTER 2: INVESTIGATIONS INTO THE FUNCTIONAL RE SPONSES OF SKIN CELLS TO THE COMPLEXES OF VITRONECTIN:GROWT H FACTORS AND HYALURONIC ACID IN THE PRESENCE OF CHRO NIC WOUND FLUID.......................................................................................................33

2.1 Introduction.......................................................................................................33

2.2 Materials and methods ......................................................................................37 2.2.1 Introduction..............................................................................................37 2.2.2 Cell Culture..............................................................................................37 2.2.3 Pre-binding VN:GF to Culture Wells ......................................................37 2.2.4 HA Solution Preparation .........................................................................38 2.2.5 Migration Assay.......................................................................................39 2.2.6 Proliferation Assay ..................................................................................40 2.2.7 Chronic Wound Fluid Collection.............................................................40 2.2.8 Statistical Analysis ...................................................................................42

2.3 Results...............................................................................................................43 2.3.1 Investigations into the Effects of VN:GF and HA on Keratinocyte Migration. ..............................................................................................43 2.3.2 Investigations into the Effects of VN:GF and HA on Keratinocyte

Page v

Proliferation. ......................................................................................... 45 2.3.3 Investigations into the Effects of VN:GF and HA on Fibroblast Migration. .............................................................................................. 47 2.3.4 Investigations into the Effects of VN:GF and HA on Fibroblast Proliferation. ......................................................................................... 49 2.3.5 Investigations into the Effects of VN:GF in the Presence of CWF on Keratinocyte Migration. ........................................................................ 51 2.3.6 Investigations into the Effects of VN:GF in the Presence of CWF on Keratinocyte Proliferation..................................................................... 53

2.4 Discussion......................................................................................................... 55

CHAPTER 3: INVESTIGATIONS INTO THE EFFECTS OF CO MPLEXES OF VN:GF AND HA ON KERATINOCYTES USING HSEs............................. 60

3.1 Introduction....................................................................................................... 60

3.2 Materials and methods...................................................................................... 63 3.2.1 Skin Collection......................................................................................... 63 3.2.2 Pre-processing of Skin Samples............................................................... 63 3.2.3 Keratinocyte Isolation.............................................................................. 64 3.2.4 Keratinocyte Culture................................................................................ 64 3.2.5 Fibroblast Isolation and Culture ............................................................. 65 3.2.6 Preparation of Dermal Equivalent ......................................................... 65 3.2.7 Construction of Human Skin Equivalent ................................................ 66 3.2.8 Assessment of the Potential of VN:GF and HA Using HSEs................... 67 3.2.9 Measurements of Epidermis Outgrowth (MTT Assay)............................. 68 3.2.10 Assessment of the Proliferative and Differentiated Layers of the HSEs ...................................................................................................... 70 3.2.11 Blocking VN:GF Activity in the HSE Model......................................... 71 3.2.12 Assessment of the Effects of VN:GF and HA on Incorporation of

Fibroblasts into the DED........................................................................ 70 3.2.13 Statistical Analysis................................................................................ 71

3.3 Results .............................................................................................................. 72 3.3.1 Investigations into the Effects of VN:GF and HA on the Outgrowth of Keratinocytes Using HSEs Cultured at the Air:Liquid Interface .......... 72 3.3.2 Investigations into the Effects of VN:GF and HA on the Proliferative and Differentiating Layers of the Epidermis Using HSEs Cultured at the Air:Liquid Interface. .............................................................................. 77 3.3.3 Investigations into the Effects of VN-binding Integrins αv and the IGF-1R

Receptor on Blocking Keratinocyte Function Evoked by VN:GF using HSEs (Outgrowth) ................................................................................... 84

3.3.4 Investigations into the Effects of VN-binding Integrins αv and the IGF-1R Receptor on Blocking Keratinocyte Function Evoked by VN:GF using HSEs (Proliferative and Differentiated Layers of Epidermis) ................ 87

3.3.5 Investigations into the Effects of VN:GF and HA on Incorporation of Fibroblasts into the DEDs ...................................................................... 90

3.4 Discussion......................................................................................................... 92

CHAPTER 4: DEVELOPMENT OF 3-D HSE WOUND MODELS FOR TESTING NOVEL WOUND HEALING THERAPIES.............. ........................ 99

Page vi

4.1 Introduction.......................................................................................................99

4.2 Materials and methods ....................................................................................101 4.2.1 Skin Collection .......................................................................................101 4.2.2 Pre-processing of the Skin Samples.......................................................101 4.2.3 Keratinocyte Isolation............................................................................101 4.2.4 Keratinocyte Culture..............................................................................101 4.2.5 Fibroblast Isolation and Culture ...........................................................101 4.2.6 Preparation of Dermal Equivalent .......................................................101 4.2.7 Fabrication of a 6 mm Diameter Partial-thickness HSE Wound Model (Removal of the Epidermis) .................................................................102 4.2.8 Fabrication of a 4 mm Diameter Full-thickness HSE Would Model (Removal of the Epidermal/dermal Core)............................................102 4.2.9 Formation of Synthetic Fibrin-like Gel..................................................103 4.2.10 Assessment of the Synthetic Fibrin-like Gel Using 6 mm Diameter Partial-thickness Wounds in HSEs ..................................................105 4.2.11 Assessment of Synthetic Fibrin Gel in 4 mm Diameter Full-thickness Wounds in HSEs................................................................................106 4.2.12 Immunofluorescence ...........................................................................106 4.2.13 Wound Coverage Measurements (MTT Assay)...................................107 4.2.14 Histological Analysis ..........................................................................107 4.2.15 Immunohistochemical Analysis...........................................................108 4.2.16 Statistical Analysis ..............................................................................108

4.3 Results.............................................................................................................109 4.3.1 Development of a 6 mm Diameter Partial-thickness HSE Wound Model ..................................................................................................109 4.3.2 Validation of the 6 mm Diameter Partial-thickness HSE Wound Model (MTT)........................................................................................112 4.3.3 Validation of the 6 mm Diameter Partial-thickness HSE Wound Model (Histology). ..............................................................................116 4.3.4 Investigation of the Effect of the Topically-applied Synthetic Fibrin- like Gel on Keratinocytes in Wounded HSEs......................................120 4.3.5 Immunohistochemistry of the Topically-applied Synthetic Fibrin- like Gel on Keratinocytes in Wounded HSEs......................................126 4.3.6 Development of a Keratinocyte and Fibroblast Incorporated HSE Wound

Model ....................................................................................................129 4.3.7 Investigation of the Responses of Keratinocytes and Fibroblasts to Synthetic Fibrin-like Gel Using 4 mm Diameter Full-thickness Wounded HSEs.....................................................................................130

4.4 Discussion.......................................................................................................132

CHAPTER 5: GENERAL DISCUSSION.........................................................137

APPENDIX ............................................................................................................152

REFERENCES.......................................................................................................158

Page vii

List of Figures

Figure 1.1. Skin structure showing four layers of the epidermis................................. 4

Figure 1.2. The three overlapping phases of repair in acute wound healing................ 5

Figure 1.3. Fibrin clot formation................................................................................ 21

Figure 1.4. Factor XIIIa-catalyzed PEG hydrogel formation. .................................... 23

Figure 2.1. Migration of HaCaT keratinocytes exposed to HA in the absence and

presence of VN:GF................................................................................... 44

Figure 2.2. Proliferation of HaCaT keratinocytes exposed to HA in the absence

and presence of VN:GF. ......................................................................... 46

Figure 2.3. Migration of HFF fibroblasts exposed to HA in the absence and presence of VN:GF. ...........................................................................................48

Figure 2.4. Proliferation of HFF fibroblasts to HA in the absence and presence of VN:GF. ............................................................................................... 50

Figure 2.5. Migration of HaCaT human keratinocytes seeded in culture wells

exposed to different treatments .............................................................. 52

Figure 2.6. Proliferation of HaCaT human keratinocytes seeded in culture wells exposed to different treatments ............................................................ 54

Figure 3.1. Construction of human skin equivalent (HSE). ...................................... 67

Figure 3.2. Timeline of the experiment...................................................................... 67

Figure 3.3. MTT analysis of HSEs (3 days) . ............................................................ 73

Figure 3.4. Quantification of the outgrowth of keratinocytes in response to

VN:GF and HA (3 days) ........................................................................ 74

Figure 3.5. MTT analysis of HSEs (7 days) ............................................................. 75

Figure 3.6. Quantification of the outgrowth of keratinocytes in response to

VN:GF and HA (7 days) ......................................................................... 76

Figure 3.7. Histological and immunohistochemical analysis of HSEs (3 days) ...... 80

Figure 3.8. Quantification of the proliferative and differentiating layers of the

epidermis in response to VN:GF and HA on the HSEs (3 days) ........... 80

Figure 3.9. Histological and immunohistochemical analysis of HSEs (7 days) ....... 81

Figure 3.10. Quantification of the proliferative and differentiating layers of the

epidermis in response to VN:GF and HA on the HSEs (7 days) ........... 83

Figure 3.11. MTT analysis of HSEs treated with function blocking antibodies ....... 85

Figure 3.12. Quantification of the outgrowth of keratinocytes in response to function blocking antibodies ............................................................................... 86

Page viii

Figure 3.13. Histological and immunohistochemical analysis of HSEs treated with function blocking antibodies................................................................. 88

Figure 3.14. Quantification of the proliferative and differentiating layers of the epidermis in response to function blocking antibodies......................... 89

Figure 3.15. Histological analysis of the DED seeded with fibroblasts .................... 90

Figure 3.16. Quantification of the number of fibroblast entering DEDs treated

with VN:GF, HA and HA + VN:GF. . ................................................. 91

Figure 4.1. Factor XIIIa-catalyzed PEG hydrogel formation. .................................. 106

Figure 4.2. Photograph of approach used to create a 6 mm diameter partial-

thickness HSE wound model................................................................. 110

Figure 4.3. MTT analysis of the partial-thickness wounded HSEs and

histological analysis of the wounded HSEs........................................... 111

Figure 4.4. Representative images of the MTT analysis of the partial-thickness wounded HSEs.................................................................................... 114

Figure 4.5. Quantification of the uncovered wound area in the partial-thickness wounded HSEs.................................................................................... 115

Figure 4.6. Histological analysis of the partial-thickness wounded HSEs.............. 118

Figure 4.7. Quantification of the lateral migration of keratinocytes from the

wound edge to the centre of the partial-thickness wounded HSEs ....... 119

Figure 4.8. Representative images of the MTT staining of the partial-thickness wounded HSEs.................................................................................... 122

Figure 4.9. Quantification of the uncovered wound area in the partial-thickness wounded HSEs.................................................................................... 123

Figure 4.10. Representative images of the histological analysis of the partial-

thickness wounded HSEs ...................................................................... 124

Figure 4.11. Quantification of the lateral migration of keratinocytes from the

wound edge to the centre of the wound bed in partial-thickness

wounded HSEs ..................................................................................... 125

Figure 4.12. Marker expression in the partial-thickness wounded HSEs................ 127

Figure 4.13. Histological analysis of the DEDs seeded with keratinocytes and fibroblasts............................................................................................ 130

Figure 4.14. Photograph of approach used to create 4 mm diameter full-

thickness wounds in the HSEs. ............................................................ 129

Figure 4.15. Immunofluorescent staining of the full-thickness wounded HSEs

with a synthetic fibrin-like gel injected into the wounds. .................... 131

Page ix

List of Tables

Table 1.1. Products of tissue-engineered skin........................................................... 28

Table 1.2. Current methods used to create wounds................................................... 31

Page x

List of Abbreviations

ABAM antibiotic/antimycotic solution

Anti-IGF-IR anti-IGF-I receptor antibody

BSA bovine serum albumin

CWF chronic wound fluid

2-D 2-dimensional

3-D 3-dimensional

DED de-epidermised dermis

DMEM Dulbecco’s Modified Eagle’s Medium

ECM extracellular matrix

EDTA ethylenediamine tetraacetic acid

EGF epidermal growth factor

FBS foetal bovine serum

FGF fibroblast growth factor

FCS foetal calf serum

FG Full Green’s Medium

GAG glycosaminoglycan

Gln glutaminyl residues

HA hyaluronic acid

HaCaT human skin keratinocyte cell line

HBB HEPES binding buffer

H&E haematoxylin and eosin

HFF human foreskin fibroblasts

HSE human skin equivalent model

Page xi

IGF insulin-like growth factor

IGFBP insulin-like growth factor binding protein

IGF-1R type 1-IGF receptor

IL interleukin

Lys є-amino group of lysyl residues

K keratin

KGF keratinocyte growth factor

MMPs matrix metalloproteinases

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyletetrazolium

bromide

P1 passage 1

PBS phosphate buffered saline

PDGF platelet-derived growth factor

PEG polyethylene glycol

RGD arginine-glycine-aspartate

RHAMM receptor for HA mediated motility

SEM standard error of the mean

SFM serum-free medium

TGF transforming growth factor

Tris tris [hydroxymethyl] aminomethane

Triton X-100 iso-octylphenoxypolyethoxyethanol

VEGF vascular-endothelial growth factor

VN vitronectin

VN:GF vitronectin:growth factor complexes

Page xii

List of Publications and Presentations

Upton, Z., Cuttle, L., Noble, A., Kempf, M., Topping, G., Malda, J., Xie, Y., Mill,J.,

Harkin, D.G., Kravchuk, O., Leavesley, D.I., & Kimble, R.M. (2008). Vitronectin:growth

factor complexes hold potential as a wound therapy approach. Journal of Investigative

Dermatology, 128, 1535-1544.

Xie, Y., Rizzi, S., Dawson, R., Leavesley, D., & Upton, Z. (2008). Development of a 3D

human skin equivalent wound model: A powerful tool for testing novel wound healing

therapies. Manuscript under preparation.

Xie, Y., Rizzi, S., Dawson, R., Leavesley, D., & Upton, Z. (2008). Synthetic fibrin-like

gel in skin wound healing. Manuscript under preparation.

Xie, Y., Rizzi, S., Dawson, R., Leavesley, D., & Upton, Z. (2008). Development of a

3D human skin equivalent wound model: A powerful tool for testing novel wound

healing therapies. Proceedings of the Third Congress of the World Union of Wound

Healing Societies, Toronto, Canada.

Xie, Y., Rizzi, S., Dawson, R., Leavesley, D., & Upton, Z. (2007). In vitro evaluation

of the potential of VitroGro® and hyaluronic acid as wound healing agents.

Proceedings of Discovery Science and Biotechnology Meeting, Brisbane, Australia.

Xie, Y., Upton, Z., & Leavesley, D. (2006). The potential of VitroGro® and

hyaluronic acid in accelerating skin wound healing. Proceedings of IGFs Down

Under Conference, Brisbane, Australia.

Page xiii

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements

for an award at this or any other higher education institution. To the best of my

knowledge and belief, the thesis contains no material previously published or written by

another person except where due reference is made.

Signature: _________________________

Date: _________________________

Page xiv

Acknowledgments

Firstly, I would like to thank Tissue Therapies for supporting my work and the

Faculty of Life Sciences for my Fee Waiver Scholarship.

I would also like to express my sincere gratitude to the following people for their

invaluable assistance:

a) Thanks to my principle supervisor Professor Zee Upton for giving me the

opportunity to be a scientist, and also for her great guidance and assistance,

stimulating suggestions, continued encouragement, meticulous concern and

extraordinary patience throughout this long journey,

b) Thanks to my co-supervisor A/Professor David Leavesley for his generous,

informative and valuable directions,

c) Thanks to my co-supervisor Dr Simone Rizzi for his untiring supervision, valuable

hints and attentive instruction,

d) Thanks to Ms Rebecca Dawson for training in techniques and the collection of

skin,

e) Thanks to Dr Don Geyer for conducting H&E staining,

f) Thanks to my colleagues Gary Shooter, Brett Hollier, Tony Parker, Gemma

Topping, Danielle Borg, Erin Rayment, Melissa Fernandez, Shea Carter, Karsten

Schrobback for their kind assistance and friendship.

And last but not the least, thanks specially to the following:

a) My parents Mr and Mrs Xie for their endless and great love and extraordinary

sacrifice,

Page xv

b) My best friend, Jason Yeh for his wonderful friendship,

c) Uncle David Russell for his great support, continued encouragement, meticulous

concern through the final stage of my PhD journey,

c) Sean Richards for accompanying me side by side through the final and key stage

of my PhD journey.

Page 1

1CHAPTER 1: LITERATURE REVIEW

1.1 INTRODUCTION

Impaired skin integrity as a consequence of illness or injury may lead to acute loss of

physiologic equilibrium, such as water and electrolyte balance, and may also cause

disability or even death (Clark et al., 2007). In the U.S.A, an estimate of cases of

significant skin loss requiring major therapeutic intervention was 35.2 million in 1993,

and around 7 million of these patients ultimately suffered chronic wounds (Clark et al.,

2007). The current prevalence of chronic wounds in Australia is estimated at 200 000 –

600 000 people (1- 3% of the population), costing the Australian health care system more

than AUD 500 million annually (Baker & Stacey, 1994; Gruen et al., 1996). Treating

chronic wounds not only places a large economic burden on health services in many

countries, but also impacts on patients, causing absence from work, forced retirement and

loss of functional independence (Omar et al., 2004).

It has been found that growth factor wound healing therapies significantly reduce skin

healing time and reduce scar tissue formation when tested in clinical trials (Fu et al.,

2000). Although growth factors appear beneficial in their own right, they need to interact

with other growth promoting agents and proteins, and must be delivered appropriately in

the wound healing process (Moore, 1999). In addition, to treat intractable wounds such as

deeper burns and other full-thickness wounds, fabricated tissue-engineered skin

replacements, such as those that contain a biopolymer backbone (providing mechanical

support for cell migration and proliferation) and a bioactive agent (stimulating tissue cells

Page 2

to migrate, proliferate and differentiate), demonstrate great therapeutic potential (Clark et

al., 2007).

In vitro assessment of potential wound healing agents more often than not include studies

using skin cells grown in a 2-dimensional (2-D) monolayer culture due to the low cost

and efficient testing that results from this. However, it has been increasingly recognized

that 2-D cell culture has limitations and disadvantages (Sun et al., 2006b). In particular,

some evidence suggests that 2-D cell culture provides misleading and non-predictive data

for in vivo responses during drug testing (Birgersdotter et al., 2005; Weaver et al., 1997).

Human skin equivalent models (HSEs), which are derived by culturing skin keratinocytes

at the air:liquid interface on a dermal scaffold (Faller et al., 2002), have been proposed to

serve as useful in vitro models to recapitulate and further understand the complicated

healing process of skin. Specifically, these models have been shown to be similar to in

vivo human skin, both morphologically and biochemically (Ponec et al., 2002; Poumay et

al., 2004). The similarity that HSEs share with in vivo skin has allowed these to be used

for various purposes including skin grafts, testing toxicity, irritancy, and metabolic study

of topically applied products (Yeh et al., 2004). Thus, HSEs are believed to be a valuable

instrument for investigating the effects of new techniques and therapies on wound

healing.

As described herein, my PhD project is focussed on investigation of the potential of novel

wound healing therapies and the development of a platform technology suitable for ex

vivo testing of these therapies. This literature review therefore provides background

information on skin wound healing, the characteristics of chronic wounds, and the

Page 3

important roles that keratinocytes and fibroblasts play in the wound healing process.

Specifically, this review will cover novel wound healing therapies such as the

vitronectin:growth factor complexes (VN:GF), hyaluronic acid (HA), as well as various

approaches that have been reported for developing in vitro models of skin.

1.2 SKIN AND WOUND HEALING

1.2.1 Skin and Wounds

Skin, the membranous tissue forming the external covering of the body, is the largest

organ in the human body (Enoch & Price, 2004). Structurally, it is divided into two

distinct layers: the outer multilayered epidermis, and the underlying dermis from

which it is separated by a basement membrane (Raghavan et al., 2000). Specifically,

the epidermis is a keratinized stratified epithelium, containing two types of

proliferating keratinocytes: long term self-renewing multipotent stem cells, and

transient amplifying cells, which are committed to lose proliferative capacity and

undergo terminal differentiation (Vaughan et al., 2004). According to keratinocyte

morphology, the epidermis is divided into four or five layers: the stratum basale,

stratum spinosum, stratum granulosum, stratum lucidium (only in thick skin) and

stratum corneum (see figure 1.1). In addition to keratinocytes, melanocytes,

Langerhans’ cells and Merkel cells are also present within the epidermis. The dermis

on the other hand is connective tissue composed mostly of elastic fibres, collagens,

HA, hair follicles, glands, lymphatic and blood vessels, endothelial cells, Schwann

cells, adipocytes and fibroblasts (Gross & Schmitt, 1948). Fibroblasts are the main

cell type within the dermis that synthesize the precursors of the extracellular matrix

Page 4

(ECM), such as fibres and glycosaminoglycans (GAGs), to maintain the structural

integrity of connective tissue (Lozzo & Murdoch, 1996).

Figure 1.1. Skin structure showing four layers of the epidermis: the stratum

basale, stratum spinosum, stratum granulosum and stratum corneum.

Skin plays a very important role in life sustenance through regulating water and

electrolyte balance, thermoregulation, and provides a physical barrier to the passage

of external noxious agents, including micro-organisms (Enoch & Price, 2004).

Severe problems such as loss of homeostatic function can arise when this physical

barrier is compromised through a wound. A wound can present as a split in the

epithelial integrity of the skin, as well as with deeper punctures with disruption

extending to the dermis, subcutaneous fat, fascia, muscle, or even the bone (Enoch &

Price, 2004). Therefore it is imperative that any wound sustained by the skin be

healed to restore homeostasis.

Dermis

Stratum Basale

Stratum Spinosum

Stratum Corneum Stratum Granulosum

Page 5

1.2.2 Skin Wound Healing

Wound healing (repair) involves a series of cellular events and a cascade of co-ordinated

and systematic biochemical events. These events are the body’s natural processes that

restore the integrity and function of the epidermis and dermis (Iba et al., 2004;

Stadelmann et al., 1998). These complicated, overlapping processes may be regarded as

three successive phases of inflammation, proliferation and remodelling (Iba et al., 2004)

as shown in figure 1.2.

Figure 1.2. The three overlapping phases of repair in acute wound healing.

In the inflammatory phase, polymorphonuclear leucocytes and macrophages phagocytose

bacteria and debris and release degrading enzymes or oxygen-derived free radical species.

In addition, macrophages and lymphocytes release factors that cause the migration and

proliferation of fibroblasts and keratinocyte cells that are involved in the proliferative

Hours after wounding (Logarithmic Time Scale)

24 240 720 72000

100%

80%

40%

20%

60%

Inflammation

Proliferation & matrix deposition

Matrix remodelling

Mag

nitu

de o

f Max

imum

Bio

logi

cal R

esp

onse

Page 6

phase. One significant part of the inflammatory response is the production of exudate

(wound fluid) from plasma. Wound fluid forms a clot which has been shown to provide

an effective seal to bacteria and the external environment, as well as provide the wound

with oxygen and all the necessary constituents for the healing process to occur (Enoch &

Price, 2004).

The proliferative phase, which starts at about day three and lasts for about two weeks after

injury includes: stages of angiogenesis, collagen synthesis, fibroplasia, granulation tissue

formation, epithelialisation and wound contraction (Midwood et al., 2004). During

angiogenesis, endothelial cells establish new blood vessels to provide oxygen and

nutrients for the healing tissue (Chang et al., 2004). In fibroplasia and granulation tissue

formation, fibroblasts secrete a provisional ECM composed of collagen and fibronectin to

support cell migration (Midwood et al., 2004). In epithelialisation, epidermal cells

migrate across the wound bed and a new basement membrane is formed, which separates

the epidermis and dermis (Krawczyk, 1971). During the contraction phase the size of the

wound is reduced by the contraction of actin in myofibroblasts, pulling the edges of the

wound together (Midwood et al., 2004). Finally, in the maturation and remodelling phase,

the provisional matrix formed of type III collagen is gradually degraded and stronger type

I collagen is deposited, increasing the tensile strength of the wound (Stadelmann et al.,

1998). The decrease of activity at the wound site triggers the apoptosis and removal of

cells such as macrophages and fibroblasts; these are no longer needed for the wound

healing response (Desmoulière et al., 1995).

Page 7

1.2.3 Keratinocyte Function and Normal Wound Healing Responses

The keratinocytes apparently demonstrate spatial awareness as they divide and position

themselves within the epidermis to maintain the integrity and normal function of the skin

(O’Toole, 2001). In normal epidermis, mitotically active keratinocytes are located in the

basal layer of the epidermis, where they directly contact the basal lamina and proliferate

slowly. In the suprabasal layers the keratinocytes start terminal differentiation as they

migrate toward the surface of the skin and their death (Tomic-Canic et al., 2004).

Keratinocytes are highly dependent on autocrine and paracrine communication, and this is

an important part of the wound healing response. The keratinocytes will communicate

with each other to recognise and repair the wound, as well as communicate with other cell

types to initiate wound closure and the host defence system (Tomic-Canic et al., 1998).

A few hours after injury keratinocytes start to migrate without proliferating (Bartkova et

al., 2003). If the wound is superficial and the basement membrane is not broken, the

epithelial cells are substituted in three days in the same manner as normal skin (the cells

in the stratum basale layer divide and migrate apically) (Romo & Pearson, 2008).

Alternatively, if the wound is sufficiently deep and the dermal appendages are damaged,

cells located at the wound edge, such as keratinocytes and fibroblasts, will migrate into

and across the wound (Mulvaney & Harrington, 1994). On the second and third days after

wounding the keratinocytes continue to proliferate at the wound edge and migrate

forward in a sheet, named the epithelial tongue (Hübner et al., 1996).

During the migratory processes of re-epithelialisation, the keratinocytes can be

categorized into three different groups: (1) the highly proliferative cells located at the

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wound edge which generate the cells that crawl over the wound bed; (2) the cells located

at the front of the migrating tongue and which express integrins, matrix

metalloproteinases (MMPs) and plasminogen activators; and (3) the cells that follow the

foremost edge and are responsible for regenerating the basement membrane and

epidermis (Bechtel et al., 1998; Saarialho-Kere et al., 1994; Saarialho-Kere et al., 1995;

Sudbeck et al., 1997). In order for these cells to migrate, certain chemical signalling

molecules, such as nitric oxide, along with deficiency of cellular contact inhibition, need

to occur (Lee et al., 2001). Furthermore, keratinocytes need to dissolve their desmosomes

(cell-to-cell adhesion) and hemidesmosomes (cell-to-ECM adhesion) (Santoro &

Gaudino, 2005), disconnect from the basement membrane, and migrate into the wound

bed (Choma et al., 2004). To date the two main theories for how keratinocytes migrate

across the wound bed are:

1. “Leap-Frogging”: Keratinocytes located on the surface of the stratum basale at

the wound edges creep into the wound bed. The cells above and following will

crawl over the first cells and “lounge” on the wound bed in front of them

(Krawczyk, 1971); and

2. “Tractor tread”: After the keratinocytes synthesize integrin receptors on their cell

surface, they can migrate by using these integrin receptors to bind fibronectin,

vitronectin (VN) and other ECM proteins of the provisional wound bed (Cheresh et

al., 1989)

During this process the keratinocytes start to secrete proteins to form a new basement

membrane as they migrate across the wound bed (Krawczyk, 1971). This migration

Page 9

across the wound bed continues until the opposing keratinocytes meet and undergo

contact inhibition triggering the final stage, that is, stratification (Bartkova et al., 2003).

Stratification involves the deactivation of the keratinocyte’s migratory behaviour. The

cells then switch to proliferation and differentiation to form the various strata, such as

stratum basale, spinosum, granulosum, lucidum and corneum (Tomic-Canic et al., 2004).

The process of stratification also involves two major changes in the keratinocytes as they

move towards the outer layer of the skin. Firstly, they secrete a protective, waxy protein

called keratin, and secondly they become more squamous in morphology and eventually

die, forming the protective outer layer (Choma et al., 2004).

1.2.4 Fibroblast Function and Normal Wound Healing Responses

In addition to keratinocytes, fibroblasts also have an important role in maintaining the

structural integrity of connective tissue in skin. They continuously secrete precursors of

the ECM, that is, a variety of fibres and “ground substance,” which is mainly composed

of GAGs, proteoglycans and glycoproteins (Lozzo & Murdoch, 1996). Approximately

two to three days after injury fibroblasts start to migrate into the wound area from the

margins of the undamaged tissue, initiating the proliferative phase (Choma et al., 2004).

When fibroblasts arrive at the wound site they alter their phenotype to be more contractile

and to increase collagen production (Ågren et al., 1999). Specifically, fibroblasts migrate

across the wound bed by adhering to fibronectin in the fibrin clot formed during the

inflammatory phase (Romo & Pearson, 2008) and deposit the non-collagenous

components of ECM into the wound bed. Subsequently, the fibroblasts deposit collagen

to facilitate cell migration. Primarily, fibroblasts proliferate and migrate in the first two to

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three days after wounding, subsequently switching their function to the synthesis and

deposition of the new collagen matrix (Stadelmann et al., 1998).

Another important characteristic of the proliferative phase of wound healing is the

formation of granulation tissue. On the second to fifth day after wounding granulation

tissue appears in the wound site and continues to expand until the wound bed is covered

(Clark, 1993). Granulation tissue is poorly organised and consists mainly of new blood

vessels, inflammatory cells, endothelial cells, fibroblasts, myofibroblasts and the new

provisional ECM (Kurkinen et al., 1980). The new provisional ECM contains fibronectin,

VN, collagen, proteoglycans and GAGs, such as HA (Romo & Pearson, 2008).

Fibronectin and HA, the major constituents of granulation tissue, facilitate cell migration

by providing a well hydrated matrix for subsequent healing events (Krawczyk, 1971).

Later, ECM similar to that found in normal tissue will replace this provisional matrix.

Fibroblasts migrate across the wound depositing ECM molecules such as fibronectin,

proteoglycans, elastin, glycoproteins and GAGs, commonly found in the matrix of normal

tissue (Hübner et al., 1996).

Fibroblasts also secrete and deposit collagen two to three days after wounding (Romo &

Pearson, 2008). Before collagen is deposited, fibrin-fibronectin, which is poor at resisting

injury, provides the main structural support for the wound clot. This is replaced later by

the collagen to provide strength to the wounded area. Additionally, collagen provides a

scaffold for cells involved in inflammation, angiogenesis and the connective tissue

construction processes, to adhere to and facilitate proliferation and differentiation. The

number of fibroblasts in the wound declines as the granulation process comes to an end

(Reed et al., 2001). At the completion of the granulation phase, apoptosis of the

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fibroblasts results in the granulation tissue being predominantly comprised of collagen

(Stadelmann et al., 1998). Approximately one week later growth factors stimulate the

fibroblasts to differentiate into myofibroblasts and contract the wound. An increase in

chondroitin sulfate and a decrease in HA production lead to the reduction of migration

and proliferation of the fibroblast cells. When myofibroblasts stop contracting and start

apoptosis, the contraction phase in proliferation ceases and the remodelling stage of

wound healing commences (Eichler & Carlson, 2006).

In addition to secreting and depositing ECM components, dermal fibroblasts have

autocrine and paracrine functions (Igarashi et al., 1993). It has been demonstrated that

growth factors and cytokines such as keratinocyte growth factor (KGF), interleukin (IL)-6

and fibroblast growth factor (FGF)-10 (Boxman et al., 1993; Marchese et al., 2001),

secreted by fibroblasts, disperse into the upper layers of the epidermis affecting

keratinocyte proliferation and differentiation (Aoki et al., 2004; El-Ghalbzouri et al.,

2002). Fibroblasts play two important roles in basement membrane construction, namely:

(1) to deposit collagen types IV and VII, laminin 5 and nidogen; and (2) to secrete

cytokines such as transforming growth factor (TGF)-β that promote keratinocytes to

synthesize basement membrane components, such as collagen types IV and VII (Kahari et

al., 1991; Konig et al., 1992; Marinkovich et al., 1993).

1.2.5 Chronic Wounds

Changes in cytokines, proteases, growth factors, extracellular and cellular elements,

however, have the potential to impair normal wound healing, resulting in the

development of a chronic wound (Enoch & Price, 2004). The underlying

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pathobiology (e.g., venous insufficiency, diabetes mellitus, arterial occlusion and

high external pressure), as well as microorganism invasion, interrupt the normal

wound healing process (including haemostasis, inflammation, proliferation and

remodelling). This can stall the wound healing process in the inflammatory or

proliferation phases (Enoch & Price, 2004).

There are many differences between a normal and a chronic wound. For example,

chronic wounds have a prolonged inflammatory response possibly triggered by a

bacterial burden, which is indicated by increased levels of neutrophil proteinases

(Grinnell & Zhu, 1994). The chronic wound provides an optimal niche for many

bacteria, aerobes and anaerobes (Tomic-Canic et al., 2004). Inflammatory cytokines,

such as tumor necrosis factor-α (TNF-α) and the interleukins (IL-1, IL-6), are

secreted by cells in response to endotoxin products produced by bacteria (Staiano-

Coico et al., 2000). The resolution of prolonged inflammation that precedes wound

re-epithelialisation closely relates to the establishment of an appropriate balance

between proteinase and proteinase inhibitor levels (Tomic-Canic et al., 2004).

Increased levels of serine proteases (neutrophil elastase, the physiologic target of α1-

antitrypsin) accompanied by decreased activity of α1-proteinase inhibitor (α1-

antitrypsin, a potent regulator protecting fibronectin from degradation) are likely to

be responsible for the degradation of the temporary matrix that supports the growth

of cells during wound repair (Rao et al., 1995).

Provisional changes in the wound environment may be modulated also by

components present in the exudate released from wounds (Tomic-Canic et al., 2004).

For example, if degraded peptides from VN and fibronectin are present, this indicates

Page 13

excessive proteolytic activity (Grinnell et al., 1990). Furthermore, elevated levels of

gelatinase-A and B, observed within chronic wound fluid (CWF), result in the

degradation of denatured collagens (gelatins), basement membrane collagens, and

several other matrix proteins (Collier et al., 1988; Wilhelm et al., 1989). In addition,

reduced levels of tissue inhibitors of metalloproteinases-1 (TIMP-1), combined with

higher levels of gelatinolytic enzymes (matrix metalloproteinases MMP-2 and 9),

have the potential to destroy surrounding tissue and degrade basement membrane

components (Howard & Banda, 1991; Kjeldsen et al., 1994; Wysocki et al., 1993).

Cellular senescence, also known as the "Hayflick limit," is a natural phenomenon

associated with the aging of cells. It is characterized by reduced proliferative capability,

changes in morphology and overexpression of certain matrix proteins (fibronectin)

(Tomic-Canic et al., 2004). In chronic wounds, cellular senescence may be induced by the

wound fluid (Ågren et al., 1999) and can be identified by increased activity of beta-

galactosidase (Mendez et al., 1998). It is likely that there is an excess of senescent

keratinocytes in chronic wounds. These are less migratory and have limited replicative

potential, which in turn leads to abnormal wound responses to healing stimuli such as

growth factors (Tomic-Canic et al., 2004).

1.2.6 Keratinocyte and Fibroblast Behaviours in Chronic Wounds

Although factors stimulating keratinocyte migration and proliferation and the underlying

molecular mechanisms have been widely studied, keratinocytes in chronic wounds have

not been extensively investigated (Li et al., 2001; Pullar et al., 2003; Sharma et al., 2003).

In chronic wounds keratinocytes migrate poorly, if at all, over the ulcer bed, although they

Page 14

appear to proliferate normally (Adair, 1977; Andriessen et al., 1995; Seiler et al., 1989).

This may arise from keratinocytes at the chronic wound edge being unable to transduce

signals that result in a migratory phenotype. Alternatively, they may not receive signals at

all (Tomic-Canic et al., 2004). It has also been proposed that the abundant MMPs and

gelatinases present in the chronic wound exudate degraded basement membrane proteins,

such as laminin and type IV collagens. These are required for migration by keratinocytes,

and indeed are secreted by the keratinocytes (Larjava et al., 1993). Basement membrane

components may also be degraded by collagenase and stromelysins, factors that have

been found also to be overproduced by keratinocytes in chronic wounds (Oikarinen et al.,

1993).

Studies of fibroblasts isolated from skin adjacent to wounds from patients with chronic

venous ulcers also exhibit a reduced capability to synthesize collagen. These fibroblasts

demonstrate diminished response to TGF-β and an increased propensity to be senescent

(Kim et al., 2003; Stanley et al., 1997). Senescent cells are unresponsive to physiologic

mitogens, such as growth factors, and are unable to reinitiate DNA synthesis and cell

division (Di Felice et al., 2005). Fibroblasts cultured from patients with diabetic ulcers are

reported also to have a changed morphology and decreased proliferative capacity (Loots

et al., 1999).

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1.3 GROWTH FACTORS AND ECM FOR WOUND HEALING

1.3.1 Growth Factors and ECM

Skin repair is a complex process involving several stages, including inflammation,

protein synthesis, matrix deposition, migration, and proliferation of keratinocytes

from the wound edge (Clark, 1993). Although a great deal remains unknown about

the complex biological processes required for successful healing of wounds, it has

been found in clinical trials that growth factor wound healing therapies can accelerate

significantly skin healing and reduce scar tissue formation (Fu et al., 2000). As

growth factors play such an important role during the wound healing process, a large

amount of both in vitro and in vivo experimentation has been conducted. In the past

10 years the appearance of engineered growth factors, including platelet-derived

growth factor (PDGF), insulin-like growth factor (IGF)-I, epidermal growth factor

(EGF), basic FGF, TGF-α and TGF-β and vascular endothelial growth factor (VEGF)

have been produced through the application of recombinant DNA technologies, and

many of these factors have been tested in animal and human wound healing trials (Fu

et al., 2000). As mentioned earlier in this review, growth factors initiate the wound

healing cascade by attracting and activating fibroblasts, endothelial cells and

macrophages (Enoch & Price, 2004). The role of growth factors in the regulation of

cell proliferation, differentiation and tissue growth has been extensively documented

and several growth factors have been identified as being critical to the coordination

of healing processes (Trengove et al., 2005). However, the only growth factor

approved by the U.S. Food and Drug Administration for human use in wound healing

thus far is PDGF. PDGF has been shown in randomized, controlled clinical trials to

Page 16

accelerate the healing of neuropathic diabetic foot ulcers by about 15 percent (Smiell

et al., 1999). Recently b-FGF has also been approved for use in humans as a wound

healing therapy in Japan (Kawai et al., 2000). Moreover, granulocyte-macrophage

colony-stimulating factor (Da Costa et al., 1999) and EGF (Brown et al., 1989;

Falanga et al., 1992) demonstrated success in human trials; and TGF-β (Broadley et

al., 1989; Shah et al., 1992) and VEGF (Nissen et al., 1998) on the other hand have

demonstrated success in animal studies. However, the widespread clinical application

of growth factors has been impeded by prohibitive costs, high doses, and inconsistent

success of these treatments (Upton et al., 2008). Perhaps more importantly,

administration of individual growth factors neglects the role of the interaction

between ECM and growth factors in biological processes associated with wound

healing (Schneller et al., 1997). These various processes are coordinated by

environmental signals such as ECM molecules and peptide growth factors (Galiano

et al., 1996).

Since the temporary wound bed matrix is rich in growth factors, the interaction

between the ECM and growth factors is regarded as very important in wound healing

(Clark, 1993). Several growth factors involved in wound repair have been found to

interact with components of the ECM (Kawahara et al., 2002). For example,

fibronectin has been found to bind to insulin-like growth factor binding protein 5

(IGFBP-5) (Xu et al., 2004), TGF-β1 (Mooradian et al., 2004) and VEGF (Wijelath

et al., 2002), while collagens have been found to bind to KGF (Ruehl et al., 2002)

and TGF-β1 (Zhu et al., 1999). Moreover, vitronectin (VN) has been found to bind to

IGF-family members (Kricker et al., 2003; Upton et al., 1999), EGF (Schoppet et al.,

2002) and b-FGF (Stockmann et al., 1993). It is now thought that these growth-

Page 17

factors:ECM interactions are key regulators for most processes of tissue growth and

repair, including cell attachment, proliferation, migration, differentiation, cell

survival, and angiogenesis (Schneller et al., 1997).

1.3.2 Vitronectin: Growth Factors Complex

Recent studies in the QUT Tissue Repair and Regeneration Research Program have

investigated the functional effects of a substrate-bound complex composed of growth

factors and ECM proteins. Initially, it was discovered that the ECM protein VN can

bind to IGF-II (Upton et al., 1999). Subsequently it has been observed that while

IGF-II can bind to VN directly, IGF-I will only associate with VN through insulin-

like growth factor binding protein (IGFBP)-2, -3, -4, or -5 (Kricker et al., 2003).

Furthermore, both dimeric (IGF-II + VN) and trimeric (IGF-I + IGFBPs + VN)

complexes have been shown to enhance keratinocyte protein synthesis and migration

(Hyde et al., 2004).

Based on these encouraging experimental results, our laboratory hypothesised that

the cooperation of growth factors and VN may also promote the healing of wounds,

such as diabetic foot ulcers and chronic venous ulcers. Recent findings that VN:IGF-

I:IGFBP3 or VN:IGF-I:IGBBP-5 complexes significantly enhance the migration of

dermal keratinocytes and fibroblasts derived from diabetic skin (Noble, 2008)

support this hypothesis. These data suggest that these novel complexes hold the

potential to overcome the stalled repair cascade observed in diabetic wounds.

Furthermore, the VN component in the VN:GF complex may make growth factors at

the wound site more stable, possibly protecting them from proteolytic degradation

Page 18

(Upton, personal communication, 9 September 2007). These stabilising and

protecting effects may be useful when delivering growth factors in the chronic wound

environment. However, the wound environment is complex and consists of many

different factors and components which aid in the wound healing events. Therefore,

the VN:GF complex may be further enhanced with the addition of another important

component of the wound environment, hyaluronic acid.

1.3.3 Hyaluronic Acid

Hyaluronic acid (HA) is another biological factor that is important in the wound

healing cascade. HA was initially discovered in bovine vitreous humor by Meyer and

Palmer in 1934. It is a high molecular weight polyanionic polysaccharide component

of the extracellular matrix. HA is a simple disaccharide composed of repeated N-

acetyl-D glucosamine and beta-glucoronic acid units (Barker et al., 1964). HA is

synthesized during the early stage of the inflammatory phase of wound repair.

However, it is subsequently degraded by fibroblasts when glycosaminoglycan (GAG)

synthesis is increasing (Alexander & Donoff, 1980). HA is an important constituent

of all connective tissues in the body and is widely distributed in the skin, eye, joint

and many other tissues and organs (Barker et al., 1964). Originally HA was thought

to have mechanical functions only because it plays a protective, shock-absorbing and

structure stabilizing role in the body. However, recently the role of HA in the

mediation of physiological functions such as morphogenesis, regeneration, wound

healing (DeGrendele, 1997) and tumor invasion (Yu et al., 1997) via interaction with

binding proteins and cell surface receptors has been recognised. It also has been

reported that HA dynamically regulates cell signalling and behaviour (Hall et al.,

Page 19

1994). Interactions between HA and its cognate cell surface receptors, CD44 and

hyaladherin (RHAMM), are thought to facilitate cell adhesion, cell motility, and

cellular proliferation (Lokeshwar et al., 1996).

1.3.4 HA and Its Role in Skin Wound Healing

HA is especially enriched in mammalian skin, in both the dermis (spongious dermis

of anuran integument) (~0.5 mg/g wet tissue) and epidermis (pericellular epidermal

matrix) (~0.1 mg/g wet tissue) (Banks et al., 1976). Although the structure of HA is

very simple, it has remarkable rheological, viscoelastic and hygroscopic properties

that are critical to dermal tissue function. Through a network of helicoidal structures,

HA is believed to contribute to the elastic properties of the dermis (Bernstein et al.,

1996) and to epidermal differentiation (Tammi & Tammi, 1991). In particular, the

highly hydrated environment provided by HA is helpful for cell migration, an

essential event in the early phase of injury, inflammation and wound healing (Weigel

et al., 1988). It has also been reported that changes in HA concentration within the

ECM modulates several cellular functions, such as cell migration (Melrose et al.,

1996), adhesion (Klein et al., 1996) and proliferation (Wiig et al., 1996). It is believed

that HA participates in the wound healing process and scarless fetal healing through

its influence on signalling pathways (Hall et al., 1994). It is also thought to modulate

collagen synthesis during wound repair. Indeed, it has been postulated that HA may

increase type III collagen synthesis which then leads to scarless healing of deep

partial thickness burns (Yang & Ge, 1995).

Page 20

In clinical trials, healing of skin lesions has been improved by topical application of

HA. For example, acute radioepithelitis, diabetic foot lesions and venous ulcers all

respond to HA treatment. A French study reported a reduction of wound area of 23%

at 7 days and 48% at 21 days in 50 patients with venous leg ulcers through the use of

HA on a gauze pad (Ortonne, 1996). Further, it is reported that 11/14 diabetic foot

ulcers patients healed in 64 days following application of autologous keratinocytes

grown on thin sheet grafts with HA (Lobmann et al., 2003).

While these HA and VN:GF appear beneficial in their own right, they must be

delivered appropriately in the wound healing process (Moore, 1999). For example,

the synthetic fibrin-like gel mimics fibrin: that is, (1) it has adhesion sites located in

the matrix to permit cells to attach and migrate into the gel; and (2) the matrix which

reacts to cell-derived proteolytic activity may provide an appropriate delivery vehicle.

1.3.5 Fibrin

Fibrin clots provide a primary matrix for wound healing and they also occur naturally in

the body. When an injury to tissue occurs, the precursor molecule fibrinogen infiltrates

into the wound from the compromised blood vessels. The activity of thrombin catalyses

the formation of a loose gel through enzymatic cleavage and self-catalysis (Pisano et al.,

1968). The transglutaminase factor XIIIa is then covalently cross- linked by glutaminyl

(Gln) and є-amino group of lysyl (Lys) residues by enzymatic action in the presence of

Ca2+ at a physiological concentration (that can be found both in humans and animals),

resulting in a stable matrix (figure 1.3) (Weisel, 2005).

Page 21

Figure 1.3. Fibrin clot formation.

This fibrin matrix “clot” facilitates the invasion of monocytes and fibroblasts into the

wound site, as well as provides a foundation for collagen deposition (Grinnell et al.,

1981). Due to its properties, fibrin has not only been used therapeutically as surgical glue,

but has also been evaluated for use as a biodegradable and biocompatible material in

biomedical applications such as tissue engineering (Eyrich et al., 2007; Jockenhoevel et

al., 2001) and drug delivery (Bhang et al., 2007; Sakiyama-Elbert & Hubbell, 2000;

Thomas & Campbell, 2004).

One of the most important advantages of fibrin is that this matrix guides cells and permits

them to readily infiltrate and traverse the wound site (Herbert et al., 1996). There are two

key features involved in this process: (1) adhesion sites located in the matrix permit cells

to attach and migrate into the gel; and (2) the matrix reacts to cell-derived proteolytic

activity. This allows cells to degrade the matrix locally by secreting plasmin or MMPs

and then migrate into the matrix without constraint (Herbert et al., 1996; Pepper, 2001).

Fibrinogen

Thrombin

Fibrin/Clot Formation

Ca2+

Ca2+ Factor XIII

Page 22

1.3.6 Synthetic Fibrin-like Gel

With the rapid advances in synthetic biomaterials, the characteristics of natural ECMs are

capable of being imitated. For example, hydrogels have been synthesized by

copolymerization of two building blocks: (1) multi-arm polyethylene glycol (PEG)

macromers responsible for the biophysical characteristics of the network; and (2)

biologically active peptide sequences, such as protease substrates for MMPs or integrin-

binding domains (RGDSP) (Lutolf et al., 2003). The incorporation of RGD and MMP

cleavage sites therefore enables this synthetic hydrogel to mimic two essential biological

functions of an ECM: cell adhesion and protease degradation (Raeber et al., 2005). The

characteristics of the hydrogel, such as physicochemical properties, proteolytic sensitivity

of the peptide and adhesion ligand density, can independently control the cell migration

rate (Lutolf et al., 2003.).

To synthesize fibrin-like biomolecular hydrogels, a gel network is formed by cross-

linking two types of branched PEGs attached with two counter reactive substrates for

factor XIIIa respectively. Bioactive molecules are then incorporated by utilising synthetic

chemistry approaches so that they incorporate factor XIIIa substrate sequences (Ehrbar et

al., 2007b). Factor XIIIa, an activated transglutaminase cross-linking enzyme, plays an

important role in forming the fibrin clot during wound healing (Weisel, 2005). It catalyses

acyl-transfer reactions between Lys and the α-carboxamide group of protein-bound Gln,

leading to the formation of є-(α-glutamyl) lysine isopeptide side-chain bridges (Curtis et

al., 1974).

Page 23

To form multifunctional synthetic hydrogels with fibrin-like biomolecular characteristics,

two different transglutaminase peptide substrates containing Gln and Lys (as well as a

substrate for matrix MMPs) are attached to multi-arm PEG macromers in

stoichiometrically equivalent amounts, respectively, as two precursors. These two

precursors (n-PEG-Gln and n-PEG-MMP-Lys), in combination with a cell adhesion

peptide (TG-Gln-RGD), are then cross-linked by the transglutaminase enzyme factor

XIIIa in the presence of Ca2+ (figure 1.4) (Ehrbar et al., 2007b).

Figure 1.4. Factor XIIIa-catalyzed PEG hydrogel formation (Reproduced from

Ehrbar et al., 2007b, with permission from Rizzi)

In fact, Ehrbar et al. (2007a) reported that these synthetic fibrin-like gel quantitatively

incorporated and released the growth factor VEGF, which promoted angiogenesis in an

embryonic chick model. Therefore, not only might this gel provide the stimulation of

proliferation and migration of fibroblast, it may also provide an appropriate delivery

vehicle, further enhancing the effect of the VN:GF technology. In order to understand the

effect of this synthetic fibrin-like gel on both keratinocytes and fibroblasts in partial- and

Page 24

full-thickness wound healing, an appropriate 3-D in vitro model will need to be

developed.

1.4 IN VIVO AND IN VITRO MODELS

1.4.1 Animal Studies

While human studies are the most relevant and accurate means to determine the

effectiveness of wound therapies, this approach is often impractical. Difficulties in

identifying similar patients, limitations related to objective measurements of wound

healing, and ethical considerations impede early progress to clinical trials (Pocock et

al., 1987). In view of this, animal studies are used to model human wound healing

and to test the efficacy of different treatments. The selection of animal models for

wound healing studies is influenced by several factors, such as availability, cost, ease

of handling, and anatomical/functional similarity to humans. Small mammals

(rabbit, guinea pig, rat and mouse) are used frequently in wound healing studies as

they are inexpensive and easy to manage. However, they are very different to humans

in anatomy and physiology. For example, these animals have a dense layer of body

hair, thin epidermis and dermis. Pig skin is regarded as most similar to human skin in

many aspects, including: dermal-epidermal thickness ratio (Vardaxis et al., 1997);

well-developed rete ridges; dermal papillary bodies; abundant subdermal adipose

tissue (Winter, 1996); dermal collagen (Heinrich et al., 1971); size, orientation and

distribution of blood vessels in the dermis; and, the number and distribution of

adnexal structures (Meyer et al., 1978). Functionally, epidermal turnover, keratinous

proteins and lipid composition of the stratum corneum are also similar (Gray et al.,

Page 25

1982). Moreover, they have a similar expression of some antigens, including keratins

16 and 10, filaggrin, collagen IV, fibronectin and vimentin (Wollina et al., 1991).

Breuing et al. (1997) demonstrated that partial-thickness wounds in pigs can be

treated with EGF to accelerate wound healing, and this trend has also been observed

in full-thickness wounds (Jijon et al., 1989). Similarly, IL-1α significantly enhanced

healing in studies of both partial-thickness and full-thickness wounds in pigs (Mertz

et al., 1991). Although it is believed that pigs provide the best current pre-clinical

model for human wound healing, the cost, availability, and difficulty of handling

restrict the widespread use of pigs. Importantly, European Union regulation

(76/768/EEC, February, 2003) forbids using animal or animal-derived materials for

the development and assessment of consumer, cosmetic, and pharmaceutical

ingredients from 2009, and this may ultimately impact on some wound healing

treatments that fall within these categories.

1.4.2 2-Dimensional Monolayer Cell Culture System

Wound therapies are more often than not initially tested in vitro using cells grown in

monolayer culture. Skin cells grown in a 2-D monolayer culture are able to recapitulate

the responses of individual cell types in a defined environment and provide some

information on wound healing and cell function. This system has been widely used as an

in vitro assessment tool for biological factors and wound healing agents, because it is

inexpensive and easy to manipulate (Geer et al., 2004). Whilst this technique provides

important preliminary information about how cells will react to the therapy, a potential

problem arises in that the cells are not exposed to their natural geometric and mechanical

Page 26

environment (Sun et al., 2006b). Therefore, models are required that possess not only the

appropriate conditions for cell growth, but also an appropriate 3-D environment.

1.4.3 Tissue-Engineered Skin

Since the 1970s when keratinocytes were first cultured successfully in the laboratory

(Rheinwald & Green, 1975; Rheinwald & Green, 1977), and cultured epithelial autografts

(Green et al., 1979; Gallico et al., 1984) were utilized as a therapy for burn patients, there

has been great progress in generating tissue engineered skin. Nowadays, numerous tissue-

engineered products have been approved for clinical applications by the U.S. Food and

Drug Administration. Examples of currently available tissue-engineered skin are detailed

in table 1.1.

Three-dimensional (3-D) engineered skin, which recapitulates the cellular and

architectural organization of normal skin, is also currently utilized as a model for various

aspects of skin biology research. For example, these models have been used to examine

skin contraction (Chakrabarty et al., 2001; Harrison et al., 2006; Ralston et al., 1997), skin

diseases (e.g., melanoma invasion) (Meier et al., 2000), pigmentation (Bessou et al.,

1996; Hedley et al., 2002), wound healing (Falanga et al., 2002), and studies of cell-cell

and cell-extracellular-matrix interactions (Sun et al., 2006a). Engineered skin is created

using a biocompatible support matrix, such as an inert filter (Rosdy & Clauss, 1990), a

collagen matrix with GAGs and fibroblasts (Boyce et al., 1990), a fibroblast-populated

collagen lattice (Garlick & Taichman, 1994; Wilkins et al., 1994), a de-epidermised

dermis (DED) (Andreadis et al., 2001; Chakrabarty et al., 1999; Medalie et al., 1996) and

a scaffold of hyaluronan

Page 27

Name Type Scaffold Cell type Application Pros Cons Source

Epicel® Epithelial cover

None Keratino-cytes

Burn wounds

Fast pain relief, rapid coverage, decreased requirement for donor sites

Expensive, high level of skilled labour required, lack of dermal component

Gallico et al., 1984

AlloDer® Dermal replace-ment

Cadaveric acellular dermal matrix

None Abdominal hernia repair

Natural framework

Transmitting infectious disease

Misra et al., 2008; Wainwright et al., 1996

Integra® Dermal replace-ment

Artificial dermal matrix of cross-linked bovine collagen and chondroitin-6-sulfate, along with a disposable silicone (silastic) membrane

None Burn wounds

Improved cosmetic outcome Improved elasticity, reduced donor site morbidity

Expensive Berger et al., 2000

Transcyte-TM

Dermal replace-ment

Collagen-coated nylon mesh

Fibro-blasts

Burn wounds

Easier to remove, resulted in less bleeding, effective

Bio-degradable scaffold Non-permanent dermal substitute

Lukish et al., 2001; Purdue et al., 1997

Dermagraft® Dermal replace-ment

Bio-absorbable polymer scaffold

Fibro-blasts

Diabetic ulcers

Long shelf life, ease of use, lack of clinical rejection

Infections Browne et al., 2001;

Keratino-cytes

Diabetic foot ulcers

Scott Lipkin et al., 2003;

OrCel® Epidermal/ dermal replace-ment

Bilayered matrix of bovine collagen

Fibro-blasts

Burn wounds

Immediate availability and ease of use, efficient

Still et al., 2003

Apligraf® Epidermal/ dermal Replace-ment

A gel type I bovine collagen

Keratino-cytes Fibro-blasts

Ulcers Most sophisticated commercially available tissue-engineered product

Expensive and short life (5 days)

Bell et al., 1981; Karr, 2008

Perma-Derm Epidermal/dermal replace-ment

Bovine collagen

Keratino-cytes Fibro-blasts

Burn wounds

Permanent skin substitute

Time to first application, compromise of tissue biopsies, variability in materials used in cultured skin substitute fabrication

Boyce et al., 2006

Table 1.1 Products of Tissue-Engineered Skin.

derivitised with benzyl ester and seeded with fibroblasts (Zacchi et al., 1998) and donated

primary human keratinocytes. These constructs imitate the tissue architecture of human

Page 28

epidermis and provide a barrier function similar to native skin (Andreadis et al., 2001;

Medalie et al., 1996; Parenteau et al., 1996). One version of 3-D engineered skin is HSE.

HSE is a model in which human keratinocytes are seeded and cultured at the air:liquid

interface on a human-derived, de-epidermized dermal scaffold.

HSEs were produced originally to provide both dermal and epidermal tissue for grafting

on to patients suffering from full-thickness skin loss (burn patients) (Chakrabarty et al.,

1999). HSEs have been demonstrated to have morphological and biochemical features

similar to those observed in the in vivo epidermis, such as the formation of a stratum

basale, stratum spinosum, stratum granulosum, and stratum corneum, as well as the

expression of biochemical markers such as keratin 1/10/11 and 16 (Monteiro-Riviere et

al., 1997; Parnigotto et al., 1998; Ponec et al., 2002; Poumay et al., 2004; Topping et al.,

2006). Additionally, the basement membrane proteins present within the HSE models are

important for guiding epidermal keratinocyte organization (Ralston et al., 1997).

Furthermore, the natural acellular dermis maintains the microtopology of human dermis,

such as the rete ridge structure and a basement membrane containing collagens IV, VII

and laminin (Medalie et al., 1996). Within this natural collagenous ECM, cells recognise

amino acid sequences of the matrix proteins (integrin attachment) and transmit these

signals through to their cytoskeleton. This signalling information plays an important role

in organizing the cell biology of the HSE (Sun et al., 2005). The HSE’s similarities to

human epidermis suggest that this model would be an appropriate tool for clinical skin

grafts, absorption, drug transfer, irritancy, toxicity, phototoxicity, metabolic studies of

topically applied products, and modelling physiological processes in skin (Yeh et al.,

2004). HSEs are thus regarded as a better ex vivo cell culture system for studying wound

Page 29

re-epithelialisation (Geer et al., 2004), as well as for assessing a range of skin repair

treatments (Breetveld et al., 2006; Chakrabarty et al., 1999; Topping et al., 2006).

1.5 METHODS FOR WOUNDING

Currently, several methods exist for creating wounds and studying wound responses.

These are tabulated and described briefly in table 1.2. Notwithstanding these previously

reported wound-healing models, standardized models utilized to evaluate topical agents

have not been routinely established. Furthermore, all the above-mentioned methods for

generating wounds listed in table 1.2 have limitations. For example, the 2-D method of

scratching epithelial cell monolayers (Legrand et al., 2001) has unnatural geometric and

mechanical restrictions; whereas the 3-D method of tape abrasion (Reed et al., 1995) is

not accurately reproducible. The pressure employed to apply the tape, the number of

repetitive strippings and the adhesiveness of the tape, can all influence the depth of the

epidermal injury. Furthermore, the electrokeratome and mesher (Falanga et al., 2002;

Sams et al., 2004) can also create to a wound with variable depth of excision, if this

technique is operated inappropriately. For water scald burns (Cribbs et al., 1998), different

temperatures and times of exposure to the water bath also can lead to a variation in wound

depth. Thermal energy can be produced using various techniques to create partial-

thickness thermal skin injuries (Pilcher et al., 1999), thus the reproducibility of this

technique is diminished. Moreover, the thermal energy wound also results in the

denaturation of the dermal collagen in the wound bed. Recently, the laser method

(Vaughan et al., 2004) has been used to generate a wound because it was thought that it

could facilitate the creation of a wound with a given size and depth.

Page 30

Wound Models/ Tests

2D/

3D

Methods / Applications in Wound Creation

Scratch test

2D The simplest method of wounding an epithelial monolayer is scratching the epithelial cell monolayers with a pipette tip. The advantage of this method is that it is inexpensive, very simple to perform, and able to provide information of migratory and proliferative responses to wounding (Legrand et al., 2001; Turchi et al., 2002; Yamada et al., 2000), as well as to assess the potential of novel therapies (Geer & Andreadis, 2003; Yamaguchi et al., 2002).

Tape abrasion

3D The simplest method of wounding epidermis in a 3-D environment is tape abrasion. The top layers of skin cells are removed by applying adherent tape on the skin. The tape is taken away with a quick stripping movement nearly parallel to the skin. The required depth of damage is achieved by repeating this action more than a few times. The advantage of this method is that it is convenient and easy to manipulate (Reed et al., 1995; Yang et al., 1995)

Electro-keratome/dermat-ome

3D The electrokeratome, composed of a razor blade and a high-speed electric motor, has been widely used to study epidermal wound healing in animal models (Goris & Nicolai, 1982; Jong & Lin, 1995; Sams et al., 2004). An even depth of skin can be removed by stable downward pressure applied on the skin pulled in the direction of the blade. To remove just the top epithelial layer of the skin the electrokeratome can be set to a depth of 0.005 cm.

Suction blisters

3D In 1950, dry suction was used to separate epidermis from dermis for the first time (Blank & Miller, 1950). Recently, this method has been employed successfully to study epidermal wound healing in animals such as rats, guinea pigs and swine (Mousa et al., 1990; Nanchahal & Riches, 1982; Rommain et al., 1991). The epidermis is slowly separated from the dermis at their interface. This separation is induced by the application of vacuum suction to skin for a period of time and a blister is created by filling fluid in the intradermal space. This technique can minimize the damage to the underlying dermis when separating the epidermis from the dermis.

Water scald burns

3D Water scald burns have been generated successfully in animals such as mice (Cribbs et al., 1998) and pigs (Brans et al., 1994). Briefly, a fixed area of the dorsal skin is partially immersed in a constant temperature water bath for a fixed period of time, and a blister forms over the burn. The wound is then exposed by removal of the roof of a blister.

Partial-thickness thermal Injury

3D Partial-thickness thermal burns have been created successfully in pigs or piglets and employed to test topical applied reagents, such as EGF (Jones et al., 1991; Pilcher et al., 1999). A blister usually forms within an hour of the burn injury and can be removed to expose the dermis.

Mesher 3D Incisional wounds were created by using a Zimmer Meshgraft II system (Zimmer Ltd., Swindon, UK). The composite composed of DED seeded with keratinocytes was placed epidermal surface downward on a Dermacarrier II skin graft carrier with a mesh ratio of 1:1.5 (Zimmer Ltd.) and passed through the mesher (Falanga et al., 2002; Harrison et al., 2006).

Biopsy Punch

3D A full-thickness wound created by using a biopsy punch laid on the top of a non-wounded dermis. This dermis can be made of either acellular collagen gel (Stephens et al., 1996), fibroblast-contracted collagen gel (Falanga et al., 2002; Garlick & Taichman, 1994) or fibroblast/matrix sheet (Laplante et al., 2001).

Laser 3D Skin equivalents composed of collagen gel seeded with keratinocytes and fibroblasts were wounded using three different types of lasers: an erbium-YAG, a high-powered excimer, and a low-powered excimer. Each laser was set to make a wound 6 mm long, 1 mm wide and 400 µm deep (Vaughan et al., 2004).

Table 1.2 Current Methods Used to Create Wounds

Page 31

However, this method proved unable to produce a wound with a preset size and depth. In

addition, the thermal damage produced by the laser could not be well-controlled. Finally,

Moll et al. (1998) and Geer et al. (2004) have used a biopsy punch to create both partial-

thickness and full-thickness wounds, respectively (Moll et al., 1998; Geer et al., 2004).

However, Moll et al. required serum to encourage keratinocyte growth and wound healing

on their human skin model. Further, Geer et al. required their model to be grafted onto a

mouse for successful wound regeneration. Clearly, a defined, xenobiotic-free, precise and

reproducible wound, with a given size and depth, is required for the accurate investigation

of novel wound healing therapies.

1.6 PROJECT HYPOTHESIS AND AIMS

Therefore I hypothesised that the HSE model could be further refined, generated in a

defined, serum- and xenobiotic-free culture environment, to produce partial- and full-

thickness wound models with a given size and depth. The development of accurate and

improved models of human skin is important, as these would clearly facilitate the

translation of new therapies into the clinical setting. Therefore, to test this hypothesis I

initially aimed to quantitate the functional responses of human skin cell keratinocytes and

fibroblasts to vitronectin:growth factor and HA using a 2-D monolayer cell culture

system. Once this was achieved I then translated the knowledge gained using the 2-D

culture system into a 3-D HSE model to represent the native human skin environment.

Finally, I aimed to recreate and characterize reproducible defined, partial-thickness and

full-thickness “wounds” with a given size in the 3-D human skin equivalent models, and

employ these wound models to investigate the effect of novel wound healing therapies.

Page 32

2CHAPTER 2: INVESTIGATIONS INTO

THE FUNCTIONAL RESPONSES OF

SKIN CELLS TO COMPLEXES OF

VITRONECTIN:GROWTH FACTOR

AND HYALURONIC ACID

2.1 INTRODUCTION

As outlined in chapter 1, keratinocytes and fibroblasts are two of the most important

cell types that have key roles during the complicated wound healing cascade

(Latijnhouwers et al., 1997). Following tissue injury, keratinocytes are stimulated to

migrate by chemical signalling molecules that are released upon wounding, such as

IL-1 (Kupper, 1990; Tomic-Canic et al., 1998) and nitric oxide (Lee et al., 2001).

Soon after injury, keratinocytes start to migrate into the wound, proliferate to cover

the wound surface, and then regenerate and remodel the basement membrane

(Krawczyk, 1971). Fibroblasts are also attracted to the wound site in response to

chemotactic factors released by platelets and macrophages. Similarly, the fibroblasts

migrate into the wound bed, proliferate and contribute to the remodelling of matrix

components (Stadelmann et al., 1998).

Another important component in wounds is wound fluid. Wound fluid is produced in the

process of wound healing, thus the composition of wound fluid is closely related to the

Page 33

processes involved in the haemostasis, inflammatory, proliferative and remodelling

phases (Enoch & Price, 2004). Wound fluid contains many different components that

contribute to the process of wound healing. For example, this fluid contains several

degrading enzymes, such as MMP-2 and 9 which play vital roles in preparing the wound

for healing, through lysis of devitalized protein and debris (Howard & Banda, 1991;

Kjeldsen et al., 1994; Wysocki et al., 1993). Additionally, wound fluid also includes HA,

which has been reported to modulate adhesion, migration, proliferation, collagen

synthesis, and more importantly, reduced scarring during the healing process (Melrose et

al., 1996; Pajulo et al., 2001; Wiig et al., 1996; Yang et al., 1995).

Wound fluid also contains growth factors and cytokines, which play pivotal roles in

proliferation, migration and differentiation. Interestingly, growth factors such as

EGF, b-FGF, PDGF, TNF-α, TNF-β, IGF-I, and TGF-β, are found at high

concentrations in the wound fluid (Ono et al., 1995). Growth factors have been

identified as regulatory polypeptides that help orchestrate the healing processes. For

example, the wound healing cascade can be initiated through attracting/activating

fibroblasts, endothelial cells, macrophages (Enoch & Price, 2004), and through the

regulation of cell proliferation and differentiation (Fu et al., 2005). One family of

growth factors thought to occupy a central role in the complex process of skin growth

and repair is the IGF family. The IGFs have various biological functions, including:

regulation of cell proliferation and differentiation (Ernst et al., 1989; Bondy et al.,

1992); DNA synthesis (Kiess et al., 1994); and stimulation of migration of cells

located at the leading edge of the wound via actin polymerization (Leventhal &

Feldman, 1997). Another key growth factor involved in wound healing is EGF. EGF

has been shown to stimulate cell motility and promote wound healing in both in vitro

Page 34

cell culture and in vivo skin wounds (Schultz et al., 1991). It is important to keep in

mind, however, that the skin repair process is also modulated by signals from the

local environment, including extracellular matrix (ECM) molecules such as collagen,

fibronectin and vitronectin (VN), to name a few (Galiano et al., 1996).

The role of vitronectin is particularly interesting as it has been found to increase

epithelial outgrowth from skin organ explant cultures and to induce enhanced

motility of human keratinocytes in vitro (Brown et al., 1991). In addition, VN

provides an essential component of the temporary wound bed matrix that facilitates

cell migration and hence wound repair (Krawczyk, 1971). Interestingly, VN has been

found to bind not only IGF-family members (Upton et al., 1999), but also associates

with other key modulators of wound repair, such as EGF (Schoppet et al., 2002).

Studies by the Queensland University of Technology’s Tissue Repair and

Regeneration Research Program have shown that IGF-I associates with VN through

IGFBPs (Kricker et al., 2003). Furthermore, the trimeric IGF-I + IGFBPs + VN

multiprotein complex has been shown to enhance not only keratinocyte protein

synthesis and migration (Hyde et al., 2004), but also to enhance significantly the

migration of dermal keratinocytes and fibroblasts derived from diabetic skin (Noble,

2008). Critically, the response of these cells to the trimeric complex is greater than

that observed with the components assessed individually or in paired combinations.

Taken together, these data suggest that these novel complexes hold potential to

promote healing of chronic wounds. However, CWF contains high proteolytic

activity and this can result in the degradation of ECM proteins such as VN (Grinnell

et al., 1992; Wysocki et al., 1993), with this in turn leading to an impaired wound

Page 35

healing response. It is not known whether the vitronectin:growth factor (VN:GF)

complexes will be active in the presence of CWF. In this chapter I report studies

examining this using 2-D cell culture approaches and CWF obtained from patients

with chronic leg ulcers, as a first step towards investiging the potential of the VN:GF

technology as a therapy for chronic wounds. In addition, HA has also been examined

together with VN:GF to determine whether it can enhance the effect of VN:GF.

Page 36

2.2 MATERIALS AND METHODS

2.2.1 Introduction

All general reagents and chemicals were of the highest quality and were obtained from

reputable suppliers. Suppliers of method-specific reagents are detailed in the appropriate

methods sections that follow.

2.2.2 Cell Culture

The HaCaT human skin keratinocyte cell line (Hyde et al., 2004) and the human foreskin

fibroblast (HFF) cell line were obtained from Professor Norbet Fusening (DKFZ,

Heidelberg, Germany) and the American Type Culture Collection (ATCC, Manassas,

VA, America), respectively. These cells were routinely grown in Dulbecco’s Modified

Eagle’s Medium (DMEM) (Invitrogen, Mulgrave, VIC, Australia) containing 10% FBS

and 5% FBS respectively (HyClone, South Logan, UT, USA). The cells were cultured in

80 cm2 culture flasks at 37 ºC, in a 5% CO2 atmosphere. All culture media were

supplemented with 0.1 µL/mL gentamicin, 50 µL/mL streptomycin sulfate and 50

units/mL penicillin G (Invitrogen). All cultures were passaged two or three times per

week at 80% confluence by trypsin/EDTA detachment.

2.2.3 Pre-binding VN:GF to Culture Wells

In this study VN:GF were “pre-bound” to the culture dishes to reflect more accurately the

cellular environment in vivo, since cells in vivo are more likely to encounter growth

factors bound to the ECM (Hollier et al., 2008). Thus, VN:GF were precoated to 48-well

Page 37

tissue culture plates (Nagle Nunc International, Roskilde, Denmark) and to the lower

chamber and membrane surface of 12-µm pore 12-well Transwell® (Costar, New York,

NY, USA). Firstly, 150 µL of DMEM containing 162 ng VN (Promega, Annandale, New

South Wales, Australia), or 600 µL of DMEM containing 648 µg VN, were added to each

well of a 48-well plate and the lower chamber of a 12-well Transwell® plate,

respectively. These plates with VN-containing media were incubated for two hours at 37

ºC after which the media containing unbound VN were removed by aspiration. Following

the removal of the media, 125 µL or 500 µL of HEPES binding buffer (HBB) consisting

of 0.1 M HEPES, 0.12 M NaCl, 5 mM KCl, 1.2 mM MgSO4, 8 mM glucose, pH 7.6

containing IGF-I (54 ng) (Novozymes, Adelaide, South Australia, Australia), IGFBP3

(162 ng) (Upstate Biotech, Lake Placid, NY, USA), EGF (54 ng) (Novozymes, Adelaide,

South Australia, Australia), or IGF-I (216 ng), IGFBP3 (648 ng), EGF (216 ng) were

added to the 48-well plates or 12-well Transwell®, respectively, and incubated for

another two hours at 37 ºC. Lastly, the media containing the growth factor complexes

were removed and the wells were washed with 500 µL or 1 mL HBB for the 48-well

plates or 12-well Transwell® respectively, and air dried under sterile conditions.

2.2.4 HA Solution Preparation

HA powder (954 kDa) (Novozymes A/S, Bagsvaerd, Denmark) was dissolved in double

distilled water (10 mg/mL) for 2 hr at 37 ºC. This solution was then filtered, sterilised

(0.22 µm) (3 mg/mL) and stored at -20 ºC.

Page 38

2.2.5 Migration Assay

Migration assays were performed as previously described (Hollier et al., 2005; Hyde et

al., 2004; Noble et al., 2003). HaCaT or HFF cells were grown to approximately 80%

confluence and were then washed for 4 hr in serum-free DMEM at 37 ºC, 5% CO2. The

cells were then trypsinised, diluted in serum-free DMEM, counted and seeded into the

upper chamber of pre-treated 12-µm pore 12-well Transwell® at a density of 1.5 x 105

cells/mL/well. The plates were incubated for 10 hr at 37 ºC, 5% CO2. Cells which had not

migrated through the microporous membrane and remained on the upper surface of the

membrane of the upper chamber were gently removed using cotton tips. The migrated

cells located on the lower surface of the porous membrane were fixed in 3.7% para-

formaldehyde (Univar, Ingleburn, New South Wales, Australia) and stained with 0.01%

crystal violet (Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia) in phosphate buffered

saline (PBS). The number of migrated cells was then quantified by extracting the crystal

violet stain in 10% acetic acid (BDH Laboratory Supplies, Poole, England) and

determining the optical density of these extracts (corresponding to the number of cells) at

595 nm using a 96-well plate reader (Benchmark Plus, Bio-Rad) (Leavesley et al., 1993).

HaCaT and HFF experimental wells were set up as follows: The lower chamber and

membrane surface of the plates were pre-treated with 600 µL VN (648 ng/well) and 500

µL volumes containing IGF-I (216 ng/well), IGFBP3 (648 ng/well) and EGF (216

ng/well). Various concentrations of HA in 250 µL were then added to the lower chamber

of the wells with or without pre-bound VN:GF. Subsequently, 150 000 cells in 400 µL

serum-free medium (SFM) were added to the upper chamber. Wells were then exposed to

2, 6, 20, 60, 200, 600 and 2000 µg of HA in both the absence and presence of VN:GF in a

final volume of 1 mL/well.

Page 39

2.2.6 Proliferation Assay

HaCaT or HFF cells were grown to approximately 80% confluence and then washed for 4

hr in serum-free DMEM at 37 0C, 5% CO2. The cells were trypsinised, diluted in serum-

free DMEM, counted and seeded into the 48-well plates at a density of 2.5 x 104

cells/500 µL/well. The 48-well plates were then incubated for 48 hr at 37 0C, 5% CO2.

The dead cells were removed by washing and the number of living cells was quantitated

with the CyQUANT® NF Proliferation Assay (Invitrogen) as per the manufacturer’s

instructions. Fluorescence was determined at λex 485nm, λem 530 nm in black 96-well

plates (PerkinElmer, Waltham, MA, USA) using a POLAR star Optima Microplate

Reader (BMG LABTECH GmbH, Offenburg, Germany). HaCaT and HFF experimental

wells were set up as follows: Plates were pre-treated with 150 µL VN (162 ng/well) and

125 µL volumes containing IGF-I (54 ng/well), IGFBP3 (162 ng/well) and EGF (54

ng/well). SFM containing 2.5 x 104 cells in 375 µL were then added to wells with or

without pre-bound VN:GF. Subsequently, various concentrations of HA in 125 µL were

added on the top. Wells were therefore exposed to 1 µg, 3 µg, 10 µg, 30 µg, 100 µg, 300

µg and 1000 µg of HA in both the absence and presence of VN:GF in a final volume of

500 µL/well.

2.2.7 Chronic Wound Fluid (CWF)

Collection

CWF samples were collected from consenting patients suffering from chronic venous leg

ulcers and undergoing compression therapies at a community leg ulcer clinic (St. Luke’s

Nursing Services, Brisbane, Queensland, Australia). Human ethical approval was

Page 40

obtained from St. Luke’s Nursing Services and the Queensland University of Technology

(3673H). Wound fluid collection was performed by the staff at St. Luke’s Nursing

Services. Briefly, wounds were washed using sterile water prior to collecting wound fluid.

An occlusive dressing was then applied to the wound for 30 min to 1 hr. Subsequently,

wound fluid was recovered by washing with 1 mL of saline and then centrifugation at 14

000 g for 10 min. The supernatant was filtered using 0.22 µm cellulose acetate filters

(Agilent Technologies, Wilmington, DE, USA). Protein concentration was then

determined using the BCA protein assay kit (Pierce Chemicals, Rockford, IL, USA.). The

pooled sample was aliquoted and stored at -80ºC until further use.

CWF HaCaT Cell Migration

Experimental wells were set up as follows: Serum-starved HaCaT cells were measured 10

hr after seeding the cells into 12-µm pore 12-well Transwell®. The lower chamber and

membrane surface of the plates were pre-treated with 600 µL VN (648 ng/well) and 500

µL volumes containing IGF-I (216 ng/well), IGFBP3 (648 ng/well) and EGF (216

ng/well). The VN:GF was pre-bound to the lower chamber of the 12-µm pore 12-well

Transwell® culture plates. Subsequently, 250 µL foetal calf serum (FCS) + 750 µL of

serum-free medium (SFM) (positive control), 250 µL CWF + 750 µL of SFM or 250 µL

bovine serum albumin (BSA) + 750 µL of SFM (negative control) were added to the

lower chamber of the wells with or without pre-bound VN:GF. Finally, the upper

chambers of the wells were seeded with 1.5 x 105 cells in 400 µL SFM.

Page 41

CWF HaCaT Cell Proliferation

Proliferation was assessed after 48 hr. Serum-starved HaCaT cells (2.5 x 104) were

seeded into 48-well culture plates that had been pre-bound with; VN (162 ng/well) IGF-I

(54 ng/well), IGFBP3 (162 ng/well) and EGF (54 ng/well). Subsequently, 125 µL of FCS

+ 375 µL of SFM (positive control), CWF + 375 µL of SFM, BSA + 375 µL of SFM

(albumin control) or 500 µL SFM (negative control) were added to either, non-coated

wells or wells that were pre-bound with VN:GF.

2.2.8 Statistical Analysis

In all of the assays, triplicate treatments were tested individually with each treatment

within each assay, and the experiments were repeated three times. The data, expressed as

the average percentage of control wells containing SFM alone, were pooled from three

replicate experiments in which each treatment was tested in triplicate. One-way ANOVA

with Tukey’s post hoc tests (all groups comparisons) was used to analyse the data.

Statistically significant differences were determined as p < 0.05.

Page 42

2.3 RESULTS

2.3.1 Investigations into the Effects of VN:GF and HA on Keratinocyte Migration.

The VN:GF has previously been shown in our laboratory to enhance HaCaT keratinocyte

migration (Hyde et al., 2004). Furthermore, others have shown that HA modulates several

cellular functions, such as cell migration (Melrose et al., 1996), adhesion (Klein et al.,

1996) and proliferation (Wiig et al., 1996). Therefore, combinations of VN:GF and HA

were investigated to determine if the VN:GF-induced HaCaT cell migration could be

enhanced with the addition of HA. As is shown in figure 2.1, responses equivalent to

control treatments (SFM and SFM with VN:GF) were recorded. Thus in the absence of

VN:GF the responses obtained from wells exposed to different concentrations of HA

were indistinguishable (p > 0.05) from responses obtained in the control treatment (SFM).

In the presence of VN:GF the responses of cells to different concentrations of HA were

not significantly different to that obtained with the control SFM with VN:GF (p > 0.05).

These results therefore indicate that the HA alone treatment has little effect on the

migration of HaCaTs. Likewise, the presence of HA does not alter the effect of VN:GF

on HaCaT migration.

Page 43

0%

50%

100%

150%

200%

250%

300%

350%

% o

f con

trol (

SF

M)

SFM2 200060020060206

+VN:GF

-VN:GF

HA

Figure 2.1. Migration of HaCaT keratinocytes exposed to HA in the absence

and presence of VN:GF. Responses of HaCaTs seeded into 12-well Transwell®

containing increasing concentrations (µg/mL) of HA in the absence and presence of

VN:GF are depicted. The data, expressed as the average percentage of control wells

containing serum free medium (SFM) alone for 10 hr, were pooled from three

replicate experiments in which each treatment was tested in triplicate (n = 9). The

response of cells exposed to wells treated with HA in the absence of VN:GF (full

colour bars) are compared with the response obtained in the control SFM (orange

bar) wells. The response of cells exposed to wells treated with HA in the presence of

VN:GF (checked bars) are compared with the effects obtained from wells with the

control SFM plus VN:GF (checked orange bar). Error bars indicate SEM.

Page 44

2.3.2 Investigations into the Effects of VN:GF and HA on Keratinocyte

Proliferation.

The VN:GF have been shown to enhance HaCaT keratinocyte protein synthesis (Hyde et

al., 2004). In view of this, we examined if HA could enhance the effects of VN:GF on

HaCaT proliferation (used as a measure of cell proliferation). Wells exposed to 1, 3, 10,

30, 100, 300, and 1000 µg/500 µL of HA respectively, in both the absence and presence

of VN:GF, exhibited similar responses equivalent to the control wells (SFM, and SFM

with VN:GF respectively) (figure 2.2). In the absence of VN:GF cellular responses in

wells exposed to different concentrations of HA were not significantly different (p >

0.05) to those observed in the control treatment (SFM). Similarly, in the presence of

VN:GF the responses observed in wells with different concentrations of HA were not

significantly different (p > 0.05) to that obtained with the control treatment (SFM with

VN:GF). Thus, the HA treatment alone has little effect on the proliferation of HaCaT

cells. Likewise, the presence of HA does not alter the effect of VN:GF on HaCaT

proliferation.

Page 45

0%

50%

100%

150%

200%

250%

% o

f con

trol (

SF

M)

SFM HA1 100030010030103

+VN:GF

-VN:GF

Figure 2.2. Proliferation of HaCaT keratinocytes exposed to HA in the absence

and presence of VN:GF. Responses of HaCaTs seeded into 48-well plates

containing increasing concentrations (µg/500 µL) of HA in the absence and presence

of VN:GF are depicted. The data, expressed as the average percentage of control

wells containing SFM alone for 48 hr, were pooled from three replicate experiments

in which each treatment was tested in triplicate (n = 9). The number of living cells

was quantitated with the CyQUANT® NF Proliferation Assay. The responses of cells

exposed to wells treated with HA in the absence of VN:GF (full colour bars) are

compared with the responses obtained in the control SFM wells (orange bar). The

responses of cells exposed to wells treated with HA in the presence of VN:GF

(checked bars) are compared with the responses obtained from wells with the control

SFM plus VN:GF (checked orange bar). Error bars indicate SEM.

Page 46

2.3.3 Investigations into the Effects of VN:GF and HA on Fibroblast Migration.

In addition to keratinocytes, a second key cell type in wound healing, namely fibroblasts,

was also examined. Specifically, the effect of the combination of the VN:GF and HA on

HFF cell migration was investigated. Thus Transwell® migration chambers were exposed

to various concentrations of HA, in both the absence and presence of VN:GF. Again, it

was found that these treatments led to similar responses to those obtained from the control

wells (SFM and SFM with VN:GF, respectively) (figure 2.3). In the absence of VN:GF

the responses obtained in wells exposed to different concentrations of HA were not

significantly different (p > 0.05) to those obtained in the control SFM wells. In the

presence of VN:GF the value obtained from wells exposed to different concentrations of

HA were not significantly different from wells with the control SFM plus VN:GF (p >

0.05). These results indicate that HA alone has little effect on the migration of HFF.

Likewise, the presence of HA does not alter the effect of VN:GF on HFF migration.

Page 47

0%

200%

400%

600%

800%

1000%

1200%

1400%

1600%

1800%

% o

f con

trol

(SF

M)

SFM HA 5%FCS2 200060020060206

+VN:GF

-VN:GF #

*

Figure 2.3. Migration of HFF fibroblasts exposed to HA in the absence and

presence of VN:GF. Responses of HFF seeded into 12-well Transwell® chambers

containing increasing concentrations (µg/mL) of HA in the absence and presence of

VN:GF are depicted. The data, expressed as the average percentage of control wells

containing SFM alone for 10 hr, were pooled from three replicate experiments in

which each treatment was tested in triplicate (n = 9). The symbols (*, #) indicate

treatments which significantly increased the migration of HFF compared to the

response obtained with SFM wells, and SFM with VN:GF wells, respectively (p <

0.05). The response obtained in cells exposed to wells treated with HA in the absence

of VN:GF (full colour bars) are compared with the response obtained in the control

SFM wells (orange bar). The response obtained in cells exposed to wells treated with

HA in the presence of VN (checked bars) are compared with the response obtained

from wells with the SFM plus VN:GF (checked orange bar). Error bars indicate

SEM.

Page 48

2.3.4 Investigations into the Effects of VN:GF and HA on Fibroblast Proliferation.

The proliferative responses of HFF fibroblasts to VN:GF in the presence of various

concentrations of HA was also assessed. The results in figure 2.3.4 demonstrate that in the

absence of VN:GF, the proliferative responses from wells exposed to the highest

concentration of HA (1000 µg/500 µL) were significantly different (p < .01) to those

obtained from the control treatment (SFM), with a response of 433 ± 26 % above the

control SFM obtained (figure 2.4). In the presence of VN:GF it was observed that HA at

concentrations of 300 µg/500 µL and 1000 µg/500 µL induced responses of 203 ± 18 %

(p < 0.05) and 287 % ± 28% (p < 0.01) above the control well with SFM plus VN:GF,

respectively (figure 2.4). These data demonstrate that HA is capable of stimulating

significant increases in cellular proliferation in HFFs, as well as enhance the effect of

VN:GF on HFF proliferation, albeit only at the higher concentrations of HA tested.

Page 49

0%

200%

400%

600%

800%

1000%

1200%

1400%

% o

f cou

trol (

SF

M)

SFM

1 100030010030103 5%FCS

+VN:GF

-VN:GF

HA

* #

#

#

*

Figure 2.4. Proliferation of HFF fibroblasts to HA in the absence and presence

of VN:GF. Responses of HFFs seeded into 48-well culture plates containing

increasing concentrations (µg/500 µL) of HA in the absence and presence of VN:GF

are depicted. The data, expressed as the average percentage of control wells

containing SFM alone for 48 hr, were pooled from three replicate experiments in

which each treatment was tested in triplicate (n = 9). The number of living cells was

quantitated with the CyQUANT® NF Proliferation Assay. The symbols (*, #)

indicate treatments which significantly increased the proliferation of HFFs compared

to the response obtained in SFM wells and SFM with VN:GF, respectively (p <

0.05). The responses of cells exposed to wells treated with HA in the absence of

VN:GF (full colour bars) are compared with the responses obtained from the control

SFM wells (orange bars). The responses obtained from cells exposed to wells treated

with HA in the presence of VN (checked bars) are compared with the responses

obtained from wells treated with the control SFM plus VN:GF (checked orange bar).

Error bars show SEM.

Page 50

2.3.5 Investigations into the Effects of VN:GF in the Presence of CWF on

Keratinocyte Migration.

It has been demonstrated that ECM proteins such as VN are degradated by the

inappropriate proteolytic activity of enzymes in CWF (Grinnell et al., 1992; Wysocki

et al., 1993). To investigate the potential of the VN:GF technology as a therapy for

chronic wound healing, CWF obtained from patients with chronic leg ulcers was

added to culture plates to replicate the in vitro environment of a chronic wound.

Subsequently, HaCaT cellular migration was used to determine if VN:GF was still

functional in the presence of CWF.

When wells were exposed to 0.002, 0.006, 0.018, 0.054, and 0.162 mg/250 µL of

FCS, CWF or BSA in the absence of VN:GF, responses of; 96%, 112%, 218%, 512%

and 860% for FCS; 104%, 107%, 136%, 227% and 397% for CWF; 89%, 107%,

90%, 97% and 100% for BSA; and 100% for SFM, respectively, were observed

(figure 2.5). When wells were exposed to the same doses of FCS, CWF, and BSA in

the presence of VN:GF, effects of; 273%, 348%, 500%, 762% and 914% for FCS;

261% 260%, 278%, 388% and 494% for CWF; 336%, 322%, 324%, 346% and

356% for BSA; and 259% for SFM, respectively, were obtained. For all

concentrations of FCS, CWF and BSA tested, the responses obtained in the presence

of VN:GF were significantly higher (p < 0.05) than those obtained when VN:GF was

absent. Thus VN:GF results in significantly enhanced keratinocyte proliferation with

the effects ranging from one to three-fold greater than the effects of the different

treatments (FCS, CWF and BSA) alone.

Page 51

0%

100%

200%

300%

400%

500%

600%

700%

800%

900%

1000%

0.002m

gFC

S

0.0018m

gFC

S0162

mgF

CS

0.006m

gCW

F0.054

mgC

WF

0.002m

gBS

A

0.0018m

gBS

A0162

mgB

SA

with

VG

0.006m

gFC

S

0.054m

gFC

S

0.002m

gCW

F0.001

8mgC

WF

0162m

gCW

F

0.006m

gBS

A

0.054m

gBS

A

SF

M

-VG Treatment +VG

% o

f con

trol (

SF

M)

C C CCC CFCS CWF BSA SFM FCS CWF BSA SFM

#

#

#

#

# # #

#

#

# # # # #

#

+VN:GF

-VN:GF

Figure 2.5. Migration of HaCaT human keratinocytes seeded in culture wells

exposed to different treatments including FCS (positive control), CWF, BSA

(negative control) and SFM (negative control) in the absence and presence of

VN:GF. Responses of HaCaTs seeded into 12-well Transwell® containing

increasing concentrations (mg/mL) of FCS, CWF and BSA in the absence and

presence of pre-coated VN:GF are depicted. The data, expressed as the average

percentage of control wells containing SFM alone, were pooled from three replicate

experiments in which each treatment was tested in triplicate (n = 9). The responses of

cells exposed to wells treated with FCS, CWF, BSA and SFM in the presence of

VN:GF (checked bars) are compared with the responses obtained from wells treated

with FCS, CWF, BSA and SFM in the absence of VN:GF (full colour bars). Error

bars indicate SEM.

Page 52

2.3.6 Investigations into the Effects of VN:GF in the Presence of CWF on

Keratinocyte Proliferation.

Given that VN:GF maintained its ability to stimulate the migration of HaCaT cells in the

presence of wound fluid, we then investigated whether VN:GF could stimulate the

proliferation of HaCaT cells in the presence of CWF. When wells were exposed to

0.001, 0.003, 0.009, 0.027 and 0.081 mg/125 µL of FCS, CWF or BSA in the absence of

VN:GF, responses of; 146%, 190%, 351%, 467% and 516% for FCS; 118%, 134%,

151%, 268% and 479% for CWF; 130%, 130%, 107%, 126% and 92% for BSA; and

100% for SFM, respectively, were observed (figure 2.6). When wells were exposed to

these same doses of FCS, CWF and BSA in the presence of VN:GF, effects of; 324%,

365%, 471%, 611% and 737% for FCS; 280% 319%, 441%, 547% and 656% for CWF;

391%, 401, 397%, 471% and 469% for BSA; and 348% for SFM, respectively, were

obtained. For all concentrations of FCS, CWF and BSA tested, except for 0.009 mg/125

µL of FCS, the effect obtained in the presence of VN:GF was significantly higher (p <

0.05) than that obtained when VN:GF was absent (p < 0.05). Thus VN:GF results in

significantly enhanced keratinocyte proliferation with the effects ranging from one to two-

fold greater than the effects of the different treatments (FCS, CWF, BSA and SFM) alone.

Page 53

0%

100%

200%

300%

400%

500%

600%

700%

800%

900%

1000%

0.001m

g/125ulFC

S0.009

mg/125ulF

CS

0.081m

g/125uolF

CS

0.003m

g/125ulCW

F0.027

mg/125ulC

WF

0.001m

g/125ulBS

A0.009

mg/125ulB

SA

0.081m

g/125ulBS

A

0.003m

g/125ulFC

S0.027

mg/125ulF

CS

0.001m

g/125ulCW

F0.009

mg/125ulC

WF

0.081m

g/125ulCW

F0.003

mg/125ulB

SA

0.027m

g/125ulBS

AS

FM

Treatment-VG Treatment+VG

% c

ontro

l (S

FM

)

C C CCC CFCS CWF BSA SFM FCS CWF BSA SFM

# #

#

#

#

# # #

# #

#

#

#

#

#

+VN:GF

-VN:GF

Figure 2.6. Proliferation of HaCaT human keratinocytes seeded in culture wells

exposed to different treatments including of FCS (positive control), CWF, BSA

(negative control) and SFM (negative control) in the absence and presence of

VN:GF. Responses of HaCaTs seeded into 48-well plates containing increasing

concentrations (mg/500 µL) of FCS, CWF and BSA in the absence and presence of

pre-coated VN:GF are depicted. The number of living cells was quantitated with the

CyQUANT® NF Proliferation Assay. The data, expressed as the average percentage

of control wells containing SFM alone for 48 hr, were pooled from three replicate

experiments in which each treatment was tested in triplicate (n = 9). The responses of

cells exposed to wells treated with FCS, CWF, BSA and SFM in the presence of

VN:GF (checked bars) are compared with the responses obtained from wells treated

with FCS, CWF, BSA and SFM in the absence of VN:GF (full colour bars). Error

bars indicate SEM.

Page 54

2.4 DISCUSSION

Two-dimensional monolayer cell culture is widely used as an in vitro assessment tool for

biological factors and wound healing agents due to its ease of use, low cost and the ability

to examine cellular responses in a defined environment. In view of this, the HaCaT

human keratinocyte and the HFF fibroblast cell lines seeded in 2-D tissue culture plates

were used to test cellular responses to VN:GF and HA. Although the HaCaT cell line has

a somewhat changed and unrestricted growth potential phenotypically and functionally,

they are comparable to normal keratinocytes. For example, the differentiation-specific

keratins (K1 and K10) are expressed. In addition, HaCaT cells maintain an outstanding

ability to differentiate normally, that is as per normal skin keratinocytes, even at high

passage numbers. Moreover, these cells have the capacity to reconstruct an orderly

structured and differentiated epidermis after being transplanted onto nude mice

(Boukamp et al., 1988). In view of this, the HaCaT cell line is widely considered as a

stable and valuable model for keratinization studies (Boukamp et al., 1988). Similarly, the

HFF cell line has been widely used as feeder layers for the culture of keratinocyte cells

(Hasskarl et al., 2005), therefore these were also utilised within this study as a model of

human dermal fibroblast cells.

Recently Hyde et al. (2004) demonstrated that HaCaT cells were stimulated to proliferate

when VN:GF was added to the culture system. Furthermore, Noble (2008) showed that

dermal fibroblasts derived from diabetic skin could be stimulated to migrate in the

presence of VN:GF. Interestingly, Greco et al. (1998) suggested that human dermal

fibroblast proliferation was stimulated by HA. Therefore, the study reported in this

chapter hypothesized that HA would have a facilitating role and perhaps may even

Page 55

potentiate, or maximize, the effects of VN:GF in terms of stimulating skin cell

proliferation or migration. This chapter aimed to quantitate the functional responses of: 1)

HaCaT and HFF human skin cell lines to combinations of VN:GF and HA in 2-D

monolayer cultures; and 2) HaCaT human keratinocyte to VN:GF in the presence of

CWF.

As reported herein, we demonstrated that HA could significantly stimulate the

proliferation of HFF fibroblasts. This result confirms the previous report by Greco et al.

(1998) which suggested that human fibroblast proliferation was stimulated by HA within

a collagen matrix. Moreover, it has been found that the progression of fibroblasts through

the cell cycle is closely regulated by the interaction of the cell surface receptor, RHAMM,

with HA (Mohapatra et al., 1996). It is proposed that the HA-RHAMM signalling

maintains the level of Cdc2/cyc B1 complex kinase activity and permits cells to progress

through the G2/M phase of the cell cycle. Thus HA plays a key role in supporting

progression through the cell cycle (Greco et al., 1998). The observation that VN:GF can

also significantly enhance HFF proliferation and that the effect of VN:GF alone can be

enhanced by HA, is therefore intriguing. One possible explanation for the enhanced effect

of VN:GF by HA is that cross-talk between the RHAMM and IGF-IR occurs. It has been

reported that RHAMM mediates HA signalling and participates in growth factor-

regulated signalling (Hall et al., 1994). Future studies directed at examining the role of

RHAMM signalling may well prove insightful and help to explain the enhanced response

observed.

Given the critical importance of cell migration for the re-epithelialisation of wounds,

experiments were also undertaken using HaCaT and HFF cells to determine whether the

Page 56

HA and VN:GF combinations affect cell migration. We found no significant difference in

cell migration when HaCaTs and HFFs were exposed to different concentrations of HA.

However, the VN:GF by themselves were able to significantly enhance the migration of

HaCaTs and HFFs. Again, this is consistent with another previous study conducted in our

laboratory. Noble (2008) observed the migration of keratinocytes and fibroblasts was

significantly enhanced by VN:GF. Furthermore, the studies reported herein demonstrate

that HA was not able to stimulate HaCaT proliferation, nor enhance the effect of VN:GF

on HaCaT proliferation. Whilst there are many similarities between HaCaT and primary

keratinocyte cells, one significant difference does exist, namely, primary keratinocytes

require a fibroblast cell feeder layer for their survival and proliferation, whereas HaCaT

cells do not. This therefore suggests that perhaps HaCaT cells may not be the best model

for studying the responses of keratinocytes to HA. Moreover, HA may interact with

keratinocyte cells differently in 2-D cultures as this really is not an accurate reflection of

the 3-D in vivo situation in skin (Birgersdotter et al., 2005; Harvima et al., 2006; Ralston

et al., 1997; Witte & Kao, 2005).

The quality of the selected cell culture system is crucial to the predictability of cell-based

experiments. The potential of low quality cell culture environments to provide non-

predictive data and misinformation for in vivo responses to drugs is of concern. It has

been found that in 2-D cultures primary cells (i.e., those cells freshly isolated from tissue)

frequently express altered morphology, metabolism and gene expression that is rarely

observed in vivo (Sun et al., 2006b). For example, human fibroblasts grown in 2-D

monolayer cultures do not form a normal adhesion integrin structure and proliferate at

reduced rates (Cukierman et al., 2001). Similarly, epithelial cells fail to form polarized,

tissue-like tubes with hollow lumens between the top surface and a basement membrane

Page 57

at the basal surface in 2-D culture (Streuli et al., 1991). Further, the target-specific

signalling pathways may not be activated in a 2-D monolayer culture, and even though

the pathways are active, the signal may be transmitted in different ways under different

cell culture conditions (Plopper et al., 1995).

It is evident that a 2-D culture system provides cells with unnatural geometric, mechanical

and biochemical environmental restrictions and therefore cells studied only in 2-D

monolayer cultures may poorly reproduce important biological properties (Sun et al.,

2006b). For this reason, 3-D cell culture approaches are likely to be more valuable models

for investigating the effects of new techniques and therapies on wound healing

(Birgersdotter et al., 2005; Weaver et al., 1997).

The development of an appropriate model for the investigation of novel therapies has to

consider not only the 3-D geometric environment, but also the physiochemical

environment of a chronic wound, such as CWF. However, before assessing the

therapeutic potential of VN:GF in a 3-D environment, we therefore decided that we

would determine if the VN:GF could maintain its function in the presence of CWF. This

chapter demonstrated that VN:GF was able to maintain the ability to stimulate

proliferation and migration of the HaCaT cells in the presence of CWF. However, limited

CWF samples restricted our study to HaCaT cells only. In the future we will assess the

responses of HFFs, primary fibroblasts and keratinocytes to VN:GF in the presence of

CWF.

Taken together, the data presented in this chapter has demonstrated that VN:GF supports

the proliferation and migration of both HaCaT keratinocyte and HFF fibroblast cells,

Page 58

while HA stimulates the proliferation of only the HFF cells. It was further demonstrated

that VN:GF maintained its function in the presence of CWF. While these results are

significant, we need to bear in mind that these studies were conducted in a 2-D culture

system, which is far removed from the in vivo situation. Therefore, the next step is to

replicate the in vivo environment of skin and assess the functional responses of

keratinocytes and fibroblasts to VN:GF and HA in a 3-D model. This is described in the

next chapter.

Page 59

3CHAPTER 3: INVESTIGATIONS INTO

THE EFFECTS OF COMPLEXES OF

VN:GF AND HA ON KERATINOCYTES

USING HSEs

3.1 INTRODUCTION

Two-dimensional (2-D) monolayer cell culture is widely used in the in vitro assessment

of biological agents. This is due primarily to its ease of use and low costs. However, 2-D

culture systems provide cells with extremely unnatural geometric, mechanical and

biochemical restrictions (Sun et al., 2006b). For example, 2-D cell culture systems do not

truly replicate the intricate interactions occurring among the multiple cells present in the

intact 3-D in vivo skin microenvironment. Consequently, 2-D monolayer cell culture

systems may result in unsatisfactory, misleading and non-predictive data that has minimal

relevance to the in vivo situation (Birgersdotter et al., 2005; Weaver et al., 1997). In view

of this, numerous products that attempt to recapitulate the in vivo cellular and

architectural organization of normal skin have been approved for clinical application, as

well as for various aspects of skin biology research. Examples of these engineered skin

technologies include: Epicel® (Gallico et al., 1984), AlloDerm® (Misra et al., 2008;

Wainwright et al., 1996), Integra® (Berger et al., 2000), Transcyte™ (Lukish et al., 2001;

Purdue et al., 1997), Dermagraft® (Browne et al., 2001), OrCel® (Scott Lipkin et al., 2003;

Still et al., 2003), Apligraf® (Bell et al., 1981; Karr, 2008) and PermaDerm (Boyce et al.,

2006). However, most of these products are composed of either an artificial dermal

Page 60

matrix, or components that are either allogeneic or xenogeneic in origin, with the

exception of Epicel® (Gallico et al., 1984), which is an epidermal-only substitute.

Therefore, a skin model needs to be developed that more closely represents the in vivo

environment, as well as containing cells and proteins that are human in origin.

For the studies described in this chapter, we adopted a human skin equivalent (HSE)

model that uses a human-derived DED scaffold, as this provides cells with a

morphological and biochemical environment similar to normal in vivo skin (Monteiro-

Riviere et al., 1997; Ponec et al., 2002; Topping et al., 2006). Benefits of this HSE model

includes the fact that the DED scaffold provides a natural acellular dermis that facilitates

the maintenance of the microtopology of the human dermis, including features such as the

rete ridge structure and the biochemical elements of the basement membrane (collagens

IV, VII, and laminin) (Medalie et al., 1996). Within this natural collagen ECM, cells

recognise motifs within the matrix proteins (e.g., integrin attachment sites), and this in

turn triggers signals through to the cytoskeleton. This signalling information plays an

important role in regulating and stimulating biological activities in cells (Sun et al., 2005).

Additionally, basement membrane proteins are important for the organisation of

epidermal keratinocytes into their mature multi-layered structure (Ralston et al., 1997). In

view of this, the DED-based HSE model has been utilized for various purposes including

skin grafts, toxicity testing, irritancy, and the metabolic study of topically applied products

(Yeh et al., 2004).

The data presented in chapter 2 detailing the 2-D monolayer cell culture studies

demonstrated that: 1) VN:GF can enhance significantly the migration and proliferation of

HaCaTs; 2) HA has no effect on either HaCaT cell proliferation or migration; and 3) HA

Page 61

can enhance the effect of VN:GF on HFF cell proliferation. The studies detailed in this

chapter were designed to investigate the effects of the VN:GF and HA on keratinocytes

and fibroblasts using a 3-D HSE model. This approach has been adopted because HSEs

can provide an environment that is morphologically and biochemically similar to that

found in normal in vivo skin. Therefore, this chapter aimed to investigate the effects of 1)

VN:GF; 2) HA; and 3) VN:GF + HA on keratinocyte and fibroblast migration,

proliferation and differentiation in a 3-D system.

Page 62

3.2 MATERIALS AND METHODS

3.2.1 Skin Collection

Human skin samples were collected from consenting patients undergoing elective

abdominal and breast reduction surgeries at Wesley Private Hospital (Brisbane,

Queensland, Australia). Human ethical approval was obtained from the hospitals, as well

as the Queensland University of Technology (QUT 3865H, Wesley 2003/46). Excess fat

and blood were removed from the underside of the skin samples and the epidermal

surface was lightly cleaned using alcohol impregnated wipes ISOWIPE (Spill Control

Systems Pty, Cardiff, New South Wales, Australia). The skin samples were transported in

sterile jars containing antibiotic/antimycotic solution (ABAM) (including 10 000

units/mL of penicillin, 10 000 µg/mL of streptomycin, and 25 µg/mL of amphotericin B,

with penicillin G, streptomycin sulphate and amphotericin B as antimycotic agents in

0.85% saline (Invitrogen).

3.2.2 Pre-processing of Skin Samples

Skin explants were washed three times with decreasing concentrations of ABAM

solution. Firstly, the skin explants were washed with 500 mL PBS plus 20 mL

ABAM solution and 160 mg gentamicin sulphate. Secondly, they were washed with

500 mL PBS plus 10 mL ABAM and 80 mg gentamicin sulphate. Thirdly, they were

washed with 500 mL PBS plus 5 mL ABAM. Lastly, they were washed with PBS

and then cut into pieces of varying sizes (3~5 mm x 3~5 mm small pieces and ~1.4

Page 63

cm x 1.4 cm large pieces) depending on the thickness of the donor skin, and stored at

4 0C and 37 0C in the refrigerator and cell incubator, respectively.

3.2.3 Keratinocyte Isolation

Keratinocytes were isolated from skin using a method based on that developed by

Rheinwald and Green (1975). In brief, small size (~3-5 mm x 3-5 mm) pieces of skin

were incubated in 0.125% trypsin (Invitrogen) overnight at 4 0C to facilitate

separation of the epidermis from the dermis. Cells were subsequently removed by

gentle scraping of the newly exposed upper dermal and underside of the epidermal

surfaces using a sterile scalpel. Cells were then resuspended in Full Green’s Medium

(FG) (Rheinwald & Green, 1975) composed of a 3:1 mixture of DMEM and Ham's

F12 medium (Invitrogen) and supplemented with 10% FCS (Hyclone), 1% v/v

penicillin/streptomycin solution (Invitrogen), 2 mM L-glutamine (Invitrogen), 10

ng/mL human-recombinant EGF (Invitrogen), 1 µg/mL insulin (Sigma-Aldrich,

Castle Hill, New South Wales, Australia), 0.1 µg/mL cholera toxin (Sigma-Aldrich),

0.01% v/v non-essential amino acids solution (Invitrogen), 5 µg/mL transferrin

(Sigma-Aldrich), 180 µM adenine (Sigma-Aldrich), 0.4 µg/mL hydrocortisone

(Sigma-Aldrich) and 0.2 µM triiodothyronine (Sigma-Aldrich) as described in

Chakrabarty et al. (1999).

3.2.4 Keratinocyte Culture

Keratinocytes were cultured as per the method described by Rheinwald and Green

(1975) using murine 3T3 feeder cells (J2 clone) (ATCC American Type Culture

Page 64

Collection, Manassas, VA, USA). The murine 3T3 feeder cells were routinely grown

in DMEM containing 5% FCS, 1% v/v penicillin/streptomycin solution and 2 mM L-

glutamine. The 3T3 cells were trypsinised, washed and suspended in fresh medium

and irradiated (50 Gy) at the Australian Red Cross Blood Service (Brisbane,

Queensland, Australia). The irradiated 3T3 feeder cells (i.3T3) were seeded into 80

cm² culture flasks (T80) at a density of 2 x 106 cells/flask for two hours, then the

freshly isolated passage 0 human keratinocytes were seeded at a density of 2 x 106

cells/flask in FG. The keratinocytes were cultured until they reached 80% confluence

in the flasks. The medium was changed every two to three days, and the cells were

incubated at 37 0C, 5% CO2.

3.2.5 Fibroblast Isolation and Culture

Dermal pieces, obtained from the keratinocyte isolation described in section 3.2.3,

were finely minced (0.2 × 0.2 cm) and immersed in 0.05% Collagenase A (Type I)

(Invitrogen) solution in DMEM at 37 0C, 5% CO2 for 18 hr. Subsequently, the

enzyme solution was centrifuged at 2000 rpm for 10 min. Lastly, cells were seeded

into T80 flasks in 10% FBS in DMEM and cultured at 37 0C, 5% CO2. The

fibroblasts were used from passages 2 – 5, when they were 80% confluent.

3.2.6 Preparation of Dermal Equivalent (DED)

The de-cellularised DED was prepared as described previously by Chakrabarty et al.

(1999) with modifications described by Dawson et al. (2006). Briefly, ~1.4 cm x 1.4

cm pieces of split thickness skin samples were immersed in 1 M sodium chloride

Page 65

(Sigma-Aldrich) at 37 0C for 12-18 hr. This facilitated the separation of the epidermis

from the dermis, which was detached by peeling and the use of forceps. The DED

was stored at 4 0C in antibiotic/antimycotic medium containing DMEM and 1% v/v

penicillin/streptomycin solution and the medium was changed every day. Before it

was used, the DED was submerged in the experimental media of choice at 37 0C for

2 hr to allow diffusion and equilibration.

3.2.7 Construction of Human Skin Equivalent (HSE) (figures 3.1 and 3.2)

The DED pieces (1.4 cm × 1.4 cm) were immersed in FG for 2 hr and were then

placed papillary side up into a 24-well culture plate (Nagle Nunc International,

Roskilde, Denmark). Sterile stainless-steel rings (Aix Scientific, Aachen, Germany)

with an internal diameter of 6.7 mm and a silicone washer base were placed on the

papillary side of the DEDs (Fischer, Stingl & Kirkpatrick, 1990; Topping et al.,

2006). Passage 1 (P 1) keratinocytes were grown to approximately 80% confluence,

trypsinised, counted and then seeded into the rings. For each ring, 2 x 104 P1 cells

suspended in 200 µL of FG was added in the centre of the ring, before 200 µL FG

was added in the channel on the side of the ring. The newly constructed HSEs

(keratinocytes plus DEDs) were incubated for 48 hr at 37 0C, 5% CO2. After two

days, the rings were removed and the HSEs were lifted to the air:liquid interface

using a stainless-steel grid in a six-well culture plate (Nagle Nunc International).

These were then cultured at 37 0C, 5% CO2 in approximately 6 mL of FG to allow the

underside of the DEDs to contact the liquid.

Page 66

Figure 3.1. Construction of human skin equivalent (HSE).

Figure 3.2. Timeline of the experiment

D0 (day 0): collect skin

D1 (day 1): Isolate keratinocytes or fibroblasts

D7 (day 8): Seed keratinocytes or fibroblasts on DEDs

D10 (day 10): Lift DEDs seeded with cells to the air:liquid interface

D13 (day 13): Finish 3 day time course experiment

D17 (day 17): Finish 7 day time course experiment

D22 (day 22): Histology

D25 (day 25): Quantitate data

3.2.8 Assessment of the Potential of VN:GF and HA Using HSEs

The HSEs were prepared as described above. At the time of being lifted to the air:liquid

interface, the HSEs were treated with a daily topical application of 40 µL of a 1 × dose of

VN:GF (V1) (0.3 µg VN + 0.3 µg IGFBP3 + 0.1 µg IGF + 0.1 µg EGF ), 3 × dose of

VN:GF (V3) (0.9 µg VN + 0.9 µg IGFBP3 + 0.3 µg IGF + 0.3 µg EGF), HA (60 µg)

+ Trypsin

+ 1M NaCl Dermis

epidermis Epidermis Reconstructed D

onor skin

Do D1 D8 D10 D13 D17 D22 D25

Page 67

(Novozymes, Denmark), HA + 1 × VN:GF (HV1), HA + 3 × VN:GF (HV3) and serum-

free medium (SFM) (as the negative control). After 3 days and 7 days culture at the air-

liquid interface, the HSEs were stained with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-

diphenyletetrazolium bromide (MTT) and fixed in 4% formalin (United Bisciences,

Carindale, Queensland, Australia ) for histological or immunohistochemical analysis

(figures 3.1 and 3.2).

3.2.9 Measurements of Epidermis Outgrowth (MTT Assay)

Keratinocyte migration was assessed by measuring the surface area occupied by

metabolically active cells in the reconstructed epidermis on the DED using the MTT

assay (Denizot & Lang, 1986). After 3 and 7 days of culture at the air:liquid

interface, the HSEs were stained with MTT. Specifically, the HSE samples were

immersed in 1 mL of 0.5 mg/mL MTT solution (Sigma-Aldrich) and were incubated

at 37 0C for 90 min. The purple colour on the upper surface of the HSEs indicates

metabolically active cells (newly formed epidermis). The stained samples were then

photographed using a digital camera Nikon Coolpix 4500 (Maxwell Optical,

Lidcombe, New South Wales, Australia). The purple area, which indicates the

metabolically active keratinocytes as well as an assessment of the extent of

keratinocyte outgrowth over the DED, was measured using Image J (Wayne Rasband,

NIMH, Bethesda, MD, USA).

Page 68

3.2.10 Assessment of the Proliferative and Differentiated Layers of the HSEs

Immunohistochemical analysis of the HSEs was performed as previously described by

Topping et al. (2006). Briefly, paraffin sections were cut to 3 µm thick and

deparaffinished in 100% xylene, and washed in a graded ethanol series (concentrations at

70%, 95%, 100%) and rehydrated in distilled water. The slides were then incubated with

the following primary antibodies: keratinocyte differentiation markers keratin 1/10/11

(K1,10,11) (diluted 1:200, ARP, American Research Product, Belmont, MA, U.S.A);

immature keratinocyte basal cell marker p63 (diluted 1:2000, RDI, Research Diagnostics,

Concord, MA, USA); keratinocyte proliferation marker keratin 14 (K14) (diluted 1:20,

RDI); and basement membrane marker collagen type IV (CIV) (diluted 1:10,

Developmental Studies Hydroma Bank, Iowa, IA, USA). Following a 1-2 hr incubation,

the sections were probed with a Dako Envision kit (Dako Denmark A/S, Glostrup,

Denmark) following the manufacturer’s instructions. The negative controls for the

immunohistochemical analysis excluded the primary antibody step. Finally, all sections

were counterstained with haematoxylin (Sigma-Aldrich) for 30 seconds and were

observed using a digital camera Micropublisher 3.3 RTV (QImaging, Surrey, British

Columbia, Canada) mounted on a fluorescence microscope BX41 (Olympus, Center

Valley, PA, America).

For histology, the HSE samples were fixed in 10% formalin, paraffin-embedded, and then

cut to 3 µm thick cross-sections. The sections were then stained with haematoxylin and

eosin (H&E) following standard protocols for histological analysis (Carlton & Short,

1954). For each H&E slide, eight successive images across the centre of the two sections

were photographed using a digital camera Micropublisher 3.3 RTV (QImaging) mounted

Page 69

on a microscope BX41 (Olympus). The thicknesses of the proliferative layer and

differentiated layer of the newly formed epidermis were quantitated using Scion Image

software (Scion Corp, Frederick, MD, USA) from the average of 10 measurements for

each image in duplicate sections from HSEs treated with each treatment from three

separate experiments.

3.2.11 Blocking VN:GF Activity in the HSE Model.

The HSEs were prepared as described above in 3.2.7. At the time of being lifted to the air:liquid

interface, the HSEs were treated with a topical applications of either: 40 µL (0.6 µg VN + 0.6

µg IGFBP3 + 0.2 µg IGF + 0.2 µg EGF ); 36 µl of (0.54 µg VN + 0.54 µg IGFBP3 + 0.18

µg IGF + 0.18 µg EGF) and 4 µL of anti integrin αv antibody (anti-αv) (1:10 dilution,

Chemicon, Temecula, CA, USA); 36 µl of (0.54 µg VN + 0.54 µg IGFBP3 + 0.18 µg IGF +

0.18 µg EGF) + anti-IGF-1 receptor antibody (anti-IGF-1R) (1:10 dilution, Calbiochem,

Darmstadt, Germany); 32 µl of (0..48 µg VN + 0.48 µg IGFBP3 + 0.16 µg IGF + 0.16 µg

EGF) + anti-αv (1:10 dilution) + anti-IGF-1R (1:10 dilution); or 40 µl of (0.6 µg VN + 0.6 µg

IGFBP3 + 0.2 µg IGF + 0.2 µg EGF )+ non-specific IgG1 subtype (IgG) (1: 500 dilution,

Chemicon, Temecula, CA, USA). After 7 days of culture at the air:liquid interface, the HSEs

were stained with MTT and fixed for histological or immunohistochemical analysis.

3.2.12 Assessment of the Effects of VN:GF and HA on Incorporation of Fibroblasts

into the DED.

Fibroblasts were seeded as keratinocytes decribed in 3.2.7, except that they were seeded on the

reticular side of the DEDs. At the time of being lifted to the air:liquid interface, the DEDs

seeded with fibroblasts were treated with either; 1) 20 µL of VN:GF (V) (0.015 µg / µL

Page 70

VN + 0.015 µg / µL IGFBP3 + 0.005 µg / µL IGF + 0.005 µg / µL EGF ); 2) 20 µL of HA

(HA) (1.5 µg / µL) (Novozymes, Denmark); 3) HA + VN:GF (HV) (0.015 µg / µL VN +

0.015 µg / µL IGFBP3 + 0.005 µg / µL IGF + 0.005 µg / µL EGF + 1.5 µg / µL HA); or 4)

serum-free medium (SFM) (as the negative control). After 3 days culture at the air-liquid

interface, the HSEs were fixed in 4% formalin (United Bisciences) for histological

analysis as described in 3.2.10. The number of fibroblasts was quantitated using Scion

Image software (Scion Corp.). Cell numbers for each treatment were obtained from

counting eight regions of the duplicated treatments for two different donor samples. These

cell counts were then pooled, averaged and analysed using the Turkey’s post hoc test.

3.2.13 Statistical Analysis

Duplicate HSEs were tested individually with each treatment within each assay, and

the experiments were repeated three times, unless otherwise indicated. The DEDs

and keratinocytes were from three different patients. These data were expressed as

the average percentage of control wells containing SFM, or as the original thickness

of the epidermis. One-way ANOVA with Tukey’s post hoc tests (all groups

comparisons) were used to analyse the data. Statistically significant differences were

determined as p < 0.05.

Page 71

3.3 RESULTS

3.3.1 Investigation into the Effect of VN:GF and HA on the Outgrowth of

Keratinocytes Using HSEs Cultured at the Air:Liquid Interface

The 3-D HSE model was used to investigate the influence of the VN:GF and HA on

keratinocyte outgrowth. Daily treatments of VN:GF, HA and HA + VN:GF were applied

topically to the HSEs for 3 days and 7 days, and then the HSEs were stained with MTT

(figures 3.3 and 3.5). The area stained by MTT revealed that there was an outgrowth of

keratinocytes from the centre seeding region to the outer edge of the HSEs.

When the HSEs were treated daily with 1 × dose of VN:GF, 3 × dose of VN:GF, HA, HA

+ 1 × VN:GF, HA + 3 × VN:GF and the negative control serum-free medium (SFM) for

3 days and 7 days, similar responses were obtained, except for the HSEs treated with

HV3. The area stained by MTT was quantitated (figures 3.4 and 3.6) and revealed that at

day 3, the stained area obtained from HV3, was significantly less than that obtained with

the control treatment (SFM), with the surface area 27.6 ± 8.0% less than the control (p <

0.05). At day 7, similar responses to day 3 were obtained. The outgrowth surface area

measured in the HSEs treated with HV3 was significantly less than that observed for the

control (SFM), with values of 28.3 ± 2.00% less than the control (p < 0.05).

Page 72

Figure 3.3. MTT analysis of HSEs (3 days). From left to right the DEDs were

seeded with primary keratinocytes for 48 hr and then topically treated with: serum-

free media (SFM), 1 × VN:GF (V1), hyaluronic acid (HA), HA + 1 × VN:GF (HV1),

3 × VN:GF (V3), HA + 3 × VN:GF (HV3). At day 7 the models were stained with

MTT to visualise the outgrowth of the keratinocytes on the DED models. Scale bar =

3 mm.

M SFM V1 HV1

HA V3 HV3

Page 73

0%

20%

40%

60%

80%

100%

120%

140%

SFM V1 HV1 HA V3 HV3

% o

f con

trol (

SF

M)

*

Figure 3.4. Quantification of the outgrowth of keratinocytes in response to

VN:GF and HA (3 days). Responses of HSEs treated with HA and different

concentrations of VN:GF. The data, expressed as the average percentage of the

outgrowth observed with the control SFM alone over 3 days, were pooled from three

replicate experiments in which each treatment was tested in duplicate. Each replicate

experiment used cells and DEDs derived from a different donor (n = 6). The asterisks

(*) indicate treatments exhibiting a significant decrease in the outgrowth compared to

the control SFM (p < 0.05). Error bars indicate SD.

Page 74

Figure 3.5. MTT analysis of HSEs (7 days). From left to right the DEDs were

seeded with primary keratinocytes for 48 hr and then topically treated with: SFM,

V1, HA, HV1, V3 and HV3. At day 7 the models were stained with MTT to visualise

the outgrowth of the keratinocytes on DED models. Scale bar = 3mm.

SFM V1 HV1

HA V3 HV3

Page 75

0%

20%

40%

60%

80%

100%

120%

140%

SFM V1 HV1 HA V3 HV3

% o

f con

trol (

SF

M)

*

Figure 3.6. Quantification of the outgrowth of keratinocytes in response to

VN:GF and HA (7 days). Responses of HSEs treated with HA and different

concentrations of VN:GF. The data, expressed as the average percentage of the

outgrowth observed with the control serum free treatment SFM alone over 7 days,

were pooled from three replicate experiments in which each treatment was tested in

duplicate (n = 6). Each replicate experiment used cells and DEDs derived from a

different donor. The asterisks (*) indicate treatments which significantly increased or

decreased the outgrowth compared to the response observed with the control SFM (p

< 0.05). The effects obtained in HSEs treated with V1, HA, HV1, V3 or HV3 are

compared with the effect obtained from control SFM. Error bars indicate SD.

Page 76

3.3.2 Investigation into the Effect of VN:GF and HA on the Proliferative and

Differentiating Layers of the Epidermis Using HSEs Cultured at the

Air:Liquid Interface.

In order to investigate the influence of the VN:GF complex and HA on the proliferative

and differentiating layers of the epidermis, different treatments (VN:GF, HA and HA +

VN:GF) were applied to the HSEs at the air:liquid interface for 3 and 7 days. The HSEs

were then analysed with histology and immnohistochemistry for examination of HSE

morphology and to enable examination of the expression of specific cell-surface markers,

namely p63, K 1/10/11 and K14 and CIV (figures 3.7 and 3.9).

The immunohistochemistry revealed at day 3, positive immunoreactivity of nuclear

transcription factor p63 (a marker for undifferentiated proliferating cells), K1/10/11 (a

marker for stratifying differentiated cells), K14 (a marker of hemidesmosome formation

in basal cells) and CIV (a basement membrane marker). This immunoreactivity was

observed in their expected locations within the epidermis (figure 3.7). This was also true

for the HSE models tested at day 7 (figure 3.9).

The immunohistochemistry revealed that the epidermis was partitioned into two layers:

the proliferative layer and the differentiated layer. The thickness of the proliferative layer

and differentiated layer of the HSEs were therefore quantitated (figures 3.8 and 3.10).

After 3 days of topical treatment, the thickness of the proliferative layers and

differentiated layers of the HSEs treated with VN:GF, HA and HA + VN:GF were

significantly different (p < 0.05) to that obtained with the control treatment SFM. The

thickness of the proliferative layers of the HSEs treated with 1 × VN:GF, HA + 1 ×

Page 77

VN:GF, HA, 3 × VN:GF and HA + 3 × VN:GF were significantly thicker than the control

SFM, with thicknesses of 24.43 ± 1.33 µm, 20.15 ± 4.80 µm, 16.51 ± 8.99 µm, 31.59 ±

13.99 µm and 23.05 ± 6.56 µm above the control SFM (p < 0.05) treatment, respectively,

observed. The thickness of the differentiated layers of HSEs treated with 1 × VN:GF, HA

+ 1 × VN:GF, HA, 3 × VN:GF and HA + 3 × VN:GF were also significantly thicker than

the control SFM, with thicknesses of 29.23 ± 2.57 µm, 20.51 ± 1.32 µm, 12.56 ± 1.13

µm, 35.52 ± 0.13 µm and 27.78 ± 1.72 µm above the control SFM (p < 0.05),

respectively, observed (figure 3.8). These results suggest that the VN:GF, HA and HA +

VN:GF can significantly enhance keratinocyte proliferation and differentiation on HSEs

at the air:liquid interface.

After 7 days of topical treatment, the thicknesses of the proliferative layers of the HSEs

treated with VN:GF, HA and HA + VN:GF were not significantly different (p > .05) to

those treated with the control SFM. The thicknesses of the differentiated layers of the

HSEs treated with 1 × VN:GF, 3 × VN:GF and HA + 3 × VN:GF, however, were

significantly thicker than those treated with the control SFM, with thicknesses of 34.96 ±

4.99 µm, 34.95 ± 5.25 µm and 33.35 ± 4.30 µm above the control SFM (p < 0.05),

respectively, observed (figure 3.10). These results indicate that the 1 × VN:GF, 3 ×

VN:GF and HA + 3 × VN:GF can significantly enhance keratinocyte differentiation

compared to the SFM control, but not the proliferation of keratinocytes on 3-D HSEs

cultured at the air:liquid interface. In addition, it was noted that HA does not enhance the

effect of VN:GF on either keratinocyte proliferation or differentiation.

Page 78

Histology Immunohistochemistry

Figure 3.7. Histological and immunohistochemical analysis of HSEs (3 days).

From top to bottom the HSEs were topically treated with: SFM, V1, HV1, HA, V3

and HV3. From left to right, histological and immunohistochemical (p63, K1, K14

and CIV) images are depicted. Scale bar = 100 µm. All images are representative

photos obtained from three different experiments with skin from different donors.

SFM

V1

HV1

HA

V3

HV3

P63 K14 K1 CIV

Page 79

Figure 3.8. Quantification of the proliferative and differentiating layers of the

epidermis in response to VN:GF and HA on the HSEs (3 days). Responses of HSEs

were treated with HA and different concentrations of VN:GF. The data, expressed as the

thickness of proliferative layer and differentiated layer of HSEs after 3 days, were pooled

from three replicate experiments in which each treatment was tested in duplicate. Each

replicate treatment had keratinocytes and DEDs from a separate donor (n = 6). The

asterisks (#) and (*) indicate treatments which significantly increased the proliferative

(dotted bars) and differentiating layers (full colour bars) compared to the responses

observed with HSEs treated with the control SFM treatment (p < 0.05). Error bars

indicate SD.

*

020406080

100120140160180200

SFM VG1x HV1x HA VG3 HV3

Thi

ckne

ss (µ

m)

V1 H V1

*

# # #

##

* * *

V3

Page 80

Histology Immunohistochemistry

Figure 3.9. Histological and immunohistochemical analysis of HSEs (7 days). From

top to bottom the HSEs were topically treated with: SFM, V1, HV1, HA, V3 and HV3.

From left to right, histological and immunohistochemical (p63, K1, K14 and CIV) images

are depicted. Scale bar = 100 µm. All images are representative photos obtained from

three different experiments with skin from different donors.

SFM

P63

V1

HV1

HA

V3

HV3

K14 K1 CIV

Page 81

020406080

100120140160180200

SFM VG1x HV1x HA VG3 HV3

Thi

ckne

ss (µ

m)

V1 HV1 V3

* * *

Figure 3.10. Quantification of the proliferative and differentiating layers of the

epidermis in response to VN:GF and HA on the HSEs (day 7). Responses of HSEs

were treated with HA and different concentrations of VN:GF. The data, expressed as the

thickness of proliferative layer and differentiated layer of HSEs after 3 days, were pooled

from three replicate experiments in which each treatment was tested in duplicate. Each

replicate treatment had keratinocytes and DEDs from a separate donor (n = 6). The

asterisks (*) indicate treatments which significantly increased the differentiating layers

(full colour bars) compared to the responses observed with HSEs treated with the control

SFM treatment (p < 0.05). Error bars indicate SD.

Page 82

3.3.3. Investigations into the Effects of VN-binding Integrins αv and the IGF-1 receptor on Blocking Keratinocyte Function Evoked by VN:GF using HSEs (outgrowth)

To elucidate the mechanisms behind the VN:GF-complex-stimulated proliferation and

differentiation of keratinocytes seeded onto the HSEs, the role of VN-binding αv integrin

receptors and the IGF-I receptors (IGF-1R) were assessed. Wertheimer et al. (2000) have

shown that primary keratinocytes normally express both insulin and IGF-I receptors (IGF-

1R) and respond to either ligand during differentiation. We investigated the contribution

of IGF-1R, as well as the VN-binding αv integrin, on keratinocyte proliferation and

differentiation in the HSEs. This was achieved by adding monoclonal antibodies with

established function-blocking properties, together with VN:GF, on the top of the HSEs.

The HSEs were topically treated with different treatments of VN:GF + anti-IGF-1R,

VN:GF + anti-αv, VN:GF + anti-IGF-1R + anti-αv and VN:GF + IgG at the air:liquid

interface every day for 7 days, and then were stained with MTT (figure 3.11). The area

stained by MTT revealed that the keratinocytes migrated from the centre seeding region to

the edge of the HSEs.

After 7 days of topical treatment, the migration surface areas obtained from VN:GF +

anti-IGF-1R, VN:GF + anti-αv, VN:GF + anti-IGF-1R + anti-αv were not significantly

different from that obtained with VN:GF according to statistical analysis (figure 3.12).

However, a trend suggesting anti-IGF-1R and anti-αv appeared to enhance keratinocyte

migration on HSEs was observed. Nevertheless, these results indicate that the treatments

of VN:GF + anti-IGF-1R, VN:GF + anti-αv, VN:GF + anti-IGF-1R + anti-αv have no

effect on keratinocyte outgrowth.

Page 83

Figure 3.11. MTT analysis of HSEs treated with VN:GF and VN:GF + IGF-

1R/anti-αv integrin function blocking antibodies (7 days). From left to right the

HSEs were topically treated with: V, V + 1R, V + αv, V + 1R + αv and V + Ig. Scale

bar = 3 mm. All images are representative photos obtained from three different

experiments with skin from different donors.

V V+1R V+ αv V+1R+ αv V+Ig

Page 84

Figure 3.12. Quantification of the outgrowth of keratinocytes in response to

VN:GF and VN:GF + IGF-1R/αv function blocking antibodies (7 days).

Responses of HSEs were treated with V, V + 1R, V + αv, V + 1R + αv and V + Ig.

The data, expressed as the average percentage of control serum free medium (SFM)

alone over 7 days, were pooled from three replicate experiments in which each

treatment was tested in duplicate (n =6). Each replicate treatment had keratinocytes

and DEDs isolated from skin from a separate donor. The effects obtained in HSEs

treated with V + 1R, V + αv, V + 1R + αv and V + Ig are compared with the effects

obtained from control V. Error bars indicate SD.

0%

20%

40%

60%

80%

100%

120%

140%

V V+IR V+αv V+IR+αv V+Ig

% o

f con

trol

(V

)

Page 85

3.3.4. Investigations into the Effects of VN-binding Integrins αv and the IGF-1 Receptor on Blocking Keratinocyte outgrowth Evoked by VN:GF using HSEs (Proliferative and Differentiated layers of epidermis)

The HSEs were treated daily with 40 µL of VN:GF, VN:GF + anti-IGF-1R, VN:GF +

anti-αv, VN:GF + anti-IGF-1R + anti-αv and VN:GF + IgG for 7 days. These HSEs were

then analysed with histology and imunohistochemistry to examine the morphology and to

assess the expression of specific cell-surface markers (figure 3.13). For

immunohistochemistry, positive immunoreactivity of nuclear transcription factor p63,

K1/10/11, K14 and CIV was observed in their expected locations within the epidermis for

HSEs (figure 3.13).

Again, immunohistochemistry results indicated that the epidermis was divided into two

layers: the proliferative layer and the differentiated layer. The thicknesses of the

proliferative layers and differentiated layers in the HSEs were therefore quantitated

(figure 3.14). For all differently-treated HSEs, similar responses to the control were

obtained, except for the VN:GF + anti-IGF-1R and VN:GF + anti-IGF-1R + anti-αv

treatments. In HSEs treated topically over a 7 day period, only the thickness of the

differentiated layers of HSEs treated with VN:GF + anti-IGF-1R and VN:GF + anti-IGF-

1R + anti-αv were significantly different from the control VN:GF, with values of 66.96 ±

0.27 µm and 79.53 ± 6.03 µm, respectively, below the control VN:GF treatment observed

(p < 0.05). These results indicate that IGF-1R antibody can block the effect of VN:GF on

keratinocyte differentiation over 7 days. Although the IGF-1R antibody can also block the

effect of VN:GF on keratinocyte proliferation over 7 days, there is no significantly

difference in the proliferative layers of HSEs treated with VN:GF or VN:GF + anti-IGF-

1R or VN:GF + anti-IGF-1R + anti-αv VN:GF following statistical analysis (figure 3.14).

Page 86

Histology Immunohistochemistry

Figure 3.13. Histological and immunohistochemical analysis of HSEs treated with

VN:GF and VN:GF + anti-IGF-1R/αv function blocking antibodies (7 days). From

top to bottom the HSEs were topically treated with: VN:GF (V), VN:GF + anti-IGF-1R

(V + 1R), VN:GF + anti-αv (V + αv), VN:GF + anti-IGF-1R + anti-αv (V + 1R + αv) and

V + IgG (V + Ig). From left to right, histological and immunohistochemical (p63, K1,

K14 and CIV) images are depicted. Scale bar = 100 µm. All images are representative

photos obtained from three different experiments with skin from different donors.

V

P63

V+1R

V+αv

V+1R+αv

V+Ig

K14 K1 CIV

Page 87

Figure 3.14. Quantification of the proliferative and differentiating layers of the

epidermis in response to VN:GF and VN:GF + anti-IGF-1R/αv function

blocking antibodies (7 days). Responses of HSEs treated with V, V + 1R, V + αv, V

+ 1R + αv and V + Ig. The data, expressed as the thickness of proliferative layer and

differentiated layer of HSEs after 7 days, were pooled from three replicate

experiments in which each treatment was tested in duplicate (n =6). Each replicate

treatment had keratinocytes and DEDs isolated from skin from a separate donor. The

effects obtained in HSEs treated with V + 1R, V + αv, V + 1R + αv and V + Ig are

compared with the effects obtained from control V. The asterisks (*) indicate

treatments which significantly decrease differentiation of the HSEs compared to the

response observed with the control V (p <.05) treatment. Error bars indicate SD.

020406080

100120140160180200

V V+IR V+av V+IR+av V+Ig

Thi

ckne

ss (µ

m)

* *

Page 88

3.3.5 Investigation into the Effect of VN:GF and HA on incorporation of

Fibroblasts into the DEDs.

In addition to keratinocytes, fibroblasts were also examined. Specifically, the VN:GF, HA

and HA +VN:GF were applied topically on DED seeded with fibroblasts to develop a

more defined DED model. Fibroblast integration into the DED models was encouraged

using daily treatments of VN:GF, HA and HA + VN:GF for 3 days. Histology was used

to examine the most appropriate serum-free treatment to support the integration of

fibroblasts into the DED model (figure 3.15). After 3 days of topical treatment, the

numbers of fibroblasts incorporated into the DEDs treated with HA and HA + VN:GF

were significantly different (p < 0.05) to that obtained with the control treatment SFM.

The number of fibroblasts integrated into the DED models treated with VN:GF, HA and

HA + VN:GF were 6.42 ± 0.86, 7.67 ± 0.71 and 8.21 ± 0.89, respectively, compared to

the control SFM 5.54 ± 0.60 (figure 3.16). These results indicate that the HA can enhance

the integration of fibroblasts into the DED

.

Page 89

Figure 3.15. Histological analysis of the DED seeded with fibroblasts. DED

seeded with fibroblasts were topically treated with: A) SFM, B) VN:GF, C) HA and

D) HA + VN:GF. Scale bar = 100 µm. All images are representative photos obtained

from two different experiments with skin from different patients. Yellow arrows

point to cells that have migrated into the DEDs.

Page 90

0

1

2

3

4

5

6

7

8

9

10

SFM VN:GF HA HV

No.

of f

ibro

blas

ts

* *

Figure 3.16. Quantification of the number of fibroblasts entering DEDs treated

with VN:GF, HA and HA + VN:GF. Responses of fibroblasts seeded into DED

treated with VN:GF, HA and HA + VN:GF over 3 days. The data is expressed as the

number of H&E sections counted from two replicate experiments with 12 sections from each

treatment being counted in each experiment (n = 24). Each replicate treatment had

fibroblasts and DEDs from a separate donor. The asterisks indicate treatments in

which significantly greater numbers of fibroblasts were present compared to that

observed with DEDs treated with the control SFM (p < 0.05) treatment. The effects

of DEDs treated with HA and HA + VN:GF are compared with the effects obtained

with those treated with the control SFM. Error bars indicate SEM.

Page 91

3.4 DISCUSSION

As covered in the introduction to this chapter, the HSE model we used demonstrates

similar morphology, structure and functions to in vivo skin, hence provides a useful ex

vivo tool for investigating the effect of potential therapeutics (Breetveld et al., 2006;

Chakrabarty et al., 1999; Topping et al., 2006). This chapter was therefore focused on

investigating the effect of HA and VN:GF on both keratinocyte and fibroblast function

using this novel 3-D approach.

The data from the 2-D cell culture studies presented in chapter 2 of this thesis

demonstrated that the VN:GF complexes enhance keratinocyte proliferation and

migration. This is similar to previous reports by Hyde et al. (2004), in which the trimeric

complexes (VN + IGF-I + IGFBPs) were shown to enhance keratinocyte protein synthesis

and migration. However, there has been little investigation into the effect of VN:GF on

the functional responses of keratinocytes using 3-D approaches. Similarly, at the time I

conducted these experiments, little was known about the potential mechanisms

underpinning these enhanced cellular functions. As such, the studies reported in this

chapter comprise the first significant investigation into the effects of the VN:GF and HA

on keratinocytes, as well as the mechanisms underlying the VN:GF-stimulated cellular

response. Moreover, these results represent the first studies that have utilised a 3-D HSE

approach.

As reported herein, the HSEs were topically treated with two different concentrations of

VN:GF for 3 days and 7 days when cultured at the air:liquid interface. They were then

analysed with MTT, histology and immunohistochemistry (figure 3.3 to 3.10). The results

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have shown that both 1 x dose VN:GF and 3 x dose of VN:GF enhanced keratinocyte

proliferation and differentiation over 3 days, and that this effect was sustained in the

differentiation layer for another four days (7 days). Moreover, the effect appeared to be

dose-dependent. The 2-D cell culture studies from chapter 2 demonstrated that the

VN:GF complex can significantly enhance keratinocyte migration and proliferation

(figures 2.1 to 2.6). The 3-D studies in this chapter indicate that the thickness of the

proliferative and differentiating layers of the epidermis, whose formation involves

keratinocyte migration, proliferation and differentiation, were significantly increased by

application of VN:GF (figures 3.7 to 3.10). We contended that compared with 2-D

approaches, the 3-D model therefore provides more accurate information that can be

related to the in vivo situation. For example, the 2-D monolayer cell culture is usually

conducted in cell culture vessels, such as 12-well (migration assay) and 48-well

(proliferation assay) cell culture plates, where the monolayer of cells is submerged in

medium. In addition, the migration assay and the proliferation assay were undertaken

independently and using different protocols. The 3-D HSE on the other hand, which uses

a human-derived de-epidermized dermal scaffold, provides cells with a morphological

and biochemical environment resembling native in vivo skin. In addition, the HSEs were

cultured at the air:liquid interface, which more closely mimics the normal environment of

in vivo skin. Indeed, this 3-D culture system allows keratinocytes to grow as if they are a

living organ, and an epidermis composed of a stratum basale, a stratum spinosum, a

stratum granulosum and a stratum corneum, similar to in vivo skin (Breetveld et al., 2006;

Chakrabarty et al., 1999; Topping et al., 2006), is formed. In addition, this model allows

keratinocyte migration, proliferation and differentiation to be studied simultaneously.

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As to the precise mechanisms underlying the enhanced cellular responses to growth

factors in the presence of VN, it has been proposed that “cross-talk” occurs between

growth factor receptors and αν integrins (Hollier et al., 2008). Growth factors have also

been known to modulate the expression of integrin family members in tissue repair

(Danilenko et al., 1995). Indeed, recent studies from our own laboratory demonstrated

that the novel VN:IGF-I:IGFBP complexes enhance breast epithelial cellular migration

via increased and sustained activation of the PI3-K/AKT signalling pathway through co-

activation of the IGF-1R and αv-integrins (Hollier et al., 2008). In addition, it has been

found that antibody inhibition of both the VN-binding αv integrins and the IGF-1R are

critical for the enhanced keratinocyte migration observed in response to the VN:GF

complexes in cell culture (Upton et al., 2008). Of note, however, all of these experiments

were undertaken in 2-D culture systems.

In view of this, and as reported in this chapter, function blocking assays were undertaken

in the HSEs to investigate the mechanisms underlying the enhanced proliferation and

differentiation of keratinocytes in response to VN:GF. Specifically, function-blocking

anti-αv integrin antibodies and anti-IGF-1R antibodies were added together with VN:GF

on the top of the HSEs. The results reveal that the VN:GF-enhanced cellular migration,

proliferation and differentiation was significantly reduced by the IGF-1R antibody, but

not the anti-αv integrin antibody (figures 3.13 and 3.14). While the anti-αv integrin

antibody appeared to decrease keratinocyte migration, proliferation and differentiation

somewhat, the effect was not significantly different to the control (figure 3.13 and 3.14).

One possible explanation for the lack of effect of the anti-αv integrin antibody is that the

cells were allowed to attach, proliferate and migrate 48 hours before the blocking

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antibodies were added. This method of blocking may be problematic due to the fact that

the cells are already attached, therefore future studies will look at pre-binding the cells

with the blocking antibodies before adding the cells to the HSEs with the VN:GF.

Furthermore, the basement membrane of the HSE models consists of large amounts of

collagen IV, which may be providing the cells with an alternative ECM to attach to and

migrate upon (Decline & Rousselle, 2001; Zhang et al., 2006). Additionally, future

experiments examining other antibodies for VN integrin blocking, such as anti-αvβ1 and -

αvβ5, need to be undertaken to determine whether we can further inhibit the VN:GF

activity. In addition, studies examining the intracellular signalling pathways activated

would be beneficial.

In this chapter, the effects of HA and HA + VN:GF on functional responses of

keratinocytes were also investigated using 3-D HSEs. The results reported herein

demonstrate that HA can significantly stimulate keratinocyte migration, proliferation and

differentiation (figure 3.7 and 3.8). These results are similar to previous reports which

suggested that HA facilitates keratinocyte migration, proliferation and differentiation

through interactions between HA and the cell surface HA receptors CD44 and

hyaladherin RHAMM (Ahrens et al., 2001; Bourguignon et al., 2004; Lokeshwar et al.,

1996; Masellis-Smith et al., 1996). When HA was mixed together with VN:GF,

keratinocyte outgrowth as measured by MTT was inhibited, especially in the HA + 3 x

VN:GF treatment (figures 3.3 to 3.6). However, immunohistochemistry confirmed that

VN:GF alone, and in conjunction with HA, were able to significantly enhance the

development of both the proliferative and differentiating layers in the HSE models (figure

3.7 and 3.9). This suggests that there is an interaction between HA and VN:GF. However,

a previous study by Prisell et al. (1992) showed that HA can retard the release rate of

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peptide growth factors such as IGF-I, and this effect is dose dependent. Thus HA may

introduce temporal effects that were missed in our experiment. The precise mechanisms

behind this interaction is unknown, but one hypothesis is that this arises from the

complexity of the long chains of HA and/or ionic interactions between HA and the

peptide (Prisell et al., 1992). It would be beneficial to perform further experiments such as

a diffusion study, or in vitro and in vivo release studies, to examine the interactions

between HA and VN:GF.

In addition to keratinocytes, fibroblasts were also examined for responses to the effect of

VN:GF and HA using 3-D HSEs. More specifically, the studies reported in this chapter

demonstrated that VN:GF and HA significantly enhanced the integration of dermal

fibroblasts into the DEDs; levels of integration were greater than that observed with the

serum-free controls (figures 3.15 and 3.16). These results suggest that VN:GF and HA

may have therapeutic potential in healing skin wounds. Furthermore, these proteins may

also be useful in terms of the development of a defined HSE model that is constructed

and cultured in the absence of serum.

Due to the need for a 3-D model for the accurate study of skin biology, as well as dermal-

epidermal constructs for clinical use, numerous products that recapitulate the in vivo

cellular and architectural organization of normal skin have been developed, such as

Integra® (Berger et al., 2000), Dermagraft® (Browne et al., 2001), PermaDerm (Boyce et

al., 2006) and so on. However, most of these skin technologies rely on components that

are either allogeneic and/or xenogeneic in origin. Moreover, all of these ex vivo

constructs are cultured with media containing serum, which provides cells with a non-

defined environment. This poses problems in terms of variability, as well as presents

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risks, as serum has significant batch-to-batch variation and potentially also carries

pathogens (Gibbs et al., 1997). Furthermore, with constructs which incorporate a

synthetic matrix, such as a bio-absorbable polymer scaffold, it has been demonstrated that

the synthetic components may falsely activate cells to digest and re-organise their

environment, for example via the production of host collagen (Wood et al., 2007).

Therefore, a defined, serum-free HSE model needs to be developed so that it more closely

represents the in vivo environment. Generation of such a model in the absence of serum

also provides a feasible construct with potential for clinical use.

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4 CHAPTER 4: DEVELOPMENT OF 3-D

HSE WOUND MODELS FOR TESTING

NOVEL WOUND HEALING THERAPIES

4.1 INTRODUCTION

Wound healing of the skin involves a cascade of co-ordinated and systematic

biochemical and cellular events. These natural processes restore the integrity and

function of the epidermis and dermis (Iba et al., 2004; Stadelmann et al., 1998).

Often external help, in the form of wound dressings and wound therapies, are

required to restore this integrity. While human studies are the most relevant and

accurate way to determine the effectiveness of wound therapies, this approach is

often impractical. Difficulties in this process include recruiting similar patients with

identical wounds, limitations with objective measurements of wound healing, and

ethical considerations (Pocock et al., 1987). In order to advance our understanding of

the complexity of wound healing, development of ex vivo models mimicking the in

vivo physiology are therefore required (Geer et al., 2004).

One example of a wound model involves the wounding or scratching of keratinocyte

monolayers in culture with a pipette tip. This has been used to study keratinocyte

cellular responses to wounding, including cell migration and proliferation (Legrand et

al., 2001; Turchi et al., 2002; Yamada et al., 2000), as well as to assess wound

healing therapies (Geer & Andreadis, 2003; Yamaguchi et al., 2002). However, as

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noted in the previous chapter, it is increasingly recognized that 2-D cell culture

approaches have limitations and disadvantages, such as the unnatural geometric and

mechanical restrictions of the 2-D environment, and more often than not is limited to

a single cell type (Sun et al., 2006b). In view of this, animal studies are widely used

to model human wound healing and to test the efficacy of different treatments. Pig

skin is regarded as being the most similar to human skin in many aspects, such as:

structure; dermal-epidermal thickness ratio (Vardaxis et al., 1997); possession of

well-developed rete ridges (Winter, 1996); the size and distribution of blood vessels

in the dermis (Meyer et al., 1978); functions, such as epidermal turnover time (Gray

et al., 1982); and similar staining patterns for some antigens (keratins 10 and 16,

collagen IV) (Wollina et al., 1991). Although it is believed that pigs provide the best

current pre-clinical model for human wound healing, the cost, availability, ethical

considerations and difficulty of handling these animals restrict their widespread use

(Winter, 1996).

To better mimic skin in vivo, we have therefore used a 3-D HSE. As described in chapter

3, the HSE is a model containing human keratinocytes that are seeded onto a de-

epidermised dermis and which are then cultured at the air:liquid interface (Dawson et al.,

2006). This model has been shown to have histological features similar to those observed

in an in vivo epidermis, such as the formation of a stratum basale, stratum spinosum,

stratum granulosum and stratum corneum, as well as the expression of biochemical

markers such as keratin 1/10/11 and 16 (Monteiro-Riviere et al., 1997; Parnigotto et al.,

1998; Ponec et al., 2002; Poumay et al., 2004; Topping et al., 2006). In addition, the

natural acellular dermis maintains the microtopology of human dermis, such as the rete

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ridge structure and elements of the basement membrane, such as collagens IV, VII and

laminin (Medalie et al., 1996).

The similarities of HSEs to human skin in aspects of morphology, structure and function

confer upon this model the ability to be a better ex vivo cell culture system for studying

wound re-epithelialisation (Tomic-Canic et al., 2004) and for the assessment of a range of

skin repair treatments (Breetveld et al., 2006; Chakrabarty et al., 1999; Topping et al.,

2006). However, a serum- and xenobiotic-free, reproducible partial-thickness wound in

this type of model, with a given size, has yet to be reported. The data reported in this

chapter was therefore focussed on the development of a reproducible partial-thickness

wound model from the HSE models for investigating the effect of the VN:GF complex in

wound healing. In addition, this model was also employed to examine the effect of a

synthetic fibrin-like gel in partial-thickness wound healing. This synthetic fibrin-like gel

was examined since it has been found that fibrin gels containing physiological

concentrations of fibrinogen and thrombin can accelerate keratinocyte activation and

wound closure (Geer et al., 2002). In addition, the model was further refined by adding

fibroblasts to the DED and creating full-thickness wounds in the HSE. The effect of the

synthetic fibrin-like gel on keratinocytes and fibroblasts in full-thickness wound healing

was examined using this full-thickness model.

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4.2 MATERIALS AND METHODS

4.2.1 Skin Collection

Skin was collected as described in chapter 3 (3.2.1).

4.2.2 Pre-processing of the Skin Samples

Skin samples were pre-processed as described in chapter 3 (3.2.2).

4.2.3 Keratinocyte Isolation

Keratinocytes were isolated as described in chapter 3 (3.2.3).

4.2.4 Keratinocyte Culture

Keratinocytes were cultured as described in chapter 3 (3.2.4).

4.2.5 Fibroblast Isolation and Culture

Fibroblasts were cultured as described in chapter 3 (3.2.5).

4.2.6 Preparation of Dermal Equivalent (DED)

DEDs were prepared as described in chapter 3 (3.2.6), except that the size of the DED in

this HSE model was 1.8 cm × 1.8 cm instead of 1.4 cm × 1.4 cm.

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4.2.7 Fabrication of a 6 mm Diameter Partial-thickness HSE Wound Model

(Removal of the Epidermis)

The DED pieces (1.8 cm × 1.8 cm) were immersed in FG for 2 hr and were then

placed papillary side up in a 12-well culture plate (Costar, New York, NY, USA).

Sterile stainless-steel rings with an internal diameter of 9 mm were placed on top of

the DEDs. When P1 keratinocytes were grown to approximately 80% confluence,

they were trypsinised, counted and seeded into the rings (Dawson et al., 2006). For

each ring, 3.6 x 104 P1 cells suspended in 200 µL FG were added in the centre of the

ring, before 500 µL of FG was added around the side of the ring. The HSEs

(keratinocytes plus DEDs) were incubated for 48 hr at 37 0C, 5% CO2 in FG. After

two days, the rings were removed and the constructs were lifted to the air:liquid

interface using a stainless-steel grid in a six-well culture plate (Nagle Nunc

International, Roskilde, Denmark). The HSEs were then cultured in approximately 6

mL of either FG, VN:GF and SFM media to allow the underside of the DEDs to

contact the medium at 37 0C, 5% CO2. After 9 days’ culture at the air:liquid

interface, 6 mm partial-thickness excision wounds were created in the HSEs with a 6

mm biopsy punch (Stiefel, Castle Hill, New South Wales, Australia) cutting through

the epidermis. The epidermis was then peeled away from the DED and discarded.

4.2.8 Fabrication of a 4 mm Diameter Full-thickness HSE Would Model

(Removal of the Epidermal/dermal Core).

The DED pieces (1.8 cm x 1.8 cm) were immersed in FG for 2 hr and were then

placed papillary side (upper dermis) up (for seeding of keratinocytes) or reticular side

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(lower dermis) upward (for seeding of fibroblasts) in a 12-well culture plate (Costar).

Sterile stainless-steel rings with an internal diameter of 9 mm were placed on the

papillary or reticular side of the DEDs. When keratinocytes and fibroblasts were

grown to approximately 80% confluence, they were trypsinised, counted and seeded

into the rings. For each ring, 3.6 x 104 cells (P1 keratinocytes or P3~P5 fibroblasts)

suspended in 200 µL FG were added in the centre of the ring, before 500 µL of FG

was added on the side of the ring. The HSEs seeded with either keratinocytes alone,

fibroblasts alone, or keratinocytes and fibroblasts together, were incubated for 48 hr

(keratinocytes), 72 hr (fibroblasts) and 48 hr (keratinocytes) + 72 hr (fibroblasts),

respectively, at 37 0C, 5% CO2 days. At this time, the rings were removed and the

HSEs (seeded with keratinocytes and fibroblasts alone, or together) were lifted to the

air:liquid interface using a stainless-steel grid in a six-well culture plate (Nagle Nunc

International). These constructs were then cultured in approximately 6 mL FG at 37

0C, 5% CO2. After seven days of culture at the air:liquid interface, 4 mm diameter

full-thickness incision wounds were created in the HSEs using a 4 mm biopsy punch

(Stiefil), which cut through all the layers of the HSEs, both epidermal and dermal.

This was followed by removal of the 4 mm diameter epidermal/dermal core.

4.2.9 Formation of Synthetic Fibrin-like Gel

The synthetic fibrin gel networks were formed by factor XIIIa (Baxter BioSurgery,

Vienna, Austria) cross-linking the two factor XIIIa substrate peptides, Ac-

FKGGGPQGIWGQ-ERCG-NH2 (TG-MMP-Lys) and H-NQEQVSPL-ERCGNH2

(TG-Gln) (NeoMPS, Strasbourg, France) to Eight-Arm PEG vinylsulfone (PEG-VS)

(Nektar, Huntsville, AL and Aldrich, Buchs, Switzerland) via a Michael-Type

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addition reaction. Hydrogel precursors for the FXIII-catalysed cross-linking were

produced as previously described (Ehrbar et al., 2007b). Briefly, peptides (NeoMP

S.A., Strasbourg, France) containing complementary substrates for FXIII-catalysed

cross-linking (as indicated in bold), NQEQVSPLERCG (TG-Gln) or

FKGGGPQG↓IWGQERCG (TG-MMP-Lys), were coupled to 8-arms Poly(Ethylene

Glycol) macromolecules (8-PEG, Mw 40 kDa) via Michael-type conjugate addition

between vinyl sulfone groups of end-functionalized PEG and thiols of peptide cystein

residues (indicated in italic), yielding the hydrogel precursors 8-PEG-Gln and 8-

PEG-MMP-Lys, respectively. The TG-MMP-Lys peptide also included an MMP

substrate (underlined, ↓ indicates cleavage position) to render the final hydrogels

susceptible to proteolytic degradation. After functionalisation, the PEG precursors

were dialysed against double-distilled water and were subsequently freeze dried.

Hydrogels were formed by FXIII-catalysed cross-linking of stoichiometrically

balanced 8-PEG-Gln and 8-PEG-MMP-Lys, prepared as described above, in Tris-

Buffer (TBS, 50 mM, pH 7.6) containing 50 mM calcium chloride.

Specifically, to produce 120 µL of 2.25% w/v synthetic fibrin hydrogel the following

solutions were used: 43.17 µL (8-PEG-Gln, 30.44 mg/mL), 45.83 µL (8-PEG-MMP-

Lys 30.24 mg/mL), 19 µL spare volume (e.g., for the incorporation of the RGD-

peptide: NQEQVSPL-RGDSPG, final concentration in gels approx. 50µM), 6 µL

calcium chloride (1M) and 6 µL of activated FXIII (213 U/mL, Baxter BioSurgery).

Cross-linking reactions were performed at 37 0C for 30-40 min.

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Figure 4.1. Factor XIIIa-catalyzed PEG hydrogel formation (Reproduced from

Ehrbar et al., 2007b, with permisson from Rizzi)

4.2.10 Assessment of the Synthetic Fibrin-like Gel Using 6 mm Diameter Partial-

thickness Wounds in HSEs

The 6 mm partial-thickness wounded HSEs were prepared as described above in section

4.2.7. After the 6 mm defects were created, the wounded areas of the HSEs were either

treated with topical application of 13 µL of synthetic fibrin gel or 13 µL of FG (positive

control). After 7 days culture at the air-liquid interface, the wounded HSEs were analysed

with MTT and fixed for histology or immunohistochemistry.

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4.2.11 Assessment of Synthetic Fibrin Gel in 4 mm Diameter Full-thickness

Wounds in HSEs

The 4 mm diameter full-thickness wounded HSEs were prepared as described above in

section 4.2.8. After the 4 mm diameter wounds were created, the defects were injected

with 13 µL of synthetic fibrin gel. After 14 days of culture at the air-liquid interface, the

wounded HSEs were probed using immunofluorescence techniques.

4.2.12 Immunofluorescence

After the full-thickness wounded HSEs were injected with synthetic fibrin gel, they

were cultured at the air:liquid interface for 14 days. The wounded HSE samples were

then probed for nuclei (DAPI) (1:2000) (Vector Laboratories Inc, CA, USA), F-actin

(Phalloidin Rhodamine) (1:250) (Molecular Probes, Eugene, OR, USA) or

pancytokeratins (1:100) (Dako, Botany, New South Wales, Australia). For the

wounded HSEs seeded with keratinocytes alone, or seeded with keratinocytes and

fibroblasts together, DAPI and anti-cytokeratin were used to identify the cell nuclei

and to identify keratinocytes expressing pancytokeratin, respectively. The wounded

HSE samples were fixed and permeabilized in 4% formalin containing 0.2% Triton

X-100 in PBS for 20 min at 20 °C. Glycine (0.1 M) was added to quench free

aldehydes, thus reducing autofluorescence. After blocking with 1% (w/v) BSA/PBS

for 1 hr, the wounded HSEs were incubated with a primary antibody (e.g., anti-

cytokeratin) for 45 min, followed by incubation with a second antibody fluorescein

isothiocyanate FITC (1:200) (Dako) for 45 min. After washing these samples three

times for 5 min in PBS, the wounded HSEs were probed with DAPI for 45 min.

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Epifluorescence images were taken using TE2000 inverted microscope and Leica

TCS SP5 confocal laser scanning microscope (Nikon, San Francisco, CA, USA;

Leica Microsystems, Wetzlar, Germany) at λex = 495 nm, λem = 515 nm for

FITC/pancytokeratin visualization and λex = 356 nm, λem = 461 nm for DAPI/nuclei

visualization.

For the wounded HSEs seeded with fibroblasts only, Phalloidin and DAPI were used

to probe the actin filament and nuclei of the fibroblasts. Samples were fixed and

permeabilized in 4% formalin containing 0.2% Triton X-100 in PBS for 20 min at

room temperature (20 °C). Glycine (0.1 M) was added to stop the permeabilization

process. The wounded HSEs were stained with phalloidin for 45 min at room

temperature. After washing the samples three times for 5 min in PBS, the wounded

HSEs were probed with DAPI (1:2000) (Vector Laboratories Inc) for 45 min.

Epifluorescence images were captured using TE2000 inverted microscope (Nikon,

USA) at λex = 557 nm, λem = 574 nm for Phalloidin Rhodamine/actin filament

visualization and λex = 356 nm, λem = 461nm for DAPI/nuclei visualization.

4.2.13 Wound Coverage Measurements (MTT Assay)

The wound coverage was measured as described in the outgrowth experiment in chapter 3

(3.2.9).

4.2.14 Histological Analysis

Histological analysis is as described in chapter 3 (3.2.10).

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4.2.15 Immunohistochemical Analysis

Immunohistochemical analysis is as described in chapter 3 (3.2.10).

4.2.16 Statistical Analysis

In all of the assays described above, duplicate HSEs were tested individually with

each treatment in each assay and the whole experiment was repeated three times,

each time using DEDs, keratinocytes and fibroblasts from different donors. The data

were pooled from three replicate experiments in which each treatment was tested in

duplicate. One-way ANOVA with Tukey’s post hoc tests (all groups comparisons)

were applied to analyse the data. Statistically significant differences were determined

as p < 0.05.

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4.3 RESULTS

4.3.1 Development of a 6 mm Diameter Partial-thickness HSE Wound Model

To determine an appropriate protocol for developing a serum-free, partial-thickness

wound model (i.e., with epidermis removed), the HSEs were cultured at the air:liquid

interface for 7, 9 and 11 days, and the epidermis was peeled away from each HSE after

being cut by a 6 mm biopsy punch through the full thickness of the epidermis (figure 4.2).

We firstly found that the epidermis of HSEs cultured at the air:liquid interface for 7 days

could not be peeled away from the dermis effectively, as revealed by MTT and H&E

staining (figure 4.3). These analyses indicated that many keratinocytes remained attached

to the dermis in the area of the defect (figure 4.3 A). However, we established that if the

wound was made to the HSE after 9 days of culture at the air:liquid interface, the

epidermis could be removed successfully from the dermis (figure 4.3 B). Although the

epidermis of HSEs cultured for 11 days at the air:liquid interface could also be peeled

away as a whole piece from the dermis, the majority of the cells at the edge of the defect

displayed a differentiated phenotype as determined by histology (figure 4.3 C). In view of

these observations, we determined that the HSEs cultured for 9 days at the air:liquid

interface facilitates the successful creation of a 6 mm partial-thickness wound (figure 4.2).

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Figure 4.2. Photograph of approach used to create a 6 mm diameter partial-

thickness HSE wound model. A wound was created by excising a central portion of the

epidermis through use of a 6 mm biopsy punch, followed by the removal of the epidermis

with forceps. Scale bar = 6 mm.

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Figure 4.3. MTT analysis (purple colour) of the HSEs inflicted with a partial-

thickness wound (left panels) and histological analysis (pink colour) of the wounded

HSEs (right panels). The wounds were created in the HSEs using a biopsy punch after

they had been cultured at air:liquid interface for 7 days (A), 9 days (B) and 11 days (C).

Three replicate experiments were tested in duplicate and each experiment used

keratinocytes and DEDs from a separate donor (n = 6). d7 = day 7, d9 = day 9, d11 = day

11. Dashed lines indicate the wound boundaries. White scale bar = 3 mm and black scale

bar = 300 µm. All images are representative photos obtained from three different

experiments with skin from different donors.

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4.3.2 Validation of the 6 mm Diameter Partial-thickness HSE Wound Model

(MTT).

In order to validate the utility of the 6 mm partial-thickness HSE wound models for

assessing wound healing parameters, the wounded HSEs were cultured in either FG,

VN:GF (containing 1.4 µg VN, 1.4 µg IGFBP3, 0.47 µg IGF, 0.47 µg EGF and SFM), or

SFM media at the air:liquid interface. The models were also immersed in either FG,

VN:GF or SFM for 30 min every second day for 3, 7 and 12 days respectively. They were

then analysed with MTT, histology and immunohistochemistry approaches to monitor

cells that repopulate the wound area, as well as to detect the expression of specific

markers on the cells (figures 4.4 and 4.6 and Appendix).

When the wounded HSEs were cultured in either FG, VN:GF or SFM media at the

air:liquid interface and immersed in either FG, VN:GF or SFM media for 30 min every

second day for 3, 7 and 12 days, different responses were obtained (figure 4.4). The

uncovered wound area of each HSE cultured with different medium was therefore

quantitated (figure 4.5). After 3 days the wound area of the HSEs cultured with FG and

VN:GF medium was gradually covered by keratinocytes migrating in from the wound

edge. The original area at the wound was initially 26.49 ± 1.18 mm². Three days later, the

uncovered wound area in the HSEs cultured with FG and VN:GF medium were reduced

to 15.91 ± 4.29 mm² and 18.54 ± 3.75 mm² (mean ± SEM, n = 6), respectively. These

areas were significantly less (p < 0.05) than those found for HSEs cultured with SFM

medium (25.77 ± 1.67 mm²) (mean ± SEM, n = 6). At day 7, the uncovered wound area

measured in the HSEs cultured with FG and VN:GF medium reduced to 10.9 ± 4.07 mm²

and 9.56 ± 2.17 mm², respectively. Again, these areas were significantly less (p < 0.05)

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than found with HSEs cultured with SFM (27.24 ± 0.65 mm²). Similarly, at day 12, the

uncovered wound area measured in the wounded HSEs cultured with FG and VN:GF

medium was reduced to 7.64 ± 3.51 mm² and 6.44 ± 1.70 mm² (mean ± SEM, n = 6),

respectively. These areas were significantly less (p < 0.05) than those found for HSEs

treated with SFM (29.65 ± 0.91 mm²) (mean ± SEM, n = 6). Taken together, these data

indicate that the uncovered wound area in HSEs cultured with FG and VN:GF medium

continued to decrease, while the wounds in HSEs cultured with SFM remained uncovered.

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Figure 4.4. Representative images of the MTT analysis of the partial-thickness

wounded HSEs cultured with FG (Full Green’s Medium), VG (VN:GF medium)

and SFM (serum free medium) at day 3, day 7 and day 12 after the wounds were

created. d0 = day 0, d3 = day 3, d7 = day 7, d12 = day 12. Scale bar = 3 mm. All images

are representative photos obtained from three different experiments with skin from

different donors.

Page 114

0

5

10

15

20

25

30

35

d0 d3 d7 d12

Time (d)

unco

vere

d ar

ea (m

m²)

SFM FG VG

#

* #

*#

*

Figure 4.5. Quantification of the uncovered wound area in the partial-thickness

wounded HSEs cultured with FG, VG and SFM at day 3, day 7 and day 12 after

the wounds were created. The data, expressed as the uncovered area of the

wounded HSEs, were pooled from three replicate experiments in which each

treatment was tested in duplicate. Each replicate treatment used keratinocytes and

DEDs isolated from skin from a separate donor (n = 6). The asterisks (#) and (*)

indicate treatments which significantly decreased the uncovered wound areas

compared to the responses observed with HSEs treated with the control SFM

treatment (p < 0.05). Error bars indicate SEM.

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4.3.3 Validation of the 6 mm Diameter Partial-thickness HSE Wound Model

(Histology).

The morphological appearance of the wounded HSEs has also been investigated through

histological analysis (figure 4.6). After 3 days, the wound bed of the HSEs cultured with

either FG or VN:GF medium was partially covered by a wedge-shaped “epithelial

tongue” two to three cells in thickness. At day 7, the wound floor of the HSEs cultured

with either FG or VN:GF medium was partially covered with a monolayer or bilayer of

keratinocytes in the centre and a more stratified epithelium was evident towards the

wound margins. After day 12, the wound bed of the HSEs cultured with FG and VN:GF

medium was partially covered by a stratified epithelium. Conversely, in the HSEs

cultured with SFM, only a few keratinocytes were seen at the edge of the wound at day 3

and these had disappeared by day 7.

The lateral migration (distance from two of the wound edges to the centre of the wound in

the HSEs) was quantitated at day 3, day 7 and day 12 (figure 4.7). At day 3, the lateral

migration of keratinocytes cultured with FG and VN:GF medium were 1.73 ± 0.54 mm

and 2.10 ± 0.92 mm (mean ± SEM), respectively. These distances were significantly

greater (p < 0.05) than those found for HSEs cultured with SFM (0.23 ± 0.23 mm) (mean

± SEM). At day 7, the lateral migration obtained in HSEs cultured with FG and VN:GF

medium were 3.49 ± 0.92 mm and 3.44 ± 0.35 mm (mean ± SEM), respectively. Those

distances were significantly greater (p < 0.05) than those found for HSEs cultured with

SFM (0 ± 0 mm). At day 12, the lateral migration obtained from HSEs cultured with FG

and VN:GF medium were 4.12 ± 0.99 mm and 4.67 ± 0.47 mm (mean ± SEM),

respectively. Again, these distances were significantly greater (p < 0.05) than those

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observed on HSEs cultured with SFM (0 ± 0 mm). Taken together, these data indicate

that the lateral migration distance of HSEs cultured with FG and VN:GF medium

continued to increase, while wounds in HSEs cultured with SFM remained unrepaired.

The results from the histological analysis are consistent with the results reported above for

MTT staining.

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Figure 4.6. Histological analysis of the partial-thickness wounded HSEs

cultured with FG, VG and SFM at day 3, day 7 and day 12 after the wounds

were created. The dash lines show the margins of the wounds. Scale bar = 300 µm.

All images are representative photos obtained from experiments using skin obtained

from three different donors.

Page 118

0

1

2

3

4

5

6

d0 d3 d7 d12

Time (d)

Late

ral m

igra

tion

dist

ance

(m

m)

SFM FG VG

#

*#

*

#

*

Figure 4.7. Quantification of the lateral migration of keratinocytes from the

wound edge to the centre of the partial-thickness wounded HSEs cultured with

FG, VG and SFM at day 3, day 7 and day 12 after the wounds were created. The

data, expressed as the lateral migration, were pooled from three replicate experiments

in which each treatment was tested in duplicate. Each replicate treatment had

keratinocytes and DEDs isolated from skin from a separate donor (n = 6). The

asterisks (#) and (*) indicate treatments which significantly increased the lateral

migration compared to the responses observed with HSEs treated with the control

SFM treatment (p < 0.05). Error bars indicate SEM.

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4.3.4 Investigation of the Effect of the Topically-applied Synthetic Fibrin-like Gel

on Keratinocytes in Wounded HSEs.

It has been found that fibrin gels containing physiological concentrations of fibrinogen

and thrombin can accelerate keratinocyte activation and wound closure (Geer et al.,

2002). The synthetic fibrin-like gel, a 3-D matrix which mimics the key features of the

fibrin, has been found to enhance fibroblast migration, proliferation and eventually

formation of an interconnected cellular network in this matrix (Ehrbar et al., 2007b). We

propose that this synthetic gel may also hold the potential to accelerate keratinocyte re-

epithelialisation. To examine this, the 6 mm diameter partial-thickness wounded HSE

model as described in 4.3.1 was used to assess the effect of this synthetic fibrin-like gel on

keratinocytes in the wound healing situation. The wound area of the HSEs received

topically-applied synthetic fibrin-like gel and were then cultured at the air:liquid interface

for 7 days. The HSE samples were then examined using MTT, histology and

immunohistochemistry approaches (figures 4.8, 4.10 and 4.12).

When the wounded HSEs were treated with either 13 µL of synthetic fibrin-like gel or

with 13 µL FG for 7 days, respectively, different responses were obtained. After 7 days,

the wound area of HSEs topically treated with either the gel or the positive control FG

were mostly covered by keratinocytes which had migrated from the edge of the wounds.

The migration of metabolically active keratinocytes from the wound edge to the centre of

the wound bed, was revealed using MTT staining and quantitated (figures 4.8 and 4.9).

The original area of the wound was 26.49 ± 1.18 mm². Seven days later, the area of the

wound not covered with keratinocytes in HSEs treated with the gel and the positive

control FG were 2.13 ± 1.05 mm² and 8.33 ± 2.22 mm², respectively. Hence the

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uncovered wound surface area in HSEs with the gel was significantly less (p < 0.05) than

that observed with the HSEs treated with FG. This indicates that the synthetic fibrin-like

gel can significantly enhance keratinocyte re-epithelialisation over and above that

obtained with FG.

The morphological appearance of the wounded HSEs was also investigated through

histological analysis (figure 4.10). By day 7, the wound bed of HSEs treated with the

synthetic fibrin-like gel was almost fully covered by a stratified epithelium. The wound

areas of the HSEs treated with the positive control FG, on the other hand, were partially

covered by multilayers of keratinocytes in the centre and a more stratified epithelium

towards the wound margins.

The lateral migration (distance from two of the wound edges to the centre of the wound

bed) in HSEs treated with the synthetic gel and FG for 7 days was also quantitated (figure

4.11). After 7 days, the lateral migration of keratinocytes treated with the synthetic gel and

FG were 4.66 ± 0.54 mm and 3.73 ± 1.01 mm, respectively. Thus, there was no

significant difference in the lateral migration between wounded HSEs treated with the

synthetic gel and FG.

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Figure 4.8. Representative images of the MTT staining of the partial-thickness

wounded HSEs treated with either Gel (synthetic fibrin-like gel) or FG (Full

Green’s Medium) at day 7 after the wounds were created. Scale bar = 3 mm. All

images are representative photos obtained from experiments using skin obtained

from three different donors.

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0

10

20

30

D0 D7Time (d)

Unc

over

ed a

rea

(mm

²)

GelFG

#

Figure 4.9. Quantification of the uncovered wound area in the partial-thickness

wounded HSEs treated with either Gel or FG at day 7 after the wounds were

created. The data, expressed as the uncovered area of the wounded HSEs, were

pooled from three replicate experiments in which each treatment was tested in

duplicate. Each replicate treatment used keratinocytes and DEDs isolated from skin

from a separate donor (n = 6). The asterisk (*) indicates treatment which significantly

decreased the uncovered wound area compared to the responses observed with HSEs

treated with the control FG treatment (p < 0.05). Error bars indicate SEM.

Page 123

Figure 4.10. Representative images of the histological analysis of the partial-

thickness wounded HSEs treated with either Gel or FG, 7 days after the wounds

were created. The arrows show the margin of the wounds. Scale bar = 300 µm. All

images are representative photos obtained from three different experiments using

skin obtained from three different donors.

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0

1

2

3

4

5

6

D0 D7

Time (d)

Late

ral m

igra

tion

(mm

)GelFG

Figure 4.11. Quantification of the lateral migration of keratinocytes from the

wound edge to the centre of the wound bed in partial-thickness wounded HSEs

treated with either Gel or FG, 7 days after the wounds were created. The data,

expressed as the uncovered area of the wounded HSEs, were pooled from three

replicate experiments in which each treatment was tested in duplicate. Each replicate

treatment using keratinocytes and DEDs from a separate donor (n = 6). d0 = day 0,

d7 = day 7. Error bars indicate SEM.

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4.3.5 Immunohistochemistry of the Topically-applied Synthetic Fibrin-like Gel on

Keratinocytes in Wounded HSEs.

Immunohistochemistry was undertaken to detect the expression of specific markers on the

surface of cells in the HSEs (figure 4.12). Immunohistochemical analysis using a

monoclonal antibody raised against the nuclear transcription factor p63 and the basal cell

marker keratin 14, revealed the presence of undifferentiated proliferating cells. At day 7,

weak immunoreactivity of p63 and K14 was detected in keratinocytes located at the edge

of the wound within the HSEs treated with the synthetic fibrin-like gel, whereas more

intense immunoreactivity was present in HSEs treated with FG. Immunohistochemical

analysis using a monoclonal antibody raised against keratins 1, 10, 11 (K1/10/11)

revealed the presence of differentiated cells. Positive immunoreactivity of K1/10/11 was

evident at the edge of the wounded HSEs treated with either Gel or FG at day 7.

Immunohistochemical analysis with a type IV collagen antibody revealed the presence of

basement membrane. At day 7, intense immunoreactivity of type IV collagen was

detected in the wounded HSEs treated with either Gel or FG. These data, together,

indicate that the synthetic fibrin-like gel can facilitate keratinocyte proliferation and

differentiation and basement membrane reconstruction. Indeed the synthetic fibrin-like

gel was as effective, if not more so, than FG in facilitating wound healing in this model.

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Figure 4.12. Characterisation of partial-thickness wounded HSEs treated with

either Gel or FG at day 7 after the wounds were created. A) p63, B) K14, C)

K1/10/11, and D) CIV. The dash line shows the margin of the wound. Scale bar =

100 µm. All images are representative photos obtained from experiments using skin

obtained from three different donors.

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4.3.6 Development of a keratinocyte and fibroblast incorporated HSE model.

In addition to keratinocytes, fibroblasts are another important cell type in dermal wound

healing; hence a second series of experiments were pursued using these cells seeded into

the DED models. To determine an appropriate protocol for seeding keratinocytes and

fibroblasts into the DED, different methods were investigated including: 1) Fibroblasts

were seeded onto the reticular side of the DED for 72 hr, and then keratinocytes were

seeded on the papillary side of this same DED for 48 hr (figure 4.13 A); 2) Fibroblasts

were seeded on the papillary side of the DED for 1 hr, and then keratinocytes were seeded

on top of the fibroblasts for 120 hr (figure 4.13 B); and, 3) Fibroblasts were seeded on the

papillary side of the dermis for 72 hr, and then kerationcytes were seeded on top of the

fibroblasts for 48 hr (figure 4.13 C). These studies revealed that fibroblasts could not be

detected using method 2. However, methods 1 and 3 demonstrated fibroblast integration

through the whole layer of the DED. In view of this, we adopted the approach in which

fibroblasts were seeded on the papillary side of the DED, followed by seeding

keratinocytes on the top of the fibroblasts 72 hr later.

After seeding the fibroblasts and keratinocytes sequentially onto the DED for 72 hr and

48 hr respectively, the HSEs were cultured at the air:liquid interface. Seven days later, 4

mm diameter full-thickness wounds were created using a 4 mm biopsy punch in the

HSEs. This wound excised all the layers of the HSEs (i.e., both epidermis and dermis),

and following wounding, the 4 mm diameter epidermal/dermal core was removed (figure

4.14)

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Figure 4.13. Histological analysis of the DEDs seeded with keratinocytes and

fibroblasts using three different methods. A) Fibroblasts were seeded on the

reticular side of the DED and keratinocytes were seeded on the papillary side of the

DED. B) Fibroblasts were seeded on the papillary side of the DED for 1 hr and

keratinocytes were seeded on top of the fibroblasts. C) Fibroblasts were seeded on

the papillary side of the DED for 72 hr, keratinocytes were seeded on top of the

fibroblasts. Scale bar = 100 µm. All images are representative photos obtained from

three different experiments with skin from different donors.

Figure 4.14. Photograph of approach used to create 4 mm diameter full-

thickness wounds in the HSEs.

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4.3.7 Investigation of the Responses of Keratinocytes and Fibroblasts to Synthetic

Fibrin-like Gel Using 4 mm Diameter Full-thickness Wounded HSEs.

In order to investigate the effect of the synthetic fibrin-like gel on keratinocytes and

fibroblasts on the healing of full-thickness wounds, 4 mm diameter full-thickness

wounded HSEs were employed. The wound areas of the HSEs were injected with the

synthetic fibrin-like gel and were then cultured at the air:liquid interface for 14 days. The

HSE samples were then probed for cell nuclei (DAPI), actin filaments (Phalloidin

Rhodamine) and pancytokeratin using immunofluorescence approaches. As controls, the

wounded HSEs were also seeded with keratinocytes or fibroblasts alone.

Immunofluorescent analysis indicated that there were significant differences between

HSEs seeded with keratinocytes or fibroblasts alone, as well as when both cells were

present. When wounded HSEs were seeded with keratinocytes alone, no cells were

detected in the wounds (figure 4.15 A). Conversely, in the wounded HSEs seeded with

fibroblasts alone, a large number of fibroblasts had migrated into the wound (figure 4.15

C and D). Interestingly, for the wounded HSEs produced with keratinocytes and

fibroblasts co-cultured together, keratinocytes migrated across the wound and formed new

tissue, but the fibroblasts were not detected at this time point (figure 4.15 B). Taken

together, these images indicate that the synthetic fibrin gel can significantly enhance the

migration and proliferation of fibroblasts, as well as keratinocytes, albeit only in the

presence of fibroblasts.

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Figure 4.15. Immunofluorescent staining of the full-thickness wounded HSEs

with a synthetic fibrin-like gel injected into the wounds. Models were then

stained for: A) DAPI; B) pancytokeratin expression in the wounded HSE seeded with

keratinocytes and fibroblasts together; C) Phalloidin Rhodamine expression in the

wounded HSE seeded with fibroblasts alone; and D) DAPI and Phalloidin

(Confocal). White arrows represent the wound edge (n = 4). Scale bar for A, B and C

is 1 mm, and scale bar for D is 250 µm. All images are representative photos

obtained from three different donors.

4mm wound

4mm wound

Wound Edge

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4.4 DISCUSSION

Currently, there are several therapies available for assisting the healing of chronic

wounds. Some examples are: 1) physical therapies, such as negative pressure

therapies (Voinchet & Magalon, 1996), therapeutic heat (Santilli et al., 1999),

electrical stimulation (Gardner et al., 1999) and laser phototherapy (Lagan et al.,

2002); 2) skin replacement therapies, such as Alloderm® (Misra et al., 2008;

Wainwright et al., 1996), TransCyte® (Lukish et al., 2001; Purdue et al., 1997) and

Apligraf® (Bell et al., 1981; Karr, 2008); and 3) wound dressing therapies, such as

moist gauze (Jensen et al., 1998), hydrogels (Flanagan, 1995), hydrocolloids and

alginates (Berry et al., 1996).

Of pertinence to this thesis, growth factors have also been examined for their

potential as therapeutics for skin wounds. Scientific evidence has shown that TGF-β

and VEGF can accelerate the healing of wounds in animal models (Broadley et al.,

1989; Galiano et al., 1996; Nissen et al., 1998; Shah et al., 1992). Moreover, Smiell

et al. (1999) demonstrated that PDGF could accelerate the healing of human diabetic

foot ulcers. Additionally, basic FGF (bFGF) has been approved as a therapeutic for

the healing of wounds in Japan (Kawai et al., 2000). Several other growth factors

have also demonstrated success in clinical trials, for example, granulocyte-

macrophage colony-stimulating factor (GM-CSF) and epidermal growth factor (EGF)

(Brown et al., 1989; Da Costa et al., 1999; Falanga et al., 1992). However, all these

growth factor technologies rely on using high doses to obtain successful wound

healing results. Additionally, most of these therapies ignore the coordinate role of the

ECM /growth factor interactions in mediating crucial cellular events in the wound

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healing response. Therefore, in this chapter we sought to investigate the effect of the

novel ECM/growth factor technology, VN:GF, for the healing of wounds. This was

achieved by developing a serum-free, reproducible partial-thickness HSE wound

model that produced a defined environment in which to study the potential of the

VN:GF technology as a novel therapy. We created a 6 mm diameter partial-thickness

wound in the HSE by means of a 6 mm diameter biopsy punch that cut through the

epidermis only. We then removed the epidermis to create the partial-thickness

excisional wound (figure 4.2). Interestingly, this model demonstrated that VN:GF

was successful in facilitating enhanced healing of wounds, and that the repaired

epithelial layer formed had a similar morphology and keratin marker expression

profile (figures 4.4 to 4.7 and Appendix) to that reported in other studies in humans

(Geer et al., 2002; Odland & Ross, 1968). Odland and Ross (1968) created

superficial skin incisions (8-10 mm in length and 0.5 mm in depth) in humans to

study wound repair. They demonstrated that the cells at edges of the incision started

to migrate as an “epithelial tongue” within 48 hr after wounding. The re-

epitheliazation was reported to be complete within 72 hr and a well-stratified

epithelium was formed by 96 hr after wounding (Odland & Ross, 1968). This is

somewhat faster than the healing response we observed in the HSEs. However, this

difference is most likely due to the fact that they used an incisional wound in

humans, and our studies used excisional wounds in HSEs. All the same it may well

be that the healing process in the HSEs is slower than that observed in normal human

skin in vivo. This may arise from the fact that the HSE ex vivo model does not

contain inflammatory cells and a blood supply. Nevertheless, it is clear that the re-

epithelialisation data reported with this partial-thickness wound model is consistent

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and demonstrates that this model can be used to study wound responses in a way that

mimics to some extent in vivo wound healing.

The success achieved using the partial thickness model to assess VN:GF as a therapeutic,

prompted us to use this model to investigate a potential gel carrier for the VN:GF

molecule. Therefore, a synthetic fibrin-like gel was applied to the partial thickness

wounds to assess its ability to initiate and accelerate wound healing. Interestingly, figures

4.8 and 4.9 demonstrate that the synthetic fibrin-like gel could accelerate wound healing

and re-epithelialisation above the levels observed in the FG control. Indeed, it has been

found that fibrin gel containing physiological concentrations of fibrinogen and thrombin

can accelerate keratinocyte activation and wound closure (Geer et al., 2002). In particular,

the synthetic fibrin-like gel we used contained a 3-D matrix that mimics the key features

of the fibrin and has been found to enhance fibroblast migration, proliferation and

eventually formation of an interconnected cellular network in this matrix (Ehrbar et al.,

2007b). Our results might be explained by the fact that the presence of the synthetic fibrin

gel in the wound transforms the air:liquid interface of the 3-D wounded HSE to a

liquid:liquid interface through the absorption of FG into the gel. This moist environment

may then stimulate the migration of keratinocytes. Indeed, Gilje reported in 1948 that

ulcers covered with adhesive tape to provide a moist environment for wounds, healed

faster than those covered with gauze. Since this early discovery, enhanced wound healing

has been found in many preclinical and clinical studies on both partial-thickness and full-

thickness acute (Madden et al., 1989) and chronic (Kerstein et al., 2001) wounds dressed

with moisture-retentive dressings.

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Whilst our results demonstrated that both the VN:GF and synthetic fibrin-like gels were

able to enhance wound healing in the partial thickness wound models, we needed to

determine whether these potential therapies could be used for deeper wound therapies.

The HSE models were therefore wounded using a 4 mm biopsy punch to remove both the

epidermal and dermal sections of the skin (figure 4.14). After the full thickness wounded

models were de-cellularised, keratinocytes and fibroblasts were seeded into the models to

mimic the cellular micro-environment of a wound. Synthetic fibrin gel (13 µL) was then

injected into the wound area and the wounded HSEs were lifted to the air:liquid interface

culture for two weeks. When the wounded HSEs were seeded with fibroblasts, the wound

area was filled with a large number of cells (figures 4.15 C and D). This result is

consistent with a previous report by Ehrbar et al. (2007b), in which primary human

fibroblasts were shown to migrate and proliferate extensively in this synthetic fibrin gel

containing substrates for MMPs and/or cell adhesion ligands, such as RGD peptides. In

this chapter we also investigated HSEs seeded with keratinocytes alone (figure 4.15 A).

In this situation, no cells were detected within the 4 mm wound area. However,

histological analysis reveals that cells were migrating along the dermis at the boundary of

the wound rather than migrating across the wound. Interestingly, for the wounded HSEs

co-cultured with keratinocytes and fibroblasts together, the keratinocytes migrated across

the wound and formed new tissue (figure 4.15 B). This suggests that fibroblasts play a

crucial role in maintaining and directing keratinocyte function in a wounded environment.

It has been noted by others previously that in sufficiently deep wound healing, the wound

must be filled with granulation tissue to facilitate epithelial cells to migrate across the

wound. The keratinocytes require viable tissue to facilitate their migration (Mulvaney &

Harrington, 1994). Moreover, the proliferative phase of wound healing includes stages of

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angiogenesis, collagen synthesis, fibroplasia and granulation tissue formation,

epithelialisation and wound contraction (Midwood et al., 2004). During fibroplasia and

granulation tissue formation, a new provisional ECM is formed from collagen and

fibronectin secreted by fibroblasts to provide a hydrated matrix that supports cell

migration (Midwood et al., 2004). The keratinocytes in turn, dissolve the basement

membrane, as well as the newly deposited ECM generated by the fibroblasts, to facilitate

their crawling across the wound (Santoro & Gaudino, 2005). In addition to secreting and

depositing ECM components, dermal fibroblasts also have autocrine roles and paracrine

functions (Igarashi et al., 1993). It has been shown that growth factors and cytokines,

such as KGF, IL-6 and FGF-10 that are secreted by fibroblasts, disperse into the upper

epidermis affecting keratinocyte proliferation and differentiation in a paracrine manner

(Aoki et al., 2004; E1-Ghalbzouri et al., 2002). In addition, when keratinocytes are co-

cultured with fibroblasts, increased expression of IL-1, which can initiate keratinocyte

proliferation, become migratory and express activation-specific genes (Aoki et al., 2004;

Kupper, 1990; Tomic-Canic et al., 1998). Hence, taken together, it is clear that fibroblasts

play a vital role in creating a balanced micro-environment for the maintenance and repair

of the epidermis (El-Ghalbzouri et al., 2002). This is confirmed by the data reported in

this chapter, in which healing of full-thickness wound in HSEs were evaluated.

The models developed within this chapter allowed us to investigate and establish the

potential of the VN:GF and synthetic fibrin-like gel technologies for the healing of both

partial and full-thickness wounds. Additionally, the defined nature of the VN:GF

technology allowed the development of a defined serum-free partial thickness model.

This in itself will provide a useful tool for the testing of other potential therapies.

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4CHAPTER 5: GENERAL DISCUSSION

Skin, the external covering of the body, has multiple functions, such as protection,

sensation, aesthetics and communication, water resistance, absorption, thermoregulation,

control of evaporation, storage and synthesis, as well as a key role as the body’s first line

of defence. Damage to this external covering caused by injury or illness may lead to acute

loss of physiological balance, disability or even death (Clark et al., 2007). Wounds,

particularly chronic wounds, are of particular concern. The current prevalence of chronic

wound incidents in the UK and Australia ranges from 200 000 – 600 000 (Baker &

Stacey, 1994) and costs the UK and Australia health care system £2.3 bn – 3.1 bn and

AU$ 500 million, respectively, per annum (Ramstadius, 1997). The cost of the therapies

associated with these wounds burden health services in many countries. Wounds also

affect patients significantly as they suffer both physically and psychologically, with pain,

exudate and odour, frequently coupled with sleep deprivation, decreased mobility and

social isolation (Omar et al., 2004).

Skin wound healing is a complicated process involving a variety of cells, such as

epidermal cells and fibroblasts, as well as their collaborative activities including cell

migration, proliferation, differentiation and apoptosis (Staiano-Coico et al., 2000).

Growth factors, cytokines, proteases and ECM proteins also contribute to the complexity

of the wound healing process (Kumar et al., 2004). Alterations in any of these elements

are potentially harmful for wound healing, and may eventually lead to chronic wounds

(Enoch & Price, 2004). Even though the complicated biological procedures required for

successful wound healing are not fully understood, the topical application of growth

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factors in clinical trials has demonstrated some potential benefits (Fu et al., 2000).

However, clinical trials have demonstrated inconsistent success in administration of

individual growth factors. In addition, high doses of growth factors are required to

achieve these beneficial wound healing effects, which place a large economic burden on

patients and the healthcare system (Upton et al., 2008). Most importantly, this single

growth factor therapeutic approach ignores the fact that growth factors usually exert their

functions through interactions with the ECM during the wound healing process (Schneller

et al., 1997).

Recent research undertaken by the QUT Tissue Repair and Regeneration Research

Program has demonstrated that a substrate-bound complex composed of ECM proteins

and growth factors (VN:GF) increased the protein synthesis and migration of the HaCaT

human keratinocyte cell line (Hyde et al., 2004). In addition, Noble (2008) found

significantly increased migration of dermal keratinocytes and fibroblasts isolated from

diabetic skin samples in response to these complexes. In the light of these encouraging

results, we theorized that these novel VN:GF may have potential in promoting the healing

of chronic wounds, and the studies reported in this thesis were initially directed at

examining this hypothesis. Since it is known that CWF is an environment rich in

proteases, there is little point in developing a VN:GF therapy if it is not able to function in

the presence of CWF. HA, another important biological factor associated with wound

healing, was also investigated alone and in conjunction with VN:GF. This approach was

adopted to examine whether HA can enhance the effects of VN:GF. A second aspect that

was examined was whether VN:GF is active in the presence of CWF. With this in mind,

the first aim of my thesis was to investigate the potential of VN:GF in chronic wound

healing, as well as whether HA can enhance the effect of VN:GF on cellular functions.

Page 138

As described in chapter 2, the HaCaT human keratinocyte and the HFF human fibroblast

cell lines were cultured in 2-D monolayer systems to investigate the functions of VN:GF

and HA on cellular responses, such as migration and proliferation. The 2-D monolayer

cell culture system represents a convenient and economic approach, and indeed has been

employed extensively as an in vitro evaluation instrument for novel biological factors and

wound healing therapies. In addition, cell lines are generally regarded as stable and

valuable models for in vitro studies, owing to their similarity to cells in vivo. The HaCaT

cell line, for example, retains expression of the differentiation-specific keratins K1 and

K10 (Boukamp et al., 1988). Our results demonstrated that: 1) The migration and

proliferation of HaCaTs and HFFs were considerably increased by the VN:GF treatment

(figures 2.1 to 2.4); 2) HFF, but not HaCaT cell proliferation, was increased by HA

(figure 2.4); and 3) VN:GF was able to maintain the proliferation and migration of

HaCaT cells in the presence of CWF (figure 2.5 and 2.6).

In spite of these findings, accumulating evidence suggests that 2-D monolayer cell culture

approaches do not generate accurate prognostic information and accurate indications of in

vivo responses for many drugs and treatments under evaluation (Birgersdotter et al., 2005;

Weaver et al., 1997). In particular, the 2-D cell culture system presents cells with

extremely unnatural geometric, mechanical and biochemical restrictions (Sun et al.,

2006b). Moreover, it has been found that primary cells cultured in 2-D monolayer

systems frequently express modifications in morphology, metabolism and gene

expression, which are seldom observed in native tissue (Sun et al., 2006b). Three-D cell

culture systems on the other hand appear to provide a superior in vitro system enabling

examination of the functions of novel wound healing reagents (Birgersdotter et al., 2005;

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Weaver et al., 1997). In view of this, we decided to explore the VN:GF and HA

combinations further, but this time using a 3-D HSE model.

HSEs are regarded as a superior in vitro cell culture system for evaluating a variety of

skin therapies, predominantly because of their similarities to human skin in

morphology, structure and function (Breetveld et al., 2006; Chakrabarty et al., 1999;

Topping et al., 2006). The 3-D HSE we utilized was prepared by culturing human

keratinocytes on the surface of a human-derived de-epidermized dermal scaffold at

the air:liquid interface. This scaffold provides cells with a micro-environment

comparable to normal human skin, both morphologically and biochemically

(Monteiro-Riviere et al., 1997; Ponec et al., 2002; Topping et al., 2006). For

example, the keratinocytes seeded on HSEs using this scaffold express biochemical

markers such as keratin 1/10/11 and 6 (Topping et al., 2006). Furthermore, aspects

related to the microtopology of the human dermis, such as the formation of rete ridge

structures and the basement membrane components including collagens IV, VII and

laminin, are retained within the HSE model (Medalie et al., 1996). Indeed, the

basement membrane has been shown previously to be a critical component of the

model for keratinocyte attachment in vitro (Chakrabarty et al., 1999). The other

advantage of the HSE model is that it maintains its native dermal matrix comprised

of elastic fibres, collagens and hyaluronic acid (Gross & Schmitt, 1948). The native

dermal matrix is essential for studying wounds. This is partly due to the fact that

synthetic dermal matrices, often utilised in other 3-D skin models, have been

demonstrated to falsely activate cells leading to the inappropriate digestion and re-

organisation of the micro-environment (Wood et al., 2007). In addition, the structural

integrity of this HSE model allows the absorption of heat, light or chemicals through

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the epidermis and into the dermis in a manner that is similar to the physical

obstacles/path as would be found in native skin (Dawson et al., 1996). Therefore, in

chapter 3, we examined functional responses of keratinocytes and fibroblasts, seeded

in DEDs to the VN:GF and HA treatments. Specifically, the HSEs were exposed to

daily topical application of VN:GF, HA and HA + VN:GF for 3 or 7 days. These

HSEs were then analysed using MTT metabolic activity assays, histology and

immunohistochemistry.

The results reported in chapter 3 have shown that VN:GF stimulates keratinocyte

proliferation and differentiation in this 3-D model (figures 3.7 to 3.10). These results

differ somewhat from those obtained using the 2-D cell culture approach described in

chapter 2, as the 2-D studies demonstrated that VN:GF increases the migration and

proliferation of keratinocytes (figures 2.1 and 2.2). This is not surprising, as the 2-D and

3-D cell culture approaches differ markedly. For example, in 2-D cell culture approaches

the cells are grown submerged in the culture medium. In contrast, in the 3-D approach

utilised here, the keratinocytes are cultured at the air:liquid interface and result in the

keratinocytes forming a multilayered epidermis (Breetveld et al., 2006; Chakrabarty et al.,

1999; Topping et al., 2006). In view of this, we contend that the 3-D study with HSEs is

likely to give rise to information with greater relevance to the native in vivo situation.

The 3-D HSE approach was also employed to investigate the functional responses of

keratinocytes to HA and HA + VN:GF. These studies revealed that the proliferation and

differentiation of keratinocytes were stimulated by HA (figures 3.7 and 3.8). These results

are consistent with previous clinical trials showing that skin lesions could be improved

using HA when applied topically. The mechanism behind the enhanced cellular functions

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is thought to involve interactions between HA itself and the CD44 or the hyaladherin

RHAMM cell surface receptors (Ahrens et al., 2001; Bourguignon et al., 2004;

Lokeshwar et al., 1996; Masellis-Smith et al., 1996). Future studies examining these

receptors in the 3-D HSE model are necessary to more fully examine the response of HA

in the 3-D model. Furthermore, future experiments should be directed at exploring the

proposal that HA holds potential for delivery of IGF-I, described by Prisell et al. (1992),

and can be translated for the delivery of the VN:GF technology. Interestingly, when HA

was examined in the presence of VN:GF, the migration of keratinocytes in the HSEs was

inhibited in a dose-dependant manner (figures 3.3 to 3.6). This unexpected finding

suggests that interactions between HA and VN:GF occur, either directly or indirectly. A

previous study by Prisell et al. suggested that HA impedes the release of peptide growth

factors such as IGF-I, due to ionic interactions between the long chains of HA and the

IGF-I peptide. Although HA inhibited the effect of VN:GF on keratinocyte migration, it

enhanced the integration of fibroblasts into the DEDs (figures 3.15 and 3.16).

Interestingly, Greco et al. (1998) demonstrated that HA within a collagen matrix could

stimulate fibroblast migration. Hence, this molecule may be a vital component for the

migration and integration of fibroblasts into the dermis and may facilitate the production

of a fully integrated wound model. This may be a central role for HA in vivo.

In order to investigate the effect of VN:GF in wound healing, a reproducible partial-

thickness “wound” with a given size in the 3-D HSEs was created. Previously, many

studies have reported batch-wise variations in HSEs in aspects related to lipid

composition and organisation, as well as barrier function (Asselineau et al., 1986; Bodde

et al., 1990). These variations have been ascribed to the undefined nature of the FG

(Gibbs et al., 1997). In view of this, the current “gold” standard, FG containing foetal calf

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serum, ideally needs to be replaced with a fully-defined serum free medium. This will

reduce the variability observed between different batches of donated skin used in the

creation of HSEs. The ability of VN:GF to reproduce an epithelial layer on the DED

model prompted us to develop a serum-free HSE wound model to assess potential wound

healing therapies.

Currently there are several methods for creating wounds, such as: tape abrasion (Reed et

al., 1995; Yang et al., 1995), electrokeratome (Goris & Nicolai, 1982; Jong & Lin, 1995;

Sams et al., 2004), suction blisters (Mousa et al., 1990; Nanchahal & Riches, 1982;

Rommain et al., 1991), water scald burns (Brans et al., 1994), thermal injury (Jones et al.,

1991; Pilcher et al., 1999), laser (Vaughan et al., 2004), mesher (Falanga et al., 2002;

Harrison et al., 2006) and biopsy punch (Falanga et al., 2002; Garlick & Taichman,

1994). However, most of these methods required the involvement of either animals or

human volunteers for the wound generation (Table 1.2). Hence we directed our efforts at

developing a reproducible partial-thickness HSE wound model. Six millimetre diameter

biopsy punches were used to cut through the epidermis in the HSE, after which the

epidermal layers were peeled away from the HSE to produce the required reproducible,

standard wound (figure 4.2). The HSE wound models treated with FG showed consistent

wound responses as revealed by MTT, histology and immunohistochemistry,

demonstrating the ability of this model to mimic to some extent in vivo wound healing

(figures 4.4 to 4.7 and Appendix).

Having established that the wounded HSEs do in fact “heal”, the effect of VN:GF in

wound healing was assessed. Analysis of these HSEs using MTT, histology and

immunohistochemistry revealed that enhanced migration, proliferation and differentiation

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of keratinocytes at the edges of the wounds occurred in response to VN:GF (figures 4.4 to

4.7 and Appendix). These results are similar to those observed in wounded HSEs treated

with FG and indicates the potential of VN:GF as: firstly, a wound-healing therapy; and

secondly, a potential defined serum-free medium that could replace the current FG. Moll

et al. (1998) and Geer et al. (2004) have also previously used a biopsy punch to create

wounds on human skin and HSEs respectively (Moll et al., 1998; Geer et al., 2004).

However, Moll et al. required serum to heal the wounds, whilst Geer et al. required their

model to be grafted onto a mouse to repair the defects. Similarly, another two studies

undertaken by Bhora et al. (1995) and Andreadis et al. (2001) investigated the effects of

growth factors such as KGF, FGF, IGF-I and EGF. However, both of these studies

required the presence bovine serum albumin (BSA) or foetal calf serum (FCS). Our

ability to study the healing of wounds in the absence of these non-defined factors

represents a significant advance.

The partial-thickness wounded HSE models were also used to test synthetic fibrin-like

gels, a potential delivery vehicle for the VN:GF technology. During the wound healing

process in vivo, a fibrin clot forms and provides a primary matrix for cells, assisting them

to migrate and permeate the wound site (Herbert et al., 1996). As described in chapter 1, a

synthetic fibrin-like gel mimicking a fibrin clot with attributes that facilitate cell adhesion,

as well as degradation of the gel by proteases has been developed by Ehrbar et al. (Ehrbar

et al., 2007b; Ehrbar et al., 2007a). They have shown that this synthetic fibrin-like gel

enhances the migration and proliferation of fibroblasts and enables the formation of an

interconnected cellular network (Ehrbar et al., 2007b). Based on this, we hypothesized

that keratinocyte re-epithelialisation may also be accelerated by this synthetic gel. In fact,

Ehrbar et al., (2007a) reported that this synthetic fibrin-like gel quantitatively incorporated

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and released the growth factor VEGF, which promoted angiogenesis in an embryonic

chick model. Therefore, this gel might not only provide the right environment for

stimulation of proliferation and migration of fibroblasts, but may also provide an

appropriate delivery vehicle for biological factors such as VN:GF. To explore this

concept further, synthetic fibrin-like gels were injected into the wound areas after the

partial-thickness wounds were created in the HSEs. The wounded HSEs with the

synthetic gels were then lifted to the air:liquid interface and were cultured with FG for 7

days. Results from the MTT analysis, histology and immunohistochemistry demonstrated

that this gel was able to augment the migration, proliferation and differentiation of

keratinocytes during partial-thickness wound healing (figures 4.8 to 4.12). We believe

that the enhanced cellular responses observed in the presence of the synthetic fibrin-like

gel may be aided by the creation of a liquid:liquid-type interface with the cells at the

edges of the wounds. That is, the gels, which were hydrated by the FG through

absorption, provided moist environments facilitating enhanced migration of the

keratinocytes. Indeed, the report by Gilje in 1948, suggested that ulcers provided with a

moist environment through an adhesive bandage tape have enhanced healing, which

supports our data and the overall concept of “a moist environment to facilitate optimal

wound healing”.

Investigations into the effect of this synthetic fibrin-like gel in full-thickness wound

healing were also undertaken as is described in chapter 4. In this situation a 4 mm

diameter full-thickness wound was created using a biopsy punch in HSEs seeded with

keratinocytes and fibroblasts (figure 4.14). In these studies we explored the effects of

fibroblasts and keratinocytes alone, as well as combination, on the wounded HSE. The

wound areas of these HSEs were filled with the synthetic fibrin-like gels and then

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cultured at the air:liquid interface for two weeks, and finally probed with antibodies to

visualise the two cell types.

Consistent with Ehrbar et al.’s (2007b) findings, we demonstrated that the fibroblasts

migrated into and proliferated in the synthetic fibrin-like gel (figures 4.15 C and D).

We also demonstrated that keratinocytes could migrate across the wound and form

new tissue over 14 days when co-cultured with fibroblast cells (figure 4.15 B).

However, the fibroblasts were not detected in the wound area, which may be due to

the fact that the gel was fully degraded by day 14. This may indicate that the

fibroblasts are required to stimulate keratinocytes to migrate across the gel. Notably,

as this experiment was conducted for only 14 days, further investigations are required

to assess if the response of the fibroblasts and keratinocytes changes over time. In

addition, immunofluorescence will be undertaken to detect the specific markers expressed

on keratinocytes such as K1/10/11, K6 and p63; and fibroblasts such as fibroblast-specific

monoclonal antibody MAb AS02 and fibroblast-specific protein-1 (FSP-1). Proliferating

fibroblasts versus differentiated fibroblasts will also be detected by examining the

expression of the alpha-smooth muscle actin (a differentiation marker), Ki-67 (a

proliferation marker) and PCNA (a proliferating cell nuclear antigen), see page 148.

In fibroplasia and granulation tissue formation during wound healing, which occurs from

day 1 to day 30 after wounding, fibroblasts secrete collagen and fibronectin to provide a

hydrated ECM, supporting the migration of cells such as keratinocytes (Midwood et al.,

2004). The newly deposited ECM generated by fibroblasts is then degraded by the

keratinocytes as they migrate across the wound (Santoro & Gaudino, 2005). In addition to

depositing ECM components, the dermal fibroblasts also secrete growth factors and

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cytokines such as KGF, IL-6 and FGF-10, which diffuse into the upper epidermis and

influence keratinocyte proliferation and differentiation (Aoki et al., 2004; E1-Ghalbzouri

et al., 2002). It has been reported that during the co-culture of kerationcytes with

fibroblasts the expression of IL-1 increases, thus stimulating keratinocyte proliferation

and migration through the activation of specific sets of genes (Aoki et al., 2004; Kupper,

1990; Tomic-Canic et al., 1998). In the full-thickness HSE wound model we report

herein, it appears that the synthetic fibrin-like gel can enhance the migration and

proliferation of fibroblasts, as well as keratinocytes, but the keratinocyte response only

occurs in the presence of fibroblasts. Further studies examining the secretion of key ECM

proteins and cytokines, such as collagen and IL-1, may well offer explanations for these

intriguing observations.

In summary, chapter 4 described the successful creation of a fully defined, reproducible 6

mm diameter partial-thickness model. Furthermore, these HSE wound models were

applied successfully to study re-epithelialisation during wound healing and to evaluate

novel wound healing therapies, including VN:GF and a synthetic fibrin-like gel. The data

obtained from these investigations revealed that: 1) the migration, proliferation and

differentiation of keratinocytes, as well as basement membrane remodelling by

keratinocytes in partial-thickness wound healing, were considerably increased by the

VN:GF media; 2) keratinocyte re-epithelialisation in partial-thickness wound healing is

improved by the presence of a synthetic fibrin-like gel; 3) the migration and proliferation

of fibroblasts were increased in full-thickness wound healing when the synthetic fibrin-

like gel was injected into the wound; and 4) fibroblasts have a major influence on

keratinocytes during full-thickness wound healing.

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It is apparent that taken together the studies outlined in this thesis have contributed a

range of new findings via assessment of potential new wound healing therapies, such as

VN:GF and the synthetic fibrin-like gel. Further, the studies reported herein describe the

development of relevant ex vivo 3-D human wound models. Nonetheless, many issues

have been raised that need further investigation. Most importantly, the HSE wound

model needs further development through the inclusion of additional cell types. In skin,

intricate interactions among different type of cells, such as keratinocytes, fibroblasts,

leukocytes, Langerhans’ cells, melanocytes and so forth play important roles in

maintaining either normal epidermal function or an epidermal responses to wounding.

Although keratinocytes migrate, proliferate and differentiate into the normal epidermis to

repair the epithelial barrier, they work together with other cells, such as the cells located

in the underlying mesenchymal layers and the cells of the immune system transported by

the circulation. This occurs through both physical interactions and chemical signalling

(Tomic-Canic et al., 2004). Hence in the future, additional cell types should be added to

the HSEs to further refine the model so that it more closely represents the in vivo skin

environment. Another aspect that requires further consideration relateds to the fact that

the wounds created in the HSEs reported here only represent acute excisional wounds. In

order to more closely recapitulate chronic wounds, future studies could include

examination of the healing of wounds in the HSE in the presence of CWF, as well as

maintaining the HSEs in hypoxic conditions. In addition, The HSEs could also be created

using cells obtained from the edges of ulcers. Given that chronic wounds also cause tissue

damage to the dermis and the fascia (Crovetti et al., 2004), the development of a full-

thickness wound model seeded with keratinocytes, fibroblasts, leukocytes, Langerhans’

cells, melanocytes would represent the ideal fully-integrated 3-D chronic wound model.

Page 148

This would be optimal for the pre-clinical study of potential therapies to accelerate

healing.

In addition, due to the potential of HA and VN:GF to reduce scar formation, a partial-

thickness scar model may be developed to determine if in fact these therapies lead to the

reduction of scar formation (Upton et al., 2008). A further aspect worthy of exploration is

using a new technological development, 2-photon imaging. This non-destructive

examination of the HSE would enable examination of histological information in real-

time. This technique may be employed to also monitor: cellular changes such as

morphology, transportation processes during construction of the model, and following

wounding; and detection of the migration and cellular interaction of active agents applied

to wounds in these models. Lastly, investigations of the synthetic fibrin-like gel as a

delivery vehicle for VN:GF are also warranted to determine whether we can further

enhance the healing of wounds through this novel delivery technology.

In summary, the studies undertaken throughout this research investigated novel potential

therapies for wounds, and developed approaches to create reproducible partial-thickness

and full-thickness wounds in the ex vivo HSE model. The resultant data has contributed

valuable new information that will further our understanding of the effects of these novel

wound healing treatments. Additionally, this research has resulted in the creation of the

first defined and reproducible partial-thickness HSE wound models with defects of a

specified size. These models will advance our understanding of human skin repair,

regeneration, and maintenance processes, and provide a relevant in vitro tool for the

assessment of potential wound healing therapies. Furthermore, the utility of these models

will decrease our reliance on the use of animals for scientific experimentation. This will

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become increasingly important, given the European Union regulation (76/768/EEC,

February, 2003) prohibiting the sale of consumer products developed with testing that

involves animals from 2009. Finally, the novel treatments assessed in this project, with

the ability to enhance healing, may be developed further for use in clinical applications,

and generate outcomes that will benefit patients and the community, generate commercial

and economic opportunities, and reduce health care expenditure.

Page 150

APPENDIX

In order to validate the utility of the 6 mm partial-thickness HSE wound models, the

wounded HSEs were cultured with either FG, VN:GF (containing 1.4 µg VN, 1.4 µg

IGFBP3, 0.47 µg IGF, 0.47 µg EGF and SFM) or SFM media at the air:liquid interface.

Models were also immersed in either FG, VN:GF or SFM for 30 min every second day

for 3, 7 and 12 days respectively.

Immunohistochemistry was undertaken to detect the expression of specific markers on the

surface of cells in the HSE. Immunohistochemical analysis with a monoclonal antibody

raised against the nuclear transcription factor p63 revealed the presence of

undifferentiated proliferating cells (Koster & Roop, 2004). At day 3, intense

immunoreactivity of p63 was detected in keratinocytes located at the edge of the wound

within the suprabasal and basal layers of the wounded HSEs cultured with either FG

medium or VN:GF medium. However, the immunoreactivity was decreased at day 7 and

limited to the deeper basal layers at day 12 (figure A1). In the wounded HSEs cultured

with either FG or VN:GF medium, at the edge of the wounds, weak immunoreactivity of

the basal cell marker keratin 14 (K14) (Moll et al., 1982) can be detected at day 3 at the

edge of the wounds. The immunoreactivity increased at day 7 and became most intense at

day 12 (figure A2). Immunohistochemical analysis with a monoclonal antibody raised

against keratins 1, 10, 11 (K1/10/11) revealed the presence of differentiated cells (Moll et

al., 1982). The positive immunoreactivity of K1/10/11 was present at the edge of the

wounded HSEs cultured with either FG or VN:GF medium at day 3, day 7 and day 12

(figure A3). Immunohistochemical analysis with a type IV collagen antibody revealed the

Page 151

presence of the basement membrane. Intense immunoreactivity was observed at the edge

of the wound in HSEs cultured with SFM medium (negative control) at day 3, day 7 and

day 12. For the wounded HSEs cultured with FG medium, weak immunoreactivity was

detected at the wound edge in HSEs at day 3. This immunoreactivity was increased at day

7 and became more intense at day 12. Wounded HSEs cultured with VN:GF medium,

exhibited weak immunoreactivity at the edges of the wounds at both day 3 and day 7,

while more intense immunoreactivity was detected at day 12 (figure A4). The

immunohistochemical analysis above indicated that the FG and VN:GF treatments can

enhance keratinocyte proliferation and differentiation, as well as the reconstruction of the

basement membrane.

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Figure A1. Representative images of the p63 expression in the partial-thickness

wounded HSEs cultured with FG, VG and SFM medium at day 3, day 7 and

day 12 after the wounds were created. FG = Full Green’s Medium, VG = VN:GF

medium, SFM = serum free medium. d0 = day 0, d3 = day 3, d7 = day 7, d12 = day

12. The dash line shows the margins of the wounds. Scale bar = 100 µm. All images

are representative photos obtained from three different experiments with skin from

different donors. HSEs were constructed and wounded as described in section 4.2.7

and immunohistochemistry was undertaken as described in section 3.2.10.

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Figure A2. Representative images of the K14 expression in the partial-thickness

wounded HSEs cultured with FG, VG and SFM medium at day 3, day 7 and

day 12 after the wounds were created. FG = Full Green’s Medium, VG = VN:GF

medium, SFM = serum free medium. d0 = day 0, d3 = day 3, d7 = day 7, d12 = day

12. The dash line shows the margins of the wounds. Scale bar = 100 µm. All images

are representative photos obtained from three different experiments with skin from

different donors. HSEs were constructed and wounded as described in section 4.2.7

and immunohistochemistry was undertaken as described in section 3.2.10.

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Figure A3. Representative images of the K1/10/11 expression in the partial-

thickness wounded HSEs cultured with FG, VG and SFM medium at day 3, day

7 and day 12 after the wounds were created. FG = Full Green’s Medium, VG =

VN:GF medium, SFM = serum free medium. d0 = day 0, d3 = day 3, d7 = day 7, d12

= day 12. Scale bar = 100 µm. All images are representative photos obtained from

three different experiments with skin from different donors. HSEs were constructed

and wounded as described in section 4.2.7 and immunohistochemistry was

undertaken as described in section 3.2.10.

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Figure A4. Representative images of the collagen type IV expression in the

partial-thickness wounded HSEs cultured with FG, VG and SFM medium at

day 3, day 7 and day 12 after the wounds were created. FG = Full Green’s

Medium, VG = VN:GF medium, SFM = serum free medium. d0 = day 0, d3 = day 3,

d7 = day 7, d12 = day 12. The dash line shows the margins of the wounds. Scale bar

= 100 µm. All images are representative photos obtained from three different

experiments with skin from different donors. HSEs were constructed and wounded as

described in section 4.2.7 and immunohistochemistry was undertaken as described in

section 3.2.10.

Page 156

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