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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

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    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.........................