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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
Page 8
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
Page 10
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
Page 11
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
Page 12
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).
Page 15
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.
Page 111
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.
Page 115
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.
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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.
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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.
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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
Page 141
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
Page 144
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
Page 145
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
Page 146
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.
Page 147
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
Page 149
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.
Page 152
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.
Page 153
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.
Page 154
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.
Page 155
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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