Investigation into the proteolytic activity in chronic wound …eprints.qut.edu.au/16564/1/Erin_Alexis_Rayment_Thesis.pdf · Investigation into the proteolytic activity in chronic

Investigation into the proteolytic activity in

chronic wound fluid and development of a

remediation strategy

By

Erin Alexis Rayment

Bachelor of Biotechnology Innovation (Hons)

Tissue Repair and Regeneration Program

Cells and Tissues Domain

Institute of Health and Biomedical Innovation

A thesis submitted for the degree of Doctor of Philosophy at the Queensland University of Technology

2007

ii.

KEYWORDS

Wound healing, chronic ulcer, hydrogel, matrix metalloproteinase, gelatinase,

zymography, protease inhibition, wound dressing, biomaterials, tissue engineering.

iii.

ABSTRACT

Chronic ulcers are an important and costly medical issue, causing their sufferers a

large amount of pain, immobility and decreased quality of life. The common

pathology in these chronic wounds is often characterised by excessive proteolytic

activity, leading to the degradation of both the extracellular matrix, as well as key

factors critical to the ulcer’s ability to heal. As matrix metalloproteinases (MMPs), a

large family of zinc-dependent endopeptidases, have been shown to have increased

activity in chronic wound fluid (CWF), it was hypothesised that this specific

proteolytic activity was directly related to an ulcer’s chronic nature. Although

previous studies have identified elevated proteases in CWF, many have reported

contradictory results and therefore the precise levels and species of MMPs in CWF are

poorly understood. The studies reported herein demonstrate that MMP activity is

significantly elevated in CWF compared with acute wound fluid (AWF). In particular,

these studies demonstrate that this proteolytic activity can be specifically attributed to

MMPs and not another class of proteases present in wound healing. Furthermore, it is

shown that MMP-9 is the predominant protease responsible for matrix degradation by

CWF and is an indicator of the clinical status of the wound itself. Moreover, MMP-9

can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a

functionalised analogue, and also tethered to a synthetic biocompatible hydrogel

compromised of aqueous poly (2-hydroxy methacrylate) PHEMA synthesised in the

presence of poly(ethylene glycol) (PEG). Together, these results highlight the

potential use of a tethered MMP inhibitor as an improved ulcer treatment to inhibit

protease activity in the wound fluid, while still allowing MMPs to remain active in the

wound bed where they perform vital roles in the activation of growth-promoting

agents and immune system regulation.

iv.

TABLE OF CONTENTS

Keywords …ii

Abstract …iii

Table of Contents …iv

List of Figures …viii

List of Tables …x

List of Abbreviations …xi

List of Publications and Presentations …xiv

Statement of Original Authorship …xv

Acknowledgements …xvi

CHAPTER 1: LITERATURE REVIEW

1.1 Introduction …2

1.2 Wound Healing …5

1.2.1 Matrix Metalloproteinases and their Role in Wound

Repair …7

1.2.2 Inhibitors of MMP Activity …12

1.2.2.1 Bisphosphonates …14

1.3 Polymers Suitable for Controlled Drug Release in a Wound

Dressing …18

1.3.1 Hydrogels …20

1.3.2 Crosslinking of Polymers …23

1.3.3 Functionalisation of polymers with bisphosphonates …27

1.4 Project Outline …30

1.4.1 Research Problem …30

1.4.2 Hypothesis …30

1.4.3 Aims …31

1.4.4 Project Design …31

CHAPTER 2: ANALYSIS OF MATRIX METALLOPROTEINASES

IN CHRONIC WOUND FLUID


2.2 Materials and Methods …37

v.

2.2.1 Wound fluid sample collection and preparation …37

2.2.2 Identification of protease activity using collagen

zymography …38

2.2.3 Analysis and quantitation of collagen zymography …38

2.2.4 Confirmation of MMP-specific collagen degradation

using GM6001 inhibitor …39

2.2.5 Specific identification of MMPs through

immunoprecipitation …39

2.2.6 Quantitative confirmation of MMP-9 levels through a

direct enzyme-linked immunosorbant assay (ELISA) …40

2.3 Results …41

2.3.1 Elevated proteolytic activity in chronic wound fluid …41

2.3.2 MMPs are responsible for collagen degradation as

shown through a specific inhibitor study …43

2.3.3 Initial immunoprecipitation of MMPs from CWF samples …43

2.3.4 MMP-9 is the predominant protease responsible for

matrix degradation in chronic wound fluid …45

2.3.5 MMP-9 levels present in chronic wound fluid correlate with

the clinical severity of the ulcer …54

2.4 Discussion …56

2.5 Conclusion …60

CHAPTER 3: SYNTHESIS OF HYDROGELS FOR WOUND

DRESSING APPLICATIONS



3.2.1 Chemicals …65

3.2.2 Synthesis of hydrogels …65

3.2.3 Analysis of polymerisation through NIR FT-Raman

Spectroscopy …66

3.2.4 Swelling of hydrogels …66

3.2.5 Protein loading …67

3.2.6 Protein release …67

3.2.7 Skin collection …67

vi.

3.2.8 Primary keratinocyte cell cultures …68

3.2.9 Toxicity testing using a cell number assay …68

3.2.10 Preparation of a three-dimensional human skin

equivalent …68

3.2.11 Biocompatibility testing of hydrogels using the DED

model …70

3.2.12 Immunohistochemistry …70

3.3 Results …73

3.3.1 Hydrogel preparation …73


Spectroscopy …73

3.3.3 Hydrogel swelling ratios in water …73

3.3.4 HRP release from the hydrogel sheets following

diffusion loading …75

3.3.5 Toxicity testing using a cell number assay …78

3.3.6 Biocompatibility testing using a three-dimensional

human skin equivalent …81



CHAPTER 4: SYNTHESIS AND EVALUATION OF

FUNCTIONALISED-ALENDRONATE HYDROGELS

FOR TREATMENT OF EXCESSIVE PROTEASE

ACTIVITY IN CHRONIC WOUND FLUID



4.2.1 Chemicals …95

4.2.2 Functionalisation of alendronate …95

4.2.3 Characterisation of methacrylated-alendronate through

Fourier Transform Nuclear Magnetic Resonance (FT-NMR)

Spectroscopy …95

4.2.4 Analysis of MMP-inhibition through incubation with

the methacrylated-alendronate …96

4.2.5 Synthesis of hydrogels …96

vii.


Spectroscopy …97

4.2.7 Analysis of MMP-inhibition through incubation with

the alendronate-functionalised hydrogels …97

4.2.8 Biocompatibility testing of hydrogels using the DED

model …97

4.2.9 Immunohistochemistry …98

4.3 Results …99

4.3.1 Synthesis and characterisation of methacrylated-

alendronate …99

4.3.2 Analysis of MMP-inhibitory action of methacrylated-

alendronate …105

4.3.3 Preparation and characterisation of alendronate-

functionalised hydrogels …105

4.3.4 Analysis of MMP-inhibitory action of alendronate-

functionalised hydrogels …108

4.3.5 Biocompatibility testing using a three-dimensional

human skin equivalent …108



CHAPTER 5: GENERAL DISCUSSION …117

CHAPTER 6: REFERENCES …129

viii.

LIST OF FIGURES

1.1 The domain structure of MMPs …8

1.2 Overlay of the backbone atoms from 4 separate MMPs. …11

1.3 Structure of bisphosphonates …14

1.4 Chemical structures …22

1.5 Mechanism of gamma induced polymerisation of HEMA …24

1.6 Potential mechanisms for gamma induced random grafting/

termination or polymerisation of HEMA and PEG …25

1.7 Mechanism of gamma induced crosslinking of HEMA and

EDGMA …28

2.1 Collagen Type I zymography demonstrating protease activity

present in wound fluid samples …42

2.2 Collagen Type IV zymography demonstrating protease activity

present in wound fluid samples …42

2.3 GM6001 inhibition of protease activity in CWF-1 confirms the

MMP specific degradation of Collagen Type I as revealed through

zymography …44

2.4 Initial immunoprecipitation of MMP-1, -8, -13 from CWF-1 and

analysis of enriched fractions through Collagen Type I zymography …46

2.5 Immunoprecipitation of MMP-8 from wound fluid samples and

analysis of MMP-8 enriched fractions through Collagen Type I

zymography …47


analysis of MMP-8 enriched fractions through Collagen Type I

zymography …48


analysis of MMP-9-bound fractions through Collagen Type I

zymography …50


analysis of MMP-9-unbound fractions through Collagen Type I

zymography …52

2.9 MMP-9 levels from wound fluid samples analysed through an

ELISA …55

ix.

3.1 Experimental design of the toxicity testing …69

3.2 Experimental timeline of the three-dimensional ex vivo human

skin equivalent model …71

3.3 Hydrogel sample analysis using NIR FT-Raman Spectroscopy …74

3.4 Swelling studies of hydrogels in water …76

3.5 Gravimetric study of the loss of PEG from the HEMA:PEG

hydrogels …77

3.6 Cumulative release of HRP from the hydrogel sheets into a

HEPES buffer at pH 7.4 …79

3.7 Toxicity testing of components leached from the hydrogels in

primary skin keratinocytes using the CyQUANT® NF Cell

Proliferation Assay to determine cell number …80

3.8 Histological and immunohistochemical analysis of an ex vivo

human skin model following exposure to both a commercially

available wound dressing and synthesised hydrogels …82

4.1 Reaction schemes …100

4.2 FT-NMR 1H spectrum of methacrylated-alendronate …101

4.3 FT-NMR 31P spectrum of methacrylated-alendronate …103

4.4 Potential structure of methacrylated-alendronate dimer …104

4.5 Collagen Type I zymography demonstrating inhibition of

protease activity present in wound fluid samples by alendronate

sodium salt and its methacrylated-counterpart …106

4.6 Analysis of alendronate-functionalised hydrogels using NIR

FT-Raman Spectroscopy …107

4.7 Collagen Type I zymography demonstrating inhibition of

protease activity present in wound fluid samples by alendronate-functionalised

hydrogels …109

4.8 Histological and immunohistochemical analysis of an ex vivo

human skin model following exposure to the alendronate-

functionalised hydrogel …110

x.

LIST OF TABLES

1.1 Acute versus chronic wounds …6

1.2 Distribution of MMPs among cell types in cutaneous wound

repair …9

1.3 Relative potencies of bisphosphonates in vitro …15

1.4 Bisphosphonates in clinical studies/development …16

1.5 Description of various polymers available for use in biomedical

applications and their properties …19

1.6 Hydrophilic polymers suitable for hydrogel matrices …21

1.7 The reaction scheme of the radiolysis of neutral water …27

2.1. Patient clinical data from chronic wound fluid samples …37

3.1 Hydrogel formulations prepared for analysis as potential

wound dressings …65

4.1 Functionalised hydrogel formulations prepared for analysis

as potential wound dressings …96

4.2 Features of methacrylated-alendronate FT-NMR 1H spectrum …102

4.3 Features of methacrylated-alendronate FT-NMR 31P spectrum …104

xi.

LIST OF ABBREVIATIONS

ABTS 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium

salt

ARC Australian Research Council

AWF acute wound fluid

BSE Bovine Spongiform Encephalopathy

CC-3 cleaved caspase-3

CHCl3 chloroform

CO2 carbon dioxide

CWF chronic wound fluid

DED de-epidermised dermis

DMEM Dulbecco’s Modified Eagle’s Medium

DMF N,N-Dimethylformamide or (CH3)2NC(O)H

D2O deuterated water

ECM extracellular matrix

EDTA ethylenediaminetetraacetic acid

EGDMA ethylene glycol dimethacrylate

EGF epidermal growth factor

ELISA enzyme-linked immunosorbant assay

FCS foetal calf serum

FDA Food and Drug Administration

FG Green’s media + serum

FGF fibroblast growth factor

FT Fourier Transform

GA1X Irradiated H2O:HEMA:PEG:1X methacrylated-alendronate (10 kGy)

GA10X Irradiated H2O:HEMA:PEG:10X methacrylated-alendronate (10 kGy)

G-CSF granulocyte-colony stimulating factor

GM6001 N-[(2R)-2-(hydrox- amidocarbonylmethyl)-4-methylpentanoyl]-L-

tryptophan methylamide

GRAS Generally Regarded as Safe

HCl hydrochloric acid

HEMA 2-hydroxylethyl methacrylate

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

xii.

H&E haematoxylin and eosin

H2O water

HRP horseradish peroxidise

HS human serum

IGF insulin-like growth factor

IGFBP insulin-like growth factor binding protein

IgG immunoglobulin G

IL-1β interleukin-1β

i3T3s lethally irradiated 3T3 mouse fibroblast feeder cells

K1/10/11 keratin 1/10/11

KGF keratinocyte growth factor

mAb monoclonal antibody

MMP matrix metalloproteinase

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NaAMPS 2-acrylamido-2-methyl- propanesulphonic acid sodium salt

NaCMC sodium carboxymethylcellulose

NaOH sodium hydroxide

NIR Near Infra-Red

NMR Nuclear Magnetic Resonance

nvCJD new variant Creutzfeldt Jakob Disease

PAA poly(acrylic acid)

pAb polyclonal antibody

PBS phosphate buffered saline

PDGF platelet-derived growth factor

PEO poly(ethylene oxide)

PEG poly(ethylene glycol)

PGA poly(glycolic acid)

PH-5 50:20 H2O:HEMA (5kGy)

PH-10 50:20 H2O:HEMA (10 kGy)

PHEMA poly(2-hydroxyethyl methacrylate)

PLA poly(lactic acid)

PLGA copolymer of PGA and PLA

PMA poly(methyl acrylate)

Polymer C 50:20:30 H2O:HEMA:PEG (5 kGy)

xiii.

Polymer G 50:20:30 H2O:HEMA:PEG (10 kGy)

P(PF-co-EG) poly(propylene fumarate-co-ethylene glycol)

PUSH Pressure Ulcer Scale of Healing

PVA poly(vinyl alcohol)

ROSs reactive oxygen species

SDS sodium dodecyl sulfate

SEM standard error of the mean

SFM serum-free medium

SMP skim milk powder

SPA acrylic acid (3-sulphopropyl)ester potassium salt

TBS Tris buffered saline

TBST Tris buffered saline with 0.1% Tween-20

TGF transforming growth factor

TIMP tissue inhibitors of metalloproteinase

TNF-α tumour necrosis factor-α

tPA tissue plasminogen activator

uPA urokinase plasminogen activator

xiv.

LIST OF PUBLICATIONS AND PRESENTATIONS

Rayment, E.A., Upton, Z., Shooter, G.K. ‘Increased matrix metalloproteinase-9 (MMP-9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer’, British Journal of Dermatology, accepted, 4 December, 2007.

Rayment, E.A, Dargaville, T.R., Shooter, G.K., George, G.A., Upton, Z., ‘Attenuation of protease activity in chronic wound fluid with alendronate-functionalised hydrogels’, manuscript under review, 16 July, 2007.

Rayment, E.A, Dargaville, T.R., Upton, Z., ‘Wound repair: composition and methods’, Australian Provisional Patent # 2007903101, filed 8 June, 2007.

Rayment, E.A., Fernandez, M.L. Shooter, G.K., Upton, Z., ‘The clinical severity of a chronic ulcer is associated with elevated levels of matrix metalloproteinase-9 activity observed in chronic wound fluid’, Proc. of Tissue Engineering and Regenerative Medicine International Society – North American Meeting, Toronto, Canada, June 2007.

Rayment, E.A., Shooter, G.K., Upton, Z., ‘Chronic wound fluid exhibits elevated matrix metalloproteinase activity leading to the degradation of collagen in the wound bed’, Proc. of Discovery Science and Biotechnology Meeting, Brisbane, Australia, May 2007. Rayment, E.A., Shooter, G.K., George, G.A., Upton, Z., ‘Elevated matrix-metalloproteinase activity in chronic wound fluid’, Proc. Of European Tissue Repair Society Conference, Pisa, Italy, September 2006.

Rayment, E.A., George, G.A., Upton, Z., ‘Analysis of aqueous PEG-HEMA wound dressing synthesis, protein release and biocompatibility in a three-dimensional skin model’, Proc. of Australasian Society of Medical Research Conference, Brisbane, Australia, May 2006.

Rayment, E.A., George, G.A., ‘Aqueous PEG-HEMA hydrogel synthesis and protein release for wound dressing applications’, Proc. of Australasian Society of Biomaterials Conference, Rotorua, New Zealand, February 2006.

Rayment, E.A., George, K.A., George, G.A., ‘Aqueous PEG-HEMA hydrogel synthesis using gamma irradiation for wound dressing applications’, Proc. of European Society of Biomaterials Conference, Sorrento, Italy, September 2005.

Rayment, E.A., George G.A., ‘Scaffold design for a bioactive wound dressing’, Queensland Polymer Symposium, Brisbane, Australia, February 2005.

xv.

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:

xvi.

ACKNOWLEDGEMENTS

Firstly, I would like to sincerely thank my principal supervisor, Prof. Zee Upton, for

the opportunity to embark on this project, as well as her continued encouragement,

assistance and direction through the establishment and completion of my PhD studies.

Secondly, I would also like to acknowledge my associate supervisor, Prof. Graeme

George, for his support and direction, especially in terms of the relatively new area of

polymer science for me. I would not have been able to successfully accomplish this

project without their combined help.

In terms of experimental design and practical support, Dr Gary Shooter and, in the

later stages of this project Dr Tim Dargaville, have both been exceptional. From

providing me with fool-proof protocols, to helping me design completely new

methods, I would like to thank you both sincerely. I fully believe that without the both

of you this project would not have been as successful as it has – culminating in the

recent filing of an Australian provisional patent application.

As this was such a diverse project, many people have been instrumental in terms of

specific knowledge and technical expertise. Therefore, in no particular order, I would

like to thank the following people: Rebecca Dawson, Evette Kairuz, Gemma Topping

and Danielle Borg for their help with the DED model and subsequent

immunohistochemistry; Dr Llew Rintoul for the FT-Raman spectroscopy training; Dr

Mark Wellard for his NMR expertise; Mel, James and Shea for their help and

brainstorming in relation to protein work; and the entire Tissue Repair and

Regeneration program here at QUT for their encouragement and assistance over the

years. In addition, the laboratory support team of Sonya, Scott and David have been

instrumental in ensuring everything has run smoothly.

This PhD project would not have been possible without the Australian Government

for their provision of an Australian Postgraduate Award, and the Institute of Health

and Biomedical Innovation for their top-up stipend. I would also like to thank the

numerous funding bodies who gave me money to travel and attend international

conferences and laboratories, namely: the School of Life Sciences, the TRR program,

xvii.

IHBI, QUT’s Grants-in-Aid scheme, the Australasian Society of Biomaterials and The

Australian Federation of University Women SA Inc.

I would also like to thank Mel, Gary and Shea - for their endless coffee breaks, laughs

so loud that you can hear from different levels, Friday drinks that have morphed into

Saturday mornings, and numerous inappropriate jokes – you have made my PhD an

extremely memorable experience. Thanks also to Gemma, Jacqui, Danielle, Brett and

Tony for their friendship and support, as well as everyone who has put up with my

consistently loud voice for the past three and a half years. You have all made my lifer

a richer place.

And an especially huge thanks to Abrona – your organisation of Zee, as well as the

entire TRR team (myself included!), means that things continually get done. Without

you, I can see myself getting stuck in huge piles of forms that nobody knows how to

fill out or who you have to give them to for signatures…

I would also like to thank my family for all of their support and encouragement

through my university years. I would especially like to thank my older sister,

Cassandra for spurring me on. The thought of potentially completing before her has

definitely been a motivator – and most importantly, has made me write faster…

Last, but certainly not least, I would like to thank Dave from the bottom of my heart.

You have been an incredible support and encourager through this entire project, as

well as being a practical helper with the design of numerous objects for my research.

Without you, I don’t think this entire process would have been anywhere near as fun.

And finally, I promise never to try and explain to you my project again.

xviii.

CHAPTER 1

LITERATURE REVIEW

2.

1.1 INTRODUCTION

Chronic wounds are an important and costly medical challenge. Venous insufficiency,

in the form of venous leg ulcers, is estimated to account for 80-90% of all chronic

ulcers (Shai and Halevy 2005). American figures show that more than four million

people (1.3% of the population) each year are affected by these wounds, costing

upwards of US$ 9 billion to treat (Clinicaltrials.gov 2002). Chronic wounds currently

place a significant burden on the Australian healthcare system, with prolonged

hospitalisations (at a cost of AU$ <500 per day for 2-3 months at a time) frequently

required. Current prevalence rates of chronic wounds in Australia are estimated to be

between 1-3% of the population (200,000 – 600,000 people) and cost the Australian

community upwards of AU$500 million per annum (Baker and Stacey 1994; Gruen,

Chang et al. 1996). Currently, there is no one accepted treatment for this costly

medical crisis, which causes the vast majority of lower leg amputations and

significantly compromises the patient’s quality of life. This is primarily due to the

large variety of causes, as well as many co-morbidities associated with this disease,

such as diabetes, osteomyelitis, renal failure and heart disease (MacLellan 2000).

Current treatments of chronic wounds include a number of wound management

strategies. Thus, good nursing skills, including regular washing and debridement,

application of antimicrobials to prevent infection, and daily changing of the dressing,

can play a major role in improving the patient’s outcome. However, in many cases,

the aforementioned procedures are simply not enough to promote wound closure.

Intrinsic health factors, such as diabetes, immune status, and in many cases age, are

often the major inhibitory factors to wound healing (MacLellan 2000).

The development of innovative, cost-effective treatments to increase the healing rates

of leg ulcers is a priority for improved health in the elderly. This was identified as one

of the ‘research gaps’ in a recent workshop addressing “Leg Ulcer Healing in Older

People” (Feb 5-6, 2007, Brisbane) funded by the ARC/NHMRC Research Network in

Ageing Well. Chronic wounds are a common and costly reality for the Australian

community, and have largely been overlooked by researchers in the area of

biotechnology and, specifically, tissue repair. This significant, yet neglected, research

field not only represents a significant challenge to our current understanding of wound

healing, but also involves a healthcare issue that affects our society at all levels. This

3.

is in terms of cost to the patient, the community and the economy. These wounds are a

major cause of pain and anxiety for affected individuals and also contribute

significantly to their lessening mobility, decreased social interactions and overall

diminished quality of life (MacLellan 2000). In addition, patients with an ulcer are

more likely to suffer depression, downfall risk, malnutrition and functional disorders

(Jawien, Szewczyk et al. 2006). Further, a two-year retrospective study published in

1996 identified all patients admitted to a major Australian teaching hospital with a leg

ulcer as the prime admission criteria and found that wound management of these 199

patients cost AU$2.75 million, yet achieved a healing rate of only 16% (McMullin,

Hyde et al. 1998)! Clearly, in many cases appropriate wound management strategies

and good nursing skills are simply not sufficient to promote wound healing

(MacLellan 2000).

Wound dressings currently on the market do not fully address all of the physiological

issues associated with chronic wounds. Many are simply antimicrobials, e.g. Actisorb

Plus (Johnson & Johnson) and Acticoat (Smith & Nephew), which while they control

bacterial growth do not attend to potential growth factor deficiencies (Stewart 2002).

Conversely, interactive dressings, e.g. Alginates (multiple products) and Promogran

(Johnson & Johnson), maintain a moist wound environment and even claim to interact

with cells or matrix proteins in the wound (Stewart 2002). In terms of alginates, the

presence of calcium in the dressing is reported to modify cellular responses (Stashak,

Farstvedt et al. 2004). However, these products are derived from animal and plant

products and are therefore not fully defined and may not be completely pathogen-free.

In terms of animal-derived products this presents unacceptable risks, especially

following the aftermath of the BSE (Bovine Spongiform Encephalopathy or Mad Cow

Disease) and nvCJD (new variant Creutzfeldt Jakob Disease) crises.

In 2000 the advanced wound management market was estimated at £1.2 billion, with a

growth rate of 7.5% over the preceding year (Smith and Nephew 2000). Active wound

management treatments, e.g. growth factors (Regranex, Johnson & Johnson) and

dermal substitutes (Dermagraft, Smith & Nephew), show an accelerated growth of

over 15% per annum with a market value of £200 million (Smith and Nephew 2000),

demonstrating why companies are willing to invest large amounts of money into this

type of product development. Current topical growth factors treatments include

4.

platelet-derived growth factor (PDGF, becaplermin, Regranex®), granulocyte-colony

stimulating factor (G-CSF, Neupogen® Filgrastim), while fibroblast growth factor-2

(FGF-2) is approved for use in Japan. Regranex® gel 0.01% (Ortho-McNeil

Pharmaceutical, Raritan, NJ, USA) is sold under the Johnson & Johnson Wound

Management product label and contains rhPDGF-BB in a sodium

carboxymethylcellulose (NaCMC) gel. This product was the first topically applied

growth factor treatment to show wound closure efficacy in Phase III clinical trials

(LeGrand 1998). In contrast, Filgrastim has demonstrated limited usefulness in

promoting healing in diabetic ulcers (Kästenbauer, Härnlein et al. 2003; Papanas and

Maltezos 2007). The major obstacle for patients obtaining effective growth factor

treatments is cost – Regranex® costs upwards of US$375/15 g tube (Ugarte, Roberts

et al. 2002), which considering the daily treatment regime, can equate to thousands of

dollars per wound. While this has been proven cost-effective by a number of studies

(Kantor and Margolis 2001), this treatment is still out of reach of the typical chronic

ulcer sufferer (Eldor, Raz et al. 2004).

The aging population, along with the increased prevalence of Type II diabetes, has led

many to concentrate on improving wound dressing treatments for these chronic

wounds. However, as this is an incredibly complex issue, many factors need to be

taken into account. With this in mind, this literature review will provide background

information on wound healing; thus will summarise the characteristics of chronic

ulcers, including increased protease activity and possible protease inhibitors; and will

also focus on controlled delivery of active compounds through polymeric materials.

This review will also concentrate on the interplay between these various themes.

5.

1.2 WOUND HEALING

Wound healing in the skin is a dynamic process. It consists of several stages,

including: inflammation, chemotaxis and cell division, neovascularisation, synthesis

of extracellular matrix, and remodelling of the scar tissue (Tarnuzzer and Schultz

1996). Inflammatory cells first release growth factors and cytokines that are essential

in the regulation of effective wound healing (Wagner, Coerper et al. 2003). Many

growth factors are important to the wound healing process. These include: insulin-like

growth factor (IGF), epidermal growth factor (EGF) and keratinocyte growth factor

(KGF); factors that are pivotal to epidermal migration, growth and differentiation

(Soler, Wright et al. 1999; Gibbs, Pinto et al. 2000; Allen, Asnes et al. 2002). There

are many different members of each of these families of growth factors that perform

different functions during wound repair. For example, IGF-I is essential for healing,

but insulin-like growth factor binding protein-3 (IGFBP-3) significantly decreases

repair (Wagner, Coerper et al. 2003). Another important growth factor in wound

healing, transforming growth factor-α (TGF-α), which along with IGF-I, also appears

to induce or enhance the expression of antimicrobial peptides and polypeptides

(Sorensen, Cowland et al. 2003). This demonstrates the multifunctional nature of

growth factors, as well as the complex dynamics of wound healing itself.

There are two main types of wounds – chronic and acute. While acute wounds follow

the wound healing process described above, disruption of any stage in this complex

cascade leads to the formation of chronic wounds (Trengove, Stacey et al. 1999).

Several previous studies have shown that there are significant differences between the

acute and chronic wound environments (Table 1.1) (Tarnuzzer and Schultz 1996;

Stadelmann, Digenis et al. 1998; Trengove, Stacey et al. 1999). In chronic wounds,

healing occurs with the formation of abundant granulation tissue and excessive

fibrosis, both of which lead to scar contraction and loss of function (Stadelmann,

Digenis et al. 1998). In addition, the major difference shown in chronic wound fluid

(CWF) compared with acute wound fluid (AWF) is their significant increase in

proteolytic activity (Tarnuzzer and Schultz 1996; Ladwig, Robson et al. 2002; Li and

Li 2003). Chronic wounds are characterised by elevated levels of pro-inflammatory

cytokines (e.g. tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)),

proteases (e.g. matrix metalloproteinases (MMPs)) and neutrophil elastase (Chen,

Schultz et al. 1999). These wounds also contain reduced levels of tissue inhibitors of

metalloproteinases (TIMPs) (Chen, Schultz et al. 1999; Baker and Leaper 2000).

Furthermore, TNF-α has a demonstrated ability to increase production of MMPs,

which in turn inhibits production of TIMPs. This then causes an extension in pro-

inflammatory cytokine expression, which can change a normal healing wound into an

environment rich in proteases and extremely toxic for connective tissue proteins and

growth factors (Chen, Schultz et al. 1999). Not surprisingly, chronic wounds have

reduced levels of growth factors, such as PDGF, basic fibroblast growth factor

(bFGF), EGF and TGF-β (Stadelmann, Digenis et al. 1998). Therefore, the regulation

of MMPs by their inhibitors might be necessary to promote wound healing, even

before growth factors can be applied (Tarnuzzer and Schultz 1996; Stadelmann,

Digenis et al. 1998; Trengove, Stacey et al. 1999; Cullen, Watt et al. 2002).

Table 1.1. Acute versus chronic wounds (Menke, Ward et al. 2007)

The chronic nature of ulcers is believed to be caused by certain endopeptidases,

namely serine proteases and matrix metalloproteinases as reported above (Grinnell,

Ho et al. 1992; Palohati, Lauharanta et al. 1993). Increased levels of neutrophils in

chronic wounds, along with their secreted proteases, have been implicated mediating

the tissue damage associated with this disease (Edwards and Howley 2007).

Furthermore, excessive inflammation caused by an hyper-stimulated neutrophil

response has also been suggested as a potential cause for a wound’s chronicity (Yager

and Nwomeh 1999). This can be further exacerbated by susceptibility of open wounds

to infection (Kantor and Margolis 2003). Since neutrophils also play a role in a

number of chemotactic, proteolytic and oxidative events in this environment, they

have also been focussed on a potential therapeutic target for these wounds. However,

a previous study by Weckroth et al. (1996) showed that serine proteases of

6.

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This table is not available online. Please consult the hardcopy thesis available from the QUT Library

7.

polymorphonuclear neutrophil origin, e.g. cathepsin G and neutrophil elastase, were

both low in activity in CWF (Weckroth, Vaheri et al. 1996).

Free radicals are another contributing factor to a chronic wound’s inability to heal.

The excess of oxygen free radicals found in a chronic wound environment often leads

to prolonged inflammation and also increased proteolysis as mentioned earlier. These

reactive oxygen species (ROSs) can subsequently cause protein, as well as lipid,

peroxidation, which then leads to cell disruption, tissue damage and cell death

(Cullen, Smith et al. 2002; Cullen, Watt et al. 2002). Therefore, when developing a

wound dressing there are many features to consider, including: the ability to attract

cells to the wound site; promotion of cell proliferation; the biocompatible and

bioresorbable function of the dressing; the limitation of excess proteinase activity;

and, the absorption and neutralisation of free radicals and excess metal ions (Hart,

Silcock et al. 2002).

1.2.1 Matrix Metalloproteinases and their Role in Wound Repair

MMPs, or matrixins, are a subgroup of the much larger metalloproteinase superfamily

(Parks 1999). Currently, there are more than 20 recognised members of this family of

proteases (Parks 1999), which are then divided into five subclasses based on their

substrate specificities: collagenases, gelatinases, stromelysins, membrane-type MMPs,

and other MMPs (Baker and Leaper 2000). This group of proteases also has a highly

conserved domain structure (Figure 1.1), consisting of a catalytic domain and an auto-

inhibitory pro-domain (Page-McCaw, Ewald et al. 2007).

More specifically, proteins classed as MMPs are defined as having the following five

characteristics. Firstly, they must require an active site Zn2+ that binds the conserved

histidine domain sequence, HEXXHXXGXXH, for their catalytic mechanism.

Second, a MMP’s prodomain needs to be approximately 80 amino acids in length and

contain the consensus sequence PRCXXPD. Third, a putative MMP must be

synthesised as an inactive zymogen. Fourth, it needs to be able to degrade or cleave at

least one extracellular matrix (ECM) protein, e.g. fibronectin, vitronectin. Lastly, its

proteolytic activity needs to be inhibited by TIMPs (Parks 1999).

Figure 1.1 The domain structure of MMPs (Somerville, Oblander et al. 2003)

In healthy tissue, MMP production and activation in fibroblasts is regulated by a

number of factors, namely expression of growth factors/cytokines (Ries and Petrides

1995), mechanical properties of the ECM/actin cytoskeleton (Tomasek, Halliday et al.

1997) and cell-matrix interactions (Azzam and Thompson 1992). Of relevance to this

project, urokinase plasminogen activator (uPA) and tissue plasminogen activator

(tPA) have both been associated with excessive matrix degradation and subsequent

activation of MMPs (Wysocki, Kusakabe et al. 1999). Furthermore, only specific

MMPs are present in those cells involving wound repair, and even then, only at certain

stages of this dynamic process (Table 1.2) (Parks 1999). The MMP family is quite

diverse in both its substrate specificity and role in wound healing. The two most

widely known and studied members are the collagenases and the gelatinases. MMP-1

(collagenase-1) and MMP-8 (collagenase-2, neutrophil-collagenase) appear to have a

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This figure is not available online. Please consult the hardcopy thesis available from the QUT Library

very limited substrate range, acting only on the fibrillar collagens, types I, II and III

(Parks 1999).

Table 1.2 Distribution of MMPs among cell types in cutaneous wound repair (+ indicates presence and – indicates absence in particular cell type) (Parks 1999).

Indeed, MMP-1 has been shown to be directly related to the ability of keratinocytes in

vivo to migrate from a wound edge (Soler, Wright et al. 1999). Moreover, MMP-8 is a

neutrophil-released collagenase and has substrate preference for type I collagen

(Diegelmann 2003). An important point to note is that while the presence of MMP-13

has been shown in chronic, and not acute, wounds, its contribution to the pathological

wound environment has not been identified (Fray, Dickinson et al. 2003). The

gelatinases (MMP-2, MMP-9) on the other hand cleave collagen more efficiently than

other MMPs (Parks 1999) and further degrade the cleaved type I collagen arising from

MMP-1 activity, as well as act on type IV collagen, the major form of collagen in

basement membranes (Tarnuzzer and Schultz 1996). MMP-2 (gelatinase-A) has a

vital role in ECM turnover and has been shown to have a role in collagen lattice

reorganisation in vitro (Cook, Stephens et al. 2000), as well as being one of only two

MMPs actively expressed in non-injured skin (Parks 1999). Recently, it was

demonstrated that reduced fibroblast MMP-2 production is associated with impaired

dermal healing in vivo in a diabetic mouse model (C57BL/KSJ-db/db) (Cook,

Stephens et al. 2000). MMP-9 (gelatinase-B) has also been shown to have the ability

to degrade Collagen types I, II, III, IV and V into small peptides at 37 ºC (Okada,

Gonoji et al. 1992). In addition, MMP-9 (gelatinase-B) is primarily produced by

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inflammatory cells, neutrophils, macrophages and keratinocytes (Cullen, Watt et al.

2002).

Other than collagenases and gelatinases, the MMP family consists of stromelysins,

matrilysins, macrophage metalloelastase and membrane type MMPs. With regard to

stromelysins, MMP-3 (stromelysin-1) has a broad collagen substrate preference and

can also degrade proteoglycans (Tarnuzzer and Schultz 1996). It is also known to

degrade α1-anti-trypsin, an inhibitor of neutrophil elastase, leading to increased levels

of this protease in chronic wounds (Fray, Dickinson et al. 2003). MMP-10

(stromelysin-2) is thought to be an injury-specific protease, involved in desmosome

disassembly during migration (Parks 1999). MMP-7 (matrilysin) on the other hand is

a more potent proteoglycanase than both MMP-3 and MMP-9, and has recently been

implicated in playing an essential role in innate immunity (Wielockx, Libert et al.

2004). In addition, along with MMP-2 (gelatinase-a), matrilysin is found in non-

injured skin, being actively expressed by the ductal epithelium in all sweat and eccrine

glands (Parks 1999). MMP-12, a macrophage metalloelastase, is the most elastolytic

member of this family, i.e. has a strong ability to degrade elastic tissue (Parks 1999).

Furthermore, MMP-14 (membrane-type-1 MMP, MT1-MMP) has been shown to

promote both selective invasion and increased growth of malignant tumour cells in

vivo (Iida, Wilhelmson et al. 2004).

As well as acting to degrade matrix substrates, MMPs also have a wide variety of

growth factor and immune system-related functions (Somerville, Oblander et al.

2003). Indeed, recent mouse knockout models, as well as human diseases, have

indicated many unexpected biological functions of MMPs (Hautamaki, Kobayashi et

al. 1997; Holmbeck, Bianco et al. 1999; Wilson, Ouellette et al. 1999; Bergers,

Brekken et al. 2000; Boulay, Masson et al. 2001; Cataldo, Tournoy et al. 2002),

including many phenotypes that are yet to be fully understood. In terms of growth

factors, MMPs are responsible for both their release from the extracellular matrix

(Imai, Hiramatsu et al. 1997; Suzuki, Raab et al. 1997), as well as their proteolytic

inactivation (McQuibban, Butler et al. 2001; Fujiwara, Matsukawa et al. 2002), and

also have roles in shedding of receptors from the cell membrane (Levi, Fridman et al.

1996). Therefore, in terms of wound healing, this balance between growth factor

activation and degradation has to be carefully controlled to ensure correct progression

towards healing. Furthermore, MMPs have also been implicated in influencing the

immune system, both through activating defensins through cleavage of their pro-

domain (Wilson, Ouellette et al. 1999) and also by their ability to cleave all

immunoglobulin G (IgG) proteins (Gearing, Thorpe et al. 2002). This final point is

extremely important in terms of infection in chronic ulcers, as by cleaving IgG

proteins, MMP-3 and -7 are able to prevent the initiation of the complement cascade, a

key mechanism in clearing pathogens from the host system (Gearing, Thorpe et al.

2002).

There is much debate surrounding the under- and overexpression of various MMPs in

chronic wounds. This can be attributed to the highly conserved nature of this family

(Page-McCaw, Ewald et al. 2007), which can cause incorrect identification when

using antibody-based detection strategies as shown through the superimposition of

four separate MMPs (Figure 1.2).

Figure 1.2 Overlay of the backbone atoms from 4 separate MMPs MMP-1 (green), MMP-9 (purple), MMP-12 (turquoise), MMP-13 (dark green) (Rush III and Powers 2004)

One view is that the overexpression of MMP-3 and MMP-13 correlates with non-

healing wounds, whereas active MMP-1, -2, -9, and -14 are all required for the

progression of normal wound healing (Fray, Dickinson et al. 2003). Ladwig et al.

(2002) and Wysocki et al. (1999) both report that elevated levels of MMP-2 and

MMP-9 are detrimental to wound healing when present for prolonged periods, and

should be inhibited to allow normal repair to occur (Wysocki, Kusakabe et al. 1999;

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12.

Ladwig, Robson et al. 2002). Along with MMP-2 and MMP-9, one report has shown

that MMP-8 is likely to act as a debridement enzyme and elevated levels of this have

been shown in chronic pressure ulcer wound fluid (Yager, Zhang et al. 1996). Further

studies indicate that CWF includes high levels of various MMPs, including MMP-1, -

2, -3, -8, and -9, leading to the degradation of key elements in the wound healing

process (Cullen, Watt et al. 2002; Hart, Silcock et al. 2002). Contrary to previous

studies, Cook et al. (2000) reported that significantly decreased levels of active MMP-

2 and MMP-1, corresponding with marked increases in expression of TIMP-1 and

TIMP-2, led to the chronicity of non-healing wounds (Cook, Stephens et al. 2000).

This is in direct contrast with all other previously reported findings. One possible

reason for this is the use of biopsies from the wound bed by Cook et al. (2000),

instead of wound fluids as has been used by other studies (Cook, Stephens et al.

2000). Currently, it is not known if biopsy and fluid samples from chronic wounds

show similar profiles of MMPs and TIMPs; nor is it known which sample type gives a

more accurate assessment of the chronic wound’s molecular environment (Ladwig,

Robson et al. 2002).

1.2.2 Inhibitors of MMP Activity

Current topical treatments of chronic ulcers have proven to be only slightly effective

in treating this pathology (Cullen, Watt et al. 2002). It has been postulated that the

main reason for the unanticipated ineffectiveness of topical growth factor therapy to

promote wound healing is due to the high levels of proteases found in CWF (Cullen,

Watt et al. 2002). CWF also contain reduced levels of TIMPs (Chen, Schultz et al.

1999; Baker and Leaper 2000). Interestingly, TNF-α has a demonstrated ability to

increase production of MMPs, which in turn represses production of TIMPs. This then

prolongs pro-inflammatory cytokine expression, which can change a normal healing

wound into an environment rich in proteases and hence antagonises deposition and

function of connective tissue proteins and growth factors (Chen, Schultz et al. 1999).

Not surprisingly, chronic wounds have reduced levels of growth factors, therefore the

regulation of MMPs by their endogenous inhibitors might be necessary to promote

wound healing, even before topical growth factor treatments can be used (Tarnuzzer

and Schultz 1996; Stadelmann, Digenis et al. 1998; Trengove, Stacey et al. 1999;

Cullen, Watt et al. 2002).

13.

There are three pathways that explain how the excessive levels of proteases contribute

to the chronic ulcer pathology. Firstly, in CWF, growth factors have been shown to

lack biological activity due to proteolytic degradation. Next, growth factor receptors

are also degraded in chronic wounds; thereby decreasing the cell’s ability to recognise

and use growth factors. Lastly, the provisional matrix needed for fibroblasts to migrate

across the ulcer wound bed is degraded by the proteolytic chronic wound environment

(Fray, Dickinson et al. 2003). Therefore, many authors have suggested that the

addition of a protease inhibitor prior to topical treatment of the wound would promote

healing (Tarnuzzer and Schultz 1996; Wysocki, Kusakabe et al. 1999; Yager and

Nwomeh 1999; Cullen, Smith et al. 2002).

There are a number of both biological and chemical inhibitors of MMPs (Massova,

Kotra et al. 1998). Intrinsic MMP inhibitors, TIMPs, are biological inhibitors of MMP

activity commonly found in wound sites (Klein, Anderson et al. 2002). MMPs mainly

form binary non-covalent complexes when inhibited by the TIMPs (Herouy, Trefzer et

al. 2000). In terms of chemical inhibitors, cation chelators, i.e.

ethylenediaminetetraacetic acid (EDTA), have proven successful in in vitro studies

examining growth factor degradation in CWF as they remove the zinc ion required for

MMP catalytic activity (Cullen, Smith et al. 2002). More specific inhibitors include

the following: tetracyclines and their chemically modified derivatives, i.e. doxycycline

(Ramamurthy, Kucine et al. 1998; Ramamurthy, McClain et al. 1999; Yager and

Nwomeh 1999); hydroxamic acids, i.e. (N-[(2R)-2-(hydrox- amidocarbonylmethyl)-4-

methylpentanoyl]-L-tryptophan methylamide), Ilomastat, galardin, GM6001 (Lund,

Romer et al. 1999); and bisphosphonates, i.e. clodronate (Valleala, Hanemaaijer et al.

2003). From these groups, the tetracyclines and their chemically modified derivatives

tend to develop a number of unwanted side-effects, e.g. gastrointestinal disturbances

and the possibility of potential antibiotic resistance (Teronen, Heikkila et al. 1999).

The bisphosphonate family on the other hand, has been shown to exhibit low toxicity

and have been tolerated in human use for several years, hence bisphosphonates seem

to be ideal candidates for MMP-related diseases (Teronen, Heikkila et al. 1999). From

this information, it seems a logical progression for bisphosphonates to be explored as

potential inhibitors of MMPs in chronic ulcers to promote the progression towards a

healing state.

14.

1.2.2.1 Bisphosphonates

Bisphosphonates, sometimes known incorrectly as diphosphonates, are stable

analogues of pyrophosphate characterised by two C―P bonds (Figure 1.3) (Fleisch

1997; Neville-Webbe, Holen et al. 2002). When the two bonds are found on the same

carbon atom, giving a P―C―P structure, they are called geminal bisphosphonates.

However, as only the geminal bisphosphonates have been found to exert strong

clinical activity they are commonly referred to in the literature as simply

“bisphosphonates” (Fleisch 1997). The main difference between bisphosphonates and

pyrophosphates is that pyrophosphate can be cleaved by enzymatic hydrolysis, but the

P―C―P bond found in bisphosphonates is hydrolysis-resistant (Vasikaran 2001).

Figure 1.3 Structure of bisphosphonates (a) General structure of bisphosphonate and pyrophosphate (b) First generation bisphosphonates (c) Second generation bisphosphonates (d) Third generation bisphosphonates (Heymann, Ory et al. 2004)

The bisphosphonates can be divided into two groups with different modes of action –

those that closely resemble pyrophosphate, i.e. clodronate and etidronate; and those

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that contain nitrogen, i.e. pamidronate, alendronate, risedronate and ibandronate. The

first group are able to be metabolically incorporated into nonhydrolysable analogues

of ATP that can inhibit ATP-dependent intracellular enzymes. The second more

potent group of nitrogen-containing bisphosphonates do not act in this manner, but

can instead inhibit enzymes of the mevalonate pathway, which can then inhibit the

synthesis of isoprenoid compounds essential for the post-translational modification of

small GTPases (Russell and Rogers 1999). In addition to this grouping,

bisphosphonates can be further divided into first-, second- and third-generation

compounds. The first-generation bisphosphonates have only short alkyl, halide or

hydroxyl side chains, giving them the lowest activity. Second-generation

bisphosphonates, with the one exception of tiludronate, have a nitrogen atom in their

side chain. Finally, the third-generation bisphosphonates, which are by far the most

potent, have a heterocyclic side chain containing nitrogen (Figure 1.3, Table 1.3)

(Heymann, Ory et al. 2004).

Table 1.3 Relative potencies of bisphosphonates in vitro (Vasikaran 2001)

Bisphosphonates have been shown to inhibit MMP-1, -2, -3, -8, -9, -12, -13 and -20 at

both therapeutically obtainable and non-cytotoxic concentrations (Heikkila, Teronen

et al. 2002). For example, alendronate has an IC50 value, i.e. the concentration of

alendronate required to inhibit 50% of MMP activity, of 40-70 µM (Heikkila, Teronen

et al. 2002). Currently, this group of drugs is mainly used in the management of

calcium and bone metabolism disorders (Vasikaran 2001), e.g. osteoporosis, Paget’s

disease, hypercalcaemia and metastatic cancer. These diseases have been effectively

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treated with bisphosphonates for a number of years (Table 1.4) (Russell and Rogers

1999; Teronen, Heikkila et al. 1999). However, future indications may include those

that are primarily caused by significant soft tissue destruction (Teronen, Heikkila et al.

1999), such as chronic wounds – an application that has not been previously

mentioned in the current literature.

Table 1.4 Bisphosphonates in clinical studies/development (Russell and Rogers 1999)

In terms of metastases, there have been a number of advances involving the

bisphosphonate-induced inhibition of MMPs. In early studies of patients with breast

cancer, clodronate significantly reduced the incidence of skeletal and visceral

metastases and gave a survival advantage. However, two other similar studies

produced completely different results, with no impact on visceral metastases

(Heymann, Ory et al. 2004). Another early bisphosphonate, alendronate, has proven to

be a potent inhibitor of bone resorption. It was shown that the use of alendronate to

prevent MMP-2 secretion blocks the solubilisation of collagen in metastatic human

prostate PC-3 ML cells (Stearns 1998). One important point to note, however, is that

these trials were performed using first- and second-generation bisphosphonates, the

activities of which vary from their third-generation counterparts (Heymann, Ory et al.

2004). Keeping this in mind, zoledronic acid, a third-generation bisphosphonate, has

been shown to be a potent inhibitor of both prostatic epithelial cells and aggressive

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breast cancer cells, preventing invasion and colony formation (Denoyelle, Hong et al.

2003; Montague, Hart et al. 2004). The mechanism responsible for this inhibition is

not dependent on protease modifications, but instead uses a disorganisation of the

actin skeleton due to a RhoA inhibition (Denoyelle, Hong et al. 2003).

These results show that while bisphosphonates appear to be good candidates for

clinical use, much more research still needs to be done to clarify their mechanism of

action and disease specificity. Furthermore, in terms of chronic ulcer treatments, the

delivery, or indeed simply the presentation of bisphosphonates to the site of excessive

MMP activity, has to be carefully considered. For this purpose, various polymers will

be considered for their potential ability to act as a scaffold to present this class of

molecules to the chronic ulcer.

18.

1.3 POLYMERS SUITABLE FOR CONTROLLED DRUG RELEASE IN A

WOUND DRESSING

Controlled drug release via polymers is currently widely studied, with many different

approaches depending on the material and drug of interest. Even more challenging, is

the prospect of manufacturing smart materials that release drugs by responding to a

patient’s individual therapeutic requirements (He, Cao et al. 2004). These delivery

systems would require complete drug protection, local targeting, specific controlled

release, self-regulating action, enzyme inhibition and reporting (He, Cao et al. 2004).

In addition, the biomaterial should be non-immunogenic and have flexible

modification strategies (Schmedlen, Masters et al. 2002; Leach, Bivens et al. 2003;

Ueda and Tabata 2003).

Traditional synthetic polymers include poly(ethylene glycol) (PEG), poly(2-

hydroxyethyl methacrylate) (PHEMA), poly(methyl acrylate) (PMA), poly(glycolic

acid) (PGA), poly(lactic acid) (PLA), and copolymers (PLGA) of these materials

(Hoffman 1998; Lee and Mooney 2001; Smeds and Grinstaff 2001). The major

advantages of these materials are that they can be easily synthesised and factors such

as mechanical, physical and chemical properties can be manipulated (Table 1.5)

(Holland, Tabata et al. 2003). Some limitations of synthetic polymers include:

requirement for inclusion on the Food and Drug Administration (FDA) ‘Generally

Regarded as Safe’ (GRAS) list; inadequate biodegradability; and, many are often

difficult to sterilise (Hoffman 1998).

Current commercially available wound dressings include a number of different types

of polymers, e.g. foams (Lyofoam, ConvaTec), hydrocolloids (Tegasorb, 3M),

hydrogels (IntraSite gel, Smith & Nephew), films (OpSite, Smith & Nephew),

polyurethanes (Allevyn, Smith & Nephew), alginates (Kaltostat, ConvaTec), activated

charcoal (CarboFlex, ConvaTec) and collagen dressings (Promogran, Johnson &

Johnson) (Queen, Orsted et al. 2004; Bouza, Muñoz et al. 2005). The use of these so-

called “modern” dressings has rapidly advanced in the last twenty years, with a large

economic impact on healthcare systems around the world (Cuzzell 1997). While these

“modern” dressings have improved wound treatment, they do not always completely

cover the primary concerns of local wound healing. These include: controlling wound

19.

Material Properties Semicrystalline polymer degraded by micro-organismsHigh solubility and low melting point make it an attractive biomaterial Degrades at a slow pace

Poly(caprolactone) (PCL)

Non-toxic and tissue compatible Highly biocompatible Exhibits versatile physical properties Poly(ethylene glycol) (PEG) Previously used as a porogen in hydrogels

Highly crystalline - high melting point and low solubility in organic solvents

Degradation to non-toxic monomers upon exposure to water Poly(glycolic acid) (PGA)

Tendency to lose mechanical strength rapidly Hydrophobic polymer, less hydrolysis and greater stability than PGA, Led to the development of PGA/PLA copolymers Poly(lactic acid) (PLA)

Choice of application based on crystallinity Combines the advantages of PGA and PLA Biodegradable and biocompatible

Poly(lactic-co-glycolic acid) (PLGA)

Low toxicity Water content similar to living tissue Inert to normal biological processes Shows resistance to degradation Permeable to metabolites Low thrombogenicity Not absorbed by the body Withstands heat sterilization without damage

Poly(2-hydroxyethyl methacrylate) (PHEMA)

Can be prepared in various shapes and sizes Biodegradable and bioabsorbable Good mechanical strength Poly(methylacrylate) (PMA) High tensile strength Good film forming properties High hydrophilicty Poly(vinyl alcohol) (PVA) Biocompatibility and chemical resistance

Table 1.5 Description of various polymers available for use in biomedical applications and their properties. Adapted from various sources (Hubbell 1996; Ramakrishna, Mayer et al. 2001; Hoffman 2002; Shao, Kim et al. 2003).

20.

infection and prolonged inflammation, while still promoting moist interactive wound

healing (Queen, Orsted et al. 2004). In addition, importance needs to be placed on

patient compliance, pain management and quality of life (Queen, Orsted et al. 2004).

Hydrogels are often selected as an ideal wound dressing as they are cool, do not stick

to the wound itself, and minimise pain to the patient (Queen, Orsted et al. 2004; Akita,

Akino et al. 2006). There is also the potential for novel biomaterials to be further

developed as wound dressings, e.g. alginates, but in their current form they are

unsuitable as completely defined wound dressings.

1.3.1 Hydrogels

In recent years, the polymeric structure widely investigated for use in tissue

engineering and controlled release is the hydrogel (Lee and Mooney 2001; Hoffman

2002; Drury and Mooney 2003). Hydrogels are hydrophilic polymer networks which

can absorb from 10-20% up to thousands of times their dry weight in water (Hoffman

2002). They can either be chemically stable or have the ability to degrade and

ultimately disintegrate, a fact that is often exploited in drug delivery (Hoffman 2002).

The structure of hydrogels is often determined by crosslinks formed between polymer

chains via various chemical bonds and physical interactions, e.g. H-bonding and

hydrophobic forces (Drury and Mooney 2003). Hydrogels also have the ability to be

fabricated in various forms, including: solid moulded forms; pressed powder matrices;

microparticles; coatings; membranes or sheets; encapsulated solids; and liquids

(Hoffman 2002). A major advantage with hydrogels is that they closely resemble the

chemical structure of the ECM, which can prove useful for applications in rapid

diffusion of hydrophilic nutrients and metabolites (Landers, Hubner et al. 2002).

There are many options for synthetic materials suitable for use in hydrogels (Table

1.6). These include, but are not restricted to, the following: poly(ethylene oxide)

(PEO); poly(vinyl alcohol) (PVA); poly(acrylic acid) (PAA); and poly(propylene

fumarate-co-ethylene glycol) (P(PF-co-EG)). In terms of naturally derived polymers,

there are also many options, e.g. agarose; alginate; chitosan; collagen; fibrin and

gelatin (Drury and Mooney 2003). However, as these matrices are derived from

animal and plant products that are non-defined, problems with sterility and

transmissible disease have to be considered (Drury and Mooney 2003; Tomizawa

2005). While plant-derived materials may not carry the risk of transmitting diseases,

they are still generally undefined and are often difficult to sterilise effectively before

application to the patient (Drury and Mooney 2003). In addition, some animal-derived

products are unable to be used for medical applications as the risk of disease, e.g.

Bovine Spongiform Encephalopathy (BSE), while low, is still present (Grassi, Farra et

al. 2005; Tomizawa 2005).

Table 1.6 Hydrophilic polymers suitable for hydrogel matrices (Hoffman 2002)

Recent work has suggested that it is possible to achieve controlled drug and protein

release from synthetic polymers (Hubbell 1996; Jeong, Choi et al. 1999; He, Cao et al.

2004). PEG hydrogels (molecular mass 10,000 Da) have been demonstrated to release

proteins from 6,000 to 66,000 Da while still displaying controlled release. This study

also showed that small proteins were released by diffusion without requiring

degradation, whereas larger proteins needed degradation first and then diffusion (West

and Hubbell 1995; Hubbell 1996). In cases of release without degradation, the rate of

permeation of the protein through the gel was inversely and linearly related to the

molecular mass of the protein. Of importance, the photopolymerisation process used

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to form the hydrogel has been shown to not adversely affect the incorporated protein’s

activity (West and Hubbell 1995).

In cases where the protein release is Fickian in nature, the diffusion kinetics are

governed by the rate of polymer swelling (Tomic, Micic et al. 2007). More

specifically, the protein release is controlled by the its ability to migrate through the

polymer scaffold, or even by diffusion through micropores of the polymer matrix

(Tomic, Micic et al. 2007). Protein diffusion from monolithic systems can be analysed

using Fick’s second law of diffusion:

αφ = D α2φ

αt αx2

Where: φ is the concentration in dimensions of [(amount of substance) length-3]

t is time [s]

D is the diffusion coefficient in dimensions of [length2 time-1]

x is the position [length] (Fick 1855)

The models of controlled release by diffusion are based on the principle of the

permeability of the polymeric matrix following swelling (Tomic, Micic et al. 2007).

Copolymers of PEG (Figure 1.4a) and 2-hydroxylethyl methacrylate (HEMA) (Figure

1.4b) have also been investigated for use as a hydrogel for protein release (Carenza,

Lora et al. 1993).

(a)

(b)

Figure 1.4 Chemical structures (a) PEG (b) HEMA

H2C

CH3

O

OOH

OH CH2

CH2

O CH2

CH2

OHn

23.

These two materials are advantageous for use in biomaterial applications for various

reasons (Hoffman 2002), including the fact that PHEMA has been used for a number

of years in the production of soft contact lenses and intraocular lenses, as it exhibits

high biocompatibility and low thrombogenecity (Caliceti, Veronese et al. 1992;

Carenza and Veronese 1994). In addition, PHEMA has good strength characteristics

for these applications, as well as the imbibed water content properties desirable in

hydrogels (George, Wentrup-Byrne et al. 2004). PEG is also highly biocompatible and

exhibits versatile physical properties, which are highly dependent on its weight

percentage, molecular chain length and crosslinking density (Almany and Seliktar

2005).

Furthermore, gamma irradiation is a very convenient tool for polymerisation when

considering hydrogels (Park, Nho et al. 2004), and has previously been used to form

PHEMA hydrogels, with the mechanism described in Figure 1.4. Unlike conventional

chemical initiation methods, including photo-polymerisation, radiation polymerisation

does not need the addition of catalysts or additives and also gives good control over

the degree of crosslinking (Park, Nho et al. 2004). By using gamma irradiation,

Carenza et al. claimed to crosslink these two materials, PEG and the HEMA

monomer, and obtained a complete polymerization conversion at a total dose of 25

kGy (Carenza, Lora et al. 1993), potentially using the mechanism described in Figure

1.5. Therefore, the crosslinking of polymers is extremely important when examining

their physical properties.

1.3.2 Crosslinking of polymers

Crosslinked hydrogels based on polymers or copolymers of HEMA have a long

history of clinical use in contact lenses and drug delivery systems. Primarily this is

because crosslinked hydrogels are inert, insoluble and easy to manufacture – and

therefore cannot become absorbed into the body like their water-soluble linear

analogues (Zahedi and Lee 2007). There are a number of ways to crosslink a polymer

namely, irreversible chemical crosslinking, crosslinking through ionic interactions,

electron bombardment, or gamma irradiation (Berger, Reist et al. 2004; Rouif 2005)

24.

Figure 1.5 Mechanism of gamma induced polymerisation of HEMA

CH2 C

CH3

C O

O

CH2

CH2

OH

CH2

C CH3

C O

O

CH2

CH2

OH

I

CH2

C CH3

C O

O

CH2

CH2

OH

CH2 C

CH3

C O

O

CH2

CH2

OH

I

C O

O

CH2

CH2

OH

CCH2

CH3

CH2

C CH3

C O

O

CH2

CH2

OH

C O

O

CH2

CH2

OH

C CH2

CH3

CO

O

CH2

CH2

OH

C CH2

CH3

C O

O

CH2

CH2

OH

CCH2

CH3

C

CH3

CO

O

CH2

CH2

OH

I I

Inititation

Propagation

+

Termination

.

γ

25.

Figure 1.6 Potential mechanisms for gamma induced random grafting/

termination or polymerisation of HEMA and PEG

C O

O

CH2

CH2

OH

C CH2

CH3

O PEG OH

C O

O

CH2

CH2

OH

CCH2

CH3

O PEG OH

CO

O

CH2

CH2

OH

C CH2

CH3

C O

O

CH2

CH2

OH

CCH2

CH3

O PEG O CH2 CO

O

CH2

CH2

OH

C

CH3

O

C O

O

CH2

CH2

OH

C

CH3

O PEG CH2

.

Random grafting / Termination

.

PEG copolymerisation

γ

26.

To synthesise a chemically crosslinked hydrogel, more than two functional sites are

required in each polymer chain with at least one reaction partner (Rouif 2005). This

then creates chains of polymers interconnected by crosslinkers, i.e. difunctional

crosslinking agents, resulting in the creation of a three-dimensional network (Berger,

Reist et al. 2004). However, as with any crosslinking agent, residual amounts are

always present in the ensuing polymer, often causing problems with biocompatibility

(Hassan and Peppas 2000). In contrast, crosslinking using irradiative methods

removes the need for potentially toxic additives, e.g. residual chemical initiators,

allowing a smoother transition to commercial use for products for biomedical

applications (Hassan and Peppas 2000).

It is also important to note that when irradiating aqueous polymer solutions, the

radiolysis properties of water need to be considered. Primarily, gamma irradiation of

aqueous solutions produces oxygen radicals. Free radicals, e.g. .OH and HO2.

(Leguéné, Clavère et al. 2001), are typically scavenged by the solutions during

irradiation (Namkyu, Seunghee et al. 2004). Furthermore, molecular products (H2O2,

H2) are formed by the combination of these radicals (e–aq, H., OH.) in the spurs

(Wasselin-Trupin, Baldacchino et al. 2002). The mechanism of water radiolysis is

quite complex and involves several other reactions, as shown by Table 1.7 (Pastina,

Isabey et al. 1999). In addition, these reactions are influenced by the radiation

characteristics, the temperature, and the chemical composition of the water (Pastina,

Isabey et al. 1999). For these reasons, it is very difficult to foresee the exact effect of

these parameters.

The radiolysis of water forms both molecular (H2, O2, H2O2) and radical (H, OH, e−aq,

HO2) species as shown by the following equation:

H2O H2, H2O2, .OH, H., e−aq, HO2, H3O

+ (Pastina, Isabey et al. 1999)

In terms of HEMA specifically, the monomer is synthesised through either the

transesterification of ethylene glycol, or by reacting ethylene oxide and methacrylic

acid (Montheard, Chatzopoulos et al. 1992). Both of these methods can cause

impurities in the resulting monomer, e.g. methacrylic acid from the hydrolysis of

HEMA, and ethylene glycol dimethacrylate (EGDMA) from the esterification

between methacrylic acid or HEMA and ethylene glycol (Montheard, Chatzopoulos et

al. 1992). As EGDMA is a difunctional crosslinking agent, the resulting copolymer

hydrogels (Figure 1.6) are crosslinked and swell in water (instead of dissolving) and,

even at very low percentages of the impurity (Montheard, Chatzopoulos et al. 1992).

Table 1.7 The reaction scheme of the radiolysis of neutral water. A, Arrhenius factor frequencies (A in moles-1 dm3 s-1); Ea, activation energies (Ea in kJ moles-1); Rate constants (moles-1 dm3 s-1) (Pastina, Isabey et al. 1999).

1.3.3 Functionalisation of polymers with bisphosphonates

The functionalisation of polymers has been widely used for a number of years to

improve various properties of the original polymer, e.g. cell adhesion (Buchmeiser

2007). Theoretically, functionalisation can be accomplished via three different

methods: copolymerisation of functional monomers; functionalising the material post-

polymerisation, commonly referred to as grafting; and imprinting (Buchmeiser 2007).

In terms of modifying bisphosphonates for clinical delivery, a number of bone

27.

halla


28.

Figure 1.7 Mechanism of gamma induced crosslinking of HEMA and

EDGMA

CH2

C CH3

R

CH2 C

CH3

C

O

CH2

CH2

O

O

C

C

CH3

CH2

O

CCH2

CH3

R

CH2

C

CH3

C

O

CH2

CH2

O

O

C

C

CH3

CH2

O

C O

O

CH2

CH2

OH

CCH2

CH3

R

CH2

C

CH3

C

O

CH2

CH2

O

O

C O

C

CH3

CH2

R =

EDGMA crosslinking

29.

specific applications have been developed. These include: conjugation of an amino-

bisphosphonate to bovine serum albumin to improve delivery to mineralised tissue

(Uludag and Yang 2002); copolymerisation of alendronate with PEG to act as a bone-

targeting polymeric drug delivery system (Wang, Miller et al. 2003); and, loading of

specific bisphosphonates into siliceous ordered mesoporous materials to enhance bone

implants (Balas, Manzano et al. 2006). Furthermore, alendronate is a useful model

bisphosphonate as it possesses a primary amine, which is convenient for conjugation

(Wang, Miller et al. 2003) using a number of already-established chemical reactions.

Nucleophilic acyl substitution reactions describe the substitution reaction of

nucleophiles, e.g. amines, and acyl compounds, e.g. acid halides (Otera 1993). In the

case of amines, they form a condensation reaction to then create amides. Therefore, it

seems a logical next step that this technique can be used to functionalise the

bisphosphonate, alendronate, to allow copolymerisation into a hydrogel.

30.

1.4 PROJECT OUTLINE

1.4.1 Research Problem

As outlined above, the issue of chronic non-healing ulcers is a major medical concern.

They are not only a major cause of pain and anxiety for those aged over sixty, but also

largely contribute to their lessening mobility, decreased social interactions and overall

diminished quality of life. Previous literature has shown that the chronic wound

environment is a very complex and toxic place for new cells to grow (Chen, Schultz et

al. 1999). Furthermore, many authors have suggested that inhibition of these proteases

is critical to enable correct progression of healing for these ulcers (Tarnuzzer and

Schultz 1996; Wysocki, Kusakabe et al. 1999; Yager and Nwomeh 1999; Cullen,

Smith et al. 2002). However, there is other evidence suggesting that levels of

proteases in the wound bed itself vary greatly from those levels found in the CWF

(Cook, Stephens et al. 2000). Therefore, it appears that it is important to inhibit the

proteases present in CWF, while not affecting those that show decreased activity in

the wound bed, to provide an effective treatment for these compromised wounds.

Once that inhibition has taken place, it is then possible to either use the body’s own

growth factors to heal the wound, or to release a synthetic growth factor complex, e.g.

VitroGro®, comprised of vitronectin, insulin-like growth factor and an insulin-like

growth factor binding protein, into the neutralised environment. This approach should

also allow for a more cost-effective solution, as minimal amounts of expensive growth

factors would be used, if at all, in the dressing. Therefore, this wound dressing would

be available as a viable treatment option to the majority of chronic leg ulcers sufferers,

regardless of their socioeconomic position. In conclusion, by treating this problem

quickly and effectively, improved holistic patient outcomes will be achieved.

1.4.2 Hypothesis

The overall goal of this applied research project was to examine the following

hypothesis:

Synthesis of a bioactive wound dressing that acts by inhibiting proteases in the CWF

will lead to the development of a therapy that can more effectively heal the

compromised ulcer.

31.

1.4.3 Aims

Thus, the specific aims of this project were to:

Aim 1: Use collagen zymography and a specific enzyme-linked immunosorbant assay

to analyse the increased activity of proteases in CWF, and then use this information to

identify the specific proteases responsible for the majority of collagen degradation in

the wound bed;

Aim 2: Identify a suitable synthetic and biocompatible polymer for the controlled

release of a biologically active compound into a chronic wound site; and

Aim 3: Apply both of these findings to the generation of a bioactive wound dressing

that would inhibit proteases in the wound fluid with a protease inhibitor tethered to the

polymer. This will then potentially still allow those proteases in the wound bed to

perform their vital functions in wound healing.

1.4.4 Project Design

This project has been designed to address both the biological and chemical issues that

need to be considered in the development of a bioactive wound dressing for chronic

leg ulcers. In addition, this project has deliberately blended the two disciplines of

wound healing biology and polymer chemistry to create a thesis that is

interdisciplinary in nature. Importantly, the key focus of this project was centred on

the development toward a tangible product as the key outcome, rather than a project

centred on generation of fundamental new mechanistic knowledge.

32.

CHAPTER 2

ANALYSIS OF MATRIX METALLOPROTEINASES

IN CHRONIC WOUND FLUID

34.

2.1 INTRODUCTION

As outlined in Chapter 1, wound healing is a highly complex process that can lead to

chronic ulcers if any stage in this complex series of events is interrupted (Trengove,

Stacey et al. 1999). While there is no single reported cause of these ulcers, many

factors are considered important when distinguishing chronic wounds from their acute

counterparts. Primarily, these wounds are characterised by excessive granulation

tissue and increased fibrosis (Stadelmann, Digenis et al. 1998), along with their

significant increase in proteolytic activity (Tarnuzzer and Schultz 1996; Ladwig,

Robson et al. 2002; Li and Li 2003). While it is understood that there are many

potential causes of these wounds, for the purposes of this thesis, the role of proteolytic

degradation and its relationship to the wound’s chronicity is the core focus. It is this

protease activity, primarily caused by a specific group of proteases called matrix

metalloproteinases (MMPs), that is believed to be responsible for the increased

extracellular matrix destruction in these chronic wounds (Chen, Schultz et al. 1999).

However, there are large amounts of conflicting information in the literature

concerning both the under- and over-expression of specific MMPs in chronic wounds.

Therefore, the purpose of this work was to clarify existing discrepancies in the

literature, and use multiple techniques to confirm individual MMP presence and

activity.

Gelatinases, also referred to as MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B), are

thought to have an extremely important function in normal wound healing during both

the remodelling and re-epithelialisation phases, and would therefore impair wound

healing if inhibited (Fray, Dickinson et al. 2003). However, other reports have

suggested that both MMP-2 and MMP-9, when present at the significantly higher

activity levels as reported in these chronic wounds, need to be inhibited to allow

normal healing to occur (Wysocki, Kusakabe et al. 1999; Ladwig, Robson et al. 2002).

The main reason why gelatinases are so important is that they are able to break down

collagen more effectively than other MMPs (Parks 1999), as well as cleave the major

constituent of the basement membrane – collagen type IV (Tarnuzzer and Schultz

1996). In a comprehensive study, MMP-9 was shown to be able to cleave collagen

types I-V into small peptides at the physiological temperature of 37 ºC (Okada, Gonoji

et al. 1992). In addition, MMP-2 and MMP-9 differ from other MMPs in that they

35.

contain three tandem fibronectin type II repeats within the amino-terminus of the

catalytic domain, which allows for binding to gelatin (Somerville, Oblander et al.

2003). Furthermore, MMP-9 contains an additional Collagen Type V-like insert in its

hinge region, with its function still to be determined (Somerville, Oblander et al.

2003).

MMP-9 is produced by a number of different cell types, namely inflammatory cells,

neutrophils, macrophages, monocytes and keratinocytes (Wysocki, Kusakabe et al.

1999; Cullen, Smith et al. 2002). As the chronic ulcers are classified as nonhealing,

the MMP-9 present in CWF is thought to be either neutrophil- or macrophage-derived

(Cullen, Smith et al. 2002). In terms of MMP-9 expression in chronic ulcers, one

study demonstrated that MMP-9 is temporally expressed during the healing of chronic

wounds (Wysocki, Kusakabe et al. 1999). This also correlates with another report that

showed MMP-9 activity levels in CWF were 25-fold elevated over a normal control

(Yager, Zhang et al. 1996). In contrast, Cullen et al. (2002) reported a lack of MMP-2

in CWF, which, as MMP-2 is fibroblast-derived, suggests that there must a lack of

fibroblasts in these chronic wounds, which may be associated with a lack of wound

closure (Cullen, Smith et al. 2002).

The ratio of MMPs and their natural tissue inhibitors, TIMPs, are also thought to be

important in the normal wound healing cascade (Cook, Stephens et al. 2000). Indeed,

a recent study, Ladwig et al. (2002) report that the ratio of MMP-9 to TIMP-1 is

inversely correlated with pressure ulcer healing (Ladwig, Robson et al. 2002). While

this study was performed in a different ulcer aetiology, there are still some similarities

between pressure ulcers and venous ulcers. Furthermore, it is interesting to note that

Cook et al. reported in 2000 that significantly decreased levels of active MMPs, along

with marked increases in expression of TIMPs, led to the chronicity of non-healing

wounds (Cook, Stephens et al. 2000) – directly contrasting all previous reports. The

most probable explanation for this is that Cook et al. (2000) used biopsies from the

wound bed, instead of wound fluids as used by other studies, including the one

reported in this chapter (Cook, Stephens et al. 2000). However, recent reports have

suggested that the MMPs present in the chronic wound bed may also be involved in

important biological processes, such as growth factor activation and immune system

regulation, rather than simply matrix and growth factor degradation (Levi, Fridman et

36.

al. 1996; Suzuki, Raab et al. 1997; McQuibban, Butler et al. 2001; Gearing, Thorpe et

al. 2002). Therefore, other MMP-interactions in the wound bed also need to be

considered when discussing chronic wounds (Somerville, Oblander et al. 2003).

At this time, there have been no studies that directly compare wound biopsies and

wound fluids, so therefore it is not know if they are indeed similar. Furthermore, it is

still unknown as to which sample provides a better indicator of the chronic wound’s

status (Ladwig, Robson et al. 2002). For the study reported herein, CWF was chosen

as the clinical sample to provide insight into the status of the chronic wound

environment, as the work described in this thesis is focussed on wound dressing

applications that could potentially modulate the proteolytic environment in CWF.

CWF is reported to be derived from plasma and, in a healing situation, delivers all of

the necessary components to the wound bed (Cutting 2003). Trengrove et al. (1996)

also describe similarities between human serum and CWF, indicating that CWF

displays the biochemical components expected in an extracellular fluid (Trengove,

Langton et al. 1996). Therefore, the aims of the study reported in this chapter were to:

identify proteolytic differences between AWF and CWF; to relate these to specific

proteases; and then in turn, identify protease inhibitors that would specifically inhibit

the major proteases with the goal of restoring the chronic wound towards a healing

state.

37.

2.2 MATERIALS AND METHODS

2.2.1 Wound fluid sample collection and preparation

Chronic wound fluid (CWF) samples were obtained from consenting patients of the

St. Luke’s Nursing Services (Brisbane, QLD, Australia), suffering from chronic

venous ulcers undergoing compression therapy (Table 2.1). Ethical approval to collect

these samples was obtained from both the Queensland University of Technology

(QUT) and St. Luke’s Nursing Services. No specific instructions were given to

patients prior to collection, e.g. no fasting requirements. A standard wound fluid

collection technique has been established and carried out at the clinical site. Briefly,

ulcers were washed with sterile water prior to collecting wound fluid, followed by the

application of an occlusive dressing over the wound. Exudate accumulated under the

dressing after 30 min to 1 hour was recovered by washing with 1 mL of saline.

Patient Age Sex Sample Code

Ulcer Duration (Weeks)

Ulcer Size (cm²)

% Reduction in wound area (from baseline)

Compression (mmHg)

PUSH Score

1 54 F 33 102 44 0 <10 15 2 79 F 34 102 15 -25 30 - 35 12 3 76 F 57 162 84 64 30 - 35 15 4 79 F 89 261 16 0 30 - 40 14 5 88 M 95 320 10.7 83.7 30 - 35 13 6 67 F 96 37 16 0 30 - 40 10 7 77 M 131 550 8 0 20 - 25 10 8 86 F 142 100 7.1 0 30 - 35 9 9 75 M 173 20 7 0 30 - 40 10

Table 2.1. Patient clinical data from chronic wound fluid samples.

Acute wound fluid (AWF) samples were collected from naturally occurring sub-

epidermal blisters on the feet of volunteers. The fluid was removed with 26G x 0.5”

needle and syringes (Terumo Medical, Somerset, NJ, USA) and collected in 1.5 mL

Protein LoBind tubes (Eppendorf, Hamburg, Germany). The wound fluid samples

were centrifuged at 14,000 g for 10 min, then the supernatant filtered using 0.45 µm

cellulose acetate filters (Agilent Technologies, Wilmington, DE, USA). This wound

fluid collection technique was chosen as being the most suitable approach, primarily

38.

because it does not rely on the absorption of wound fluid on to a porous, hydrophilic

wound dressing. In particular, the use of absorption techniques has been reported to

result in lower sample volume and protein amount (Moseley, Hilton et al. 2004). In

addition, other techniques that rely on this absorption approach do not take into

account that proteins may differentially adhere to these dressings, which will then

influence the CWF sample in later analyses (Cook, Stephens et al. 2000). A pooled

human serum (HS) sample was purchased commercially from Sigma-Aldrich (St

Louis, MO, USA). Protein content for all samples was quantitated and standardised

using the BCA Protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The

samples were then sub-aliquoted and stored at -80 ºC until further analysis.

2.2.2 Identification of protease activity using collagen zymography

Collagen zymography was performed as previously described (Gogly, Groult et al.

1998) using Collagen Type I and Collagen Type IV (Sigma-Aldrich) at a final

concentration of 0.5 mg/mL in 10% total acrylamide gels under non-reducing

conditions. MMP-8 (Calbiochem, San Diego, CA, USA) and MMP-9 (Calbiochem)

were run alongside the fluid samples as standards. Briefly, electrophoresis was

performed at 4 ºC under Laemmli conditions (Laemmli 1970). The gels were then

washed in 2.5% Triton X-100 for 30 minutes, then a further 60 minutes, prior to

incubation in 50 mM Tris-HCl, 10 mM CaCl2, 50 mM NaCl at pH 7.6 for 24 hours at

37 ºC. The gels were then stained using 0.25% Coomassie brilliant blue R-250 (Bio-

rad Laboratories, Hercules, CA, USA) (40% methanol, 10% acetic acid) and destained

appropriately (40% methanol, 10% acetic acid). Protease activity was visualised as

clear (unstained) bands.

2.2.3 Analysis and quantitation of collagen zymography

Gels were scanned using GeneSnap version 6.07 (SynGene, Cambridge, UK) and

analysed using GeneTools version 3.07 to determine the molecular weight and

quantitate the clear bands (SynGene). All quantitations were performed using the

analysis of 3 separate gels per treatment and values were expressed as a relative % of

the MMP standard, similar to previously published reports (Cook, Stephens et al.

2000; Lerman, Galiano et al. 2003). The data was also analysed in terms of grouping

39.

the samples based on the “PUSH” score of the wounds from which the CWF was

collected. Clinicians use the PUSH (Pressure Ulcer Scale of Healing) score to grade

the wounds (1 = best, 17 = worst) with this value taking into account a number of

factors to score the ulcer (Ratliff and Rodeheaver 2005). While this measurement

technique was originally designed by the National Pressure Ulcer Advisory Panel to

grade pressure ulcers, it has also been validated as an effective tool to monitor healing

trends in venous ulcers (Ratliff and Rodeheaver 2005). Thus, Ratliff et al. (2005)

noted that this system was a simple, valid and reliable tool for monitoring venous

ulcer clinical status (Ratliff and Rodeheaver 2005). In view of this, the data in our

study was organised in terms of this PUSH score, with five samples (CWF-1, -2, -3, -

4, -5) exhibiting a high PUSH score (≥12) and four samples (CWF-6, -7, -8, -9)

showing a lower PUSH score (≤11). While we ideally would have preferred to assess

a greater number of samples, CWF sample numbers were restricted due to patient

availability, as well as the amount of protein required from individual samples to

complete the full spectrum of experiments reported here. Statistical analysis was

performed using Tukey’s test (all group’s comparison), with statistical significance

determined as either * (p<0.05) or # (p<0.01).

2.2.4 Confirmation of MMP-specific collagen degradation using GM6001

inhibitor

To confirm that the collagen degrading activity visualised through zymography was

due to MMPs, a specific MMP inhibitor, GM6001 (Ilomastat or N-[(2R)-2-(hydrox-

amidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) (Chemicon,

Temecula, CA, USA) was used. Increasing concentrations of GM6001 (1, 3, 10, 30,

100, 300 and 1000 µM) were incubated with CWF-1 samples at 37 ºC for 24 hours.

These samples were then run on Collagen Type I zymograms and analysed as

described above.

2.2.5 Specific identification of MMPs through immunoprecipitation

Further confirmation of MMP activity was established through the

immunoprecipitation of specific MMPs followed by collagen zymography. UltraLink

Immobilized Protein A/G resin (Pierce Biotechnology) was incubated with individual

40.

MMP antibodies in a Tris-buffered saline solution with 0.1% Tween-20 (TBST) and

rotated for 5 hours at 4 ºC in 0.5 mL Protein LoBind tubes (Eppendorf). The

antibodies were: a mouse mAb for MMP-9 (Ab-7) (Calbiochem, San Diego, CA,

USA); a rabbit pAb for MMP-1 (Chemicon); a rabbit pAb for MMP-8 (Chemicon);

and a rabbit pAb for MMP-13 N-terminal (Chemicon). Following incubation, the

solution was centrifuged at 2,000 rpm for 2 minutes, the supernatant removed, and the

resin then washed twice with TBST. This “charged” resin was then incubated with the

human serum, AWF and CWF samples for 18 hours at 4 ºC, constantly rotating. As

before, following incubation the solution was centrifuged at 2,000 rpm for 2 minutes,

the supernatant removed, and the resin was washed twice with TBST. The resin was

then mixed with a 2X SDS sample buffer and left at room temperature for 1 hour. To

examine the bound proteins, these resin samples were run on Collagen Type I

zymograms and analysed as described above.

2.2.6 Quantitative confirmation of MMP-9 levels through a direct enzyme-

linked immunosorbant assay (ELISA)

Quantitative MMP-9 levels in HS, AWF and CWF samples were obtained using a

direct ELISA. Briefly, 96-well plates (Nunc) were coated with 40 ng of the

sample/well and left overnight at 4 ºC. Next, the wells were washed three times with

TBST and blocked with 5% skim milk powder (SMP) in TBST for 1 hour at room

temperature. Wells were again washed with TBST, and then probed with a rabbit

polyclonal antibody (1:1000) raised against the MMP-9 whole molecule (Abcam,

Cambridge, UK) for 1 hour at room temperature. Following washing, the wells were

probed with polyclonal goat anti-rabbit immunoglobulins/HRP (1:1000) (Dako

Denmark A/S, Glostrup, Denmark) for 1 hour at room temperature. Then, wells were

again washed and rinsed a final time with TBS before adding the substrate solution,

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Sigma-Aldrich) for

30 minutes at room temperature. The optical density of the plate was then read at 405

nm on a standard plate reader (Bio-rad Laboratories). The amount of MMP-9 in the

samples was calculated from a standard curve (MMP-9, Calbiochem) and then

converted to MMP-9 (ng)/total sample protein (μg). Data was organised in terms of

the sample’s PUSH score.

41.

2.3 RESULTS

2.3.1 Elevated proteolytic activity in chronic wound fluid

In order to analyse the proteolytic activity in wound fluid samples, CWFs from nine

separate patients were analysed using Collagen Type I and Collagen Type IV

zymography to identify protease activity. Collagens were used as they are the major

extracellular matrix constituents of the skin, contributing to approximately 70% of its

dry weight. Furthermore, Collagen Type I accounts for approximately 80% of the skin

collagens, while Collagen Type IV forms the basement membrane (Schacke, Docke et

al. 2002). Collagen degrading activity was identified as clear bands on a dark

background in the zymograms, with some smearing evident in the standards, as seen

previously by others (Stuart, Evans et al. 2007). This activity was then quantitated

through densitometric analysis. Ten times more total protein of both the HS and AWF

samples had to be loaded on the gels in order to adequately visualise the collagenase-

like activity on both substrates. As shown in Figure 2.1, the degradation profiles vary

between the patient samples in terms of both intensity and molecular weight of the

visualised bands. However, the bands appear to be consistent between the two

different substrates.

More specifically, Collagen Type I zymography revealed a high level of protease

activity in the CWF samples, especially considering the lower amount of the sample

loaded (Figure 2.1). In addition to the main band at approximately 102 kDa, there are

also smaller and potentially more active forms visible at 75 kDa, 66 kDa, 54 kDa and

48 kDa across the samples. Compared to CWF, less protease activity was evident in

both the HS and AWF samples, especially considering the increased protein loaded

for these samples. The main band in the AWF samples correlates with a mass of 66

kDa, similar to the main band seen in the HS. CWF-6 appears to have less proteolytic

activity than the other CWF samples, although, the activity present was greater than

that found with the HS and AWF samples.

Similar to the results found with the Collagen Type I zymography, Collagen Type IV

zymography also revealed increased levels of protease activity in CWF as compared

with HS and AWF samples (Figure 2.2). The main band in CWF is visualised at 102

42.

Figure 2.1 Collagen Type I zymography demonstrating protease activity present in wound fluid samples. Lane 1 is the positive control of MMP-8 (100 ng). Lane 2 is HS and lanes 3-5 are AWF samples 1-3 with 5 µg of total protein loaded per sample. Lanes 6-14 are CWF samples 1-9 with 500 ng of total protein loaded per sample.

Figure 2.2 Collagen Type IV zymography demonstrating protease activity present in wound fluid samples. Lane 1 is the positive control of MMP-9 (10 ng). Lane 2 is human serum and lanes 3-5 are AWF samples 1-3 with 10 µg of total protein loaded per sample. Lanes 6-14 are CWF samples 1-9 with 1 µg of total protein loaded per sample.

43.

kDa, suggesting that this is the same protease as identified with Collagen Type I

zymography. The main differences observed with Collagen Type IV zymography, as

compared to Collagen Type I zymography, are that firstly, there seem to be less bands

of lower molecular weight activity with this substrate, and also, the amount of

proteolytic activity is reduced when taking into account the fact that twice the sample

amount was loaded.

2.3.2 MMPs are responsible for collagen degradation as shown through a

specific inhibitor study

The MMP-specific inhibitor, GM6001, was used to confirm that the collagen

degradation revealed by zymography was indeed due to MMPs and not another group

of proteases present in the wound fluid samples. Through incubation with increasing

concentrations of GM6001 at 37 ºC over 24 hours, the proteolytic activity in CWF-1

gradually decreased as revealed by Collagen Type I zymography. Indeed, no visible

degradation of collagen was apparent at 1 mM GM6001 (Figure 2.3A). Densitometry

was used to quantify the reduction in proteolytic activity revealed by zymography, and

the levels are represented graphically in Figure 2.3B. Both the total amount of

protease activity, plus the protease activity in individual molecular weight species,

were followed across the increasing concentrations of 1, 10, 100 and 1,000 µM

concentrations of GM6001 and the data is expressed as relative % of the untreated

CWF-1 ± SEM. In all cases, the collagen degrading activity decreases with the

increasing concentrations of the MMP-specific inhibitor, confirming that the majority

of the proteases responsible for collagenase-like activity in these CWF samples are

from the MMP family.

2.3.3 Initial immunoprecipitation of MMPs from CWF samples

Following confirmation that the majority of collagen-degrading activity seen in CWF

samples was due to MMP-specific activity, individual MMPs were

immunoprecipitated from the CWF samples. Individual MMP antibodies were linked

to a protein A/G resin and then incubated with the CWF samples. In the first instance,

antibodies for MMP-1, -8 and -13 were used to identify particular species in terms of

relative abundance when compared to the complete samples (Figure 2.1). Specific

44.

(A)

(B)

Figure 2.3 GM6001 inhibition of protease activity in CWF-1 confirms the MMP specific degradation of Collagen Type I as revealed through zymography. (A) Lane 1 is the positive control of MMP-8 (100 ng). Lane 2 is the untreated sample of CWF-1 with 500 ng of total protein loaded. Lanes 3-9 are CWF-1 samples (500 ng) treated with increasing concentrations of the specific MMP-inhibitor, GM6001, at 1, 3, 10, 30, 100, 300, and 1000 µM at 37 ºC for 24 hours. (B) Relative levels of protease activity in CWF-1 samples treated with increasing concentrations of GM6001. The MMP-specific inhibition of collagen degrading activity was represented quantitatively through densitometric analysis. Levels are shown as the % collagen degrading activity, of both the total and also individual molecular weight bands, as compared to the untreated sample, ± SEM (n=3).

45.

MMPs were isolated from CWF-1 and then analysed through Collagen Type I

zymography (Figure 2.4). It appeared that there was minimal activity in all three

MMP-specific immunoprecipitations, as shown through the decreased degradation of

Collagen Type I when compared with the complete samples (Figure 2.1).

In view of this, six CWF samples were analysed using specific immunoprecipitation

of MMP-8, followed by analysis through Collagen Type I zymography as described

previously. In these preliminary optimisation studies, only six CWFs were used due to

limited sample volume. Again, there appeared to be minimal MMP-8 activity across

all 6 CWF samples containing the MMP-8 enriched fractions (Figure 2.5), confirming

the initial observation with CWF-1 (Figure 2.4). MMP-13 was also analysed across

the same 6 CWF samples and revealed similar results (Figure 2.6). Therefore, from

both the initial observations in CWF-1, followed by the analysis of 6 separate CWF

samples, it appeared that the levels of MMP-1, -8 and -13 were insufficient to cause

the majority of Collagen Type I degrading activity as revealed in the complete CWF

samples through zymography (Figure 2.1).

2.3.4 MMP-9 is the predominant protease responsible for matrix degradation

in chronic wound fluid

In light of the minimal levels of MMP-1, -8 and -13 activity detected in the initial

studies, MMP-9 was chosen due to its ability to degrade a number of substrates,

namely collagen types I-V (Okada, Gonoji et al. 1992), as well as being reported in

excessive levels in CWF in previous studies (Yager, Zhang et al. 1996). Through

incubation with a MMP-9 monoclonal antibody linked to an A/G protein resin, this

particular MMP species was able to be analysed in terms of relative abundance within

HS, AWF and CWF samples. Both the bound and unbound fractions were analysed

through Collagen Type I zymography and then quantitated through densitometric

analysis. The degradation profiles obtained via zymography varied in terms of both

abundance and molecular weight of the proteolytic activity.

The MMP-9 enriched fractions of CWF showed high levels of Collagen Type I

degrading activity (Figure 2.7A). However, there was minimal activity in the enriched

46.

Figure 2.4 Initial immunoprecipitation of MMP-1, -8, -13 from CWF-1 and analysis of enriched fractions through Collagen Type I zymography. Lanes 1-3 contains MMP-1-bound fraction, MMP-8-bound fraction and MMP-13-bound fraction respectively with 500 ng of total protein loaded per sample.

47.

Figure 2.5 Immunoprecipitation of MMP-8 from wound fluid samples and analysis of MMP-8 enriched fractions through Collagen Type I zymography. Lane 1 contains the positive control of MMP-8 (100 ng). Lanes 2 and 3 are the negative controls of Protein A/G and Protein A/G + MMP-8 pAb. Lanes 4 -9 are the bound fractions of CWF samples 1-6 with 500 ng of total protein loaded per sample

48.

Figure 2.6 Immunoprecipitation of MMP-13 from wound fluid samples and analysis of MMP-8 enriched fractions through Collagen Type I zymography. Lane 1 contains the positive control of a 27 kDa truncated version of MMP-13 (100 ng). Lanes 2 and 3 are the negative controls of Protein A/G and Protein A/G + MMP-13 pAb. Lanes 4 -9 are the bound fractions of CWF samples 1-6 with 500 ng of total protein loaded per sample

49.

fractions of human serum and AWF samples, except for AWF-2. The molecular

weight profile in these bound fractions appeared quite similar to the Collagen Type I

zymograms of the complete samples (Figure 2.1), possibly indicating that MMP-9

was indeed the major constituent of the collagen degrading activity in the wound

fluids. Quantitatively, there were significant differences between HS and AWF-2

(p<0.05), and with all CWF samples (p<0.01), except for CWF-6 (Figure 2.7B). The

main clinical difference in the patient from whom CWF-6 was collected is that the

patient had had the ulcer for a shorter duration, which may provide further insight into

wound’s clinical status. In terms of grouped samples, the CWF samples showed

statistically significant increased collagenase-like activity (p<0.01) when compared to

both HS and AWF samples (Figure 2.7C). Further detailed analysis of the samples in

terms of the PUSH scores revealed that both the lower PUSH score (PUSH ≤11)

grouped samples and the higher PUSH score (PUSH ≥12) grouped samples both had

significantly increased MMP-9 activity compared to both HS and AWF samples

(p<0.01) (Figure 2.7D).

MMP-9 depleted fractions showed lower levels of Collagen Type I degrading activity

(Figure 2.8A) than both the unfractionated samples (Figure 2.1) and enriched

fractions (Figure 2.7A). Importantly, the total protein amount loaded on the

zymograms with these depleted fractions was identical to that loaded in the

unfractionated samples described above (Figure 2.1). Furthermore, there were

increased levels of collagenase-like activity in the depleted fractions of CWF samples

1-5 and 7-9, compared with HS, AWF samples and CWF-6. Quantitatively, the levels

of MMP-9 activity were significantly increased in CWF samples 1-3 and 8-9 (p<0.01)

and CWF-7 (p<0.05), with similar trends evident in CWF samples 4 and 5, although

they were not significant (Figure 2.8B). When the samples were grouped into their

relative types, the CWF samples displayed highly significant Collagen Type I

degrading activity when compared with HS and AWF samples (p<0.01) (Figure

2.8C). In addition, analysis of the samples in terms of PUSH scores revealed that the

higher PUSH score (PUSH ≥12) group had significantly increased MMP-9 activity

compared to HS and AWF samples (p<0.01), as well as the lower PUSH score (PUSH

≤11) group (p<0.05). In addition, the lower PUSH score (PUSH ≤11) group was not

significantly different from either HS or AWF samples. The main band differences in

the zymography profiles of the bound and unbound fractions were at an approximate

50.

(A)

(B)

Figure 2.7 Immunoprecipitation of MMP-9 from wound fluid samples and analysis of MMP-9 enriched fractions through Collagen Type I zymography. (A) Lane 1 contains the positive control of MMP-9 (500 pg). Lanes 2 and 3 are the negative controls of Protein A/G and Protein A/G + MMP-9 mAb. Lanes 4 -16 are the bound fractions of HS, AWF samples 1-3 and CWF samples 1-9 with 500 ng of total protein loaded per sample (B) Relative levels of MMP-9-bound fractions in individual samples represented quantitatively through densitometric analysis. Levels are shown as the mean activity ± SEM (n=3). Statistical significance is relative to HS and shown as either * (p<0.05) or # (p<0.01) as determined by Tukey’s test.

51.

(C)

(D)

Relative levels of MMP-9 enriched fractions in samples represented quantitatively through densitometric analysis. Levels are shown as the mean activity ± SEM (n=3).(C) Data from (B) is separated into HS, AWF and CWF pooled samples. Statistical significance is relative to both HS and AWF and shown as # (p<0.01) as determined by Tukey’s test. (D) Data from (B) is separated into samples with a higher PUSH score (CWF samples 1-5, PUSH ≥12) and lower PUSH scores (CWF samples 6-9, PUSH ≤11). A higher PUSH score indicates a clinically worse ulcer. Statistical significance is relative to both HS and AWF and shown as # (p<0.01) as determined by Tukey’s test.

52.

(A)

(B)

Figure 2.8 Immunoprecipitation of MMP-9 from wound fluid samples and analysis of MMP-9 depleted factions through Collagen Type I zymography. (A) Lane 1 contains the positive control of MMP-9 (500 pg). Lanes 2 -14 are the unbound fractions of HS, AWF samples 1-3 and CWF samples 1-9 with 500 ng of total protein loaded per sample (B) Relative levels of MMP-9-unbound fractions in individual samples represented quantitatively through densitometric analysis. Levels are shown as the mean activity ± SEM (n=3). Statistical significance is relative to HS and shown as either * (p<0.05) or # (p<0.01) as determined by Tukey’s test.

53.

(C)

(D)

Relative levels of MMP-9 depleted fractions in samples represented quantitatively through densitometric analysis. Levels are shown as the mean activity ± SEM (n=3).(C) Data from (B) is separated into HS, AWF and CWF pooled samples. Statistical significance is relative to both HS and AWF and shown as # (p<0.01) as determined by Tukey’s test. (D) Data from (B) is separated into samples with a higher PUSH score (CWF samples 1-5, PUSH ≥12) and lower PUSH scores (CWF samples 6-9, PUSH ≤11). A higher PUSH score indicates a clinically worse ulcer. Statistical significance is relative to both HS and AWF and shown as # (p<0.01) as determined by Tukey’s test.

54.

molecular weight of 65 and 55 kDa respectively. Therefore, from these results it

appears that increased levels of MMP-9 activity are significantly different in CWF, as

compared with both HS and its acute counterpart, in terms of both activity and

molecular weight species. In addition, the MMP-9-unbound fractions, which had been

depleted of MMP-9, appear to display a trend towards a higher level of Collagen Type

I degrading activity correlating with a higher PUSH score, i.e. a clinically worse ulcer.

2.3.5 MMP-9 levels present in chronic wound fluid correlate with the clinical

severity of the ulcer

To confirm the findings found by immunoprecipitation, a direct ELISA was used with

a different MMP-9 antibody to ensure accuracy in antibody specificity. MMP-9 levels

for all samples ranged from approximately 70 ng/µg total protein to 370 ng/µg total

protein. In all AWF samples, there were no statistically significant differences to HS.

Concerning the CWF samples, CWF-8 displayed significantly increased levels of

MMP-9 compared with HS (p<0.05), with CWF samples 1-3, 5 and 9 also showing

statistically increased MMP-9 levels (p<0.01) (Figure 2.9A). When grouped samples

were analysed, CWF showed a statistically significant increase in MMP-9 levels

compared with both AWF and HS (p<0.01) (Figure 2.9B). In addition, when the

samples were grouped according to their clinical PUSH score, the lower PUSH score

(PUSH ≤11) group was not significantly different from HS, but did have statistically

significant differences from both AWF and the higher PUSH score (PUSH ≥12) group

(p<0.01) (Figure 2.9C). Furthermore, the higher PUSH score (PUSH ≥12) group

showed a significant increase in MMP-9 levels compared with both HS and AWF

(p<0.01) (Figure 2.9C). From these results it appears that there is a trend towards

increased levels of MMP-9 correlating with an increased PUSH score, and hence a

clinically worse ulcer.

55.

(A)

(B) (C)

Figure 2.9 MMP-9 levels from wound fluid samples analysed through an ELISA. Levels are shown as MMP-9 (ng)/total protein (µg) ± SEM (measured in triplicate and repeated).(A) Data is represented as individual samples. Statistical significance is relative to HS and shown as either * (p<0.05) or # (p<0.01) as determined by Tukey’s test. (B) Data from (A) is separated into HS, AWF and CWF pooled samples. Statistical significance is relative to both HS and AWF and shown as # (p<0.01) as determined by Tukey’s test. (C) Data from (A) is separated into samples with a higher PUSH score (CWF samples 1-5, PUSH ≥12) and lower PUSH scores (CWF samples 6-9, PUSH ≤11). A higher PUSH score indicates a clinically worse ulcer. Statistical significance is relative to both HS and AWF and shown as # (p<0.01) as determined by Tukey’s test.

56.

2.4 DISCUSSION

A major factor responsible for the chronic nature of ulcers is believed to be the tissue-

destructive events mediated by certain endopeptidases (Grinnell, Ho et al. 1992;

Palohati, Lauharanta et al. 1993). These neutral endopeptidases are able to degrade the

extracellular matrix at the ulcer’s physiological pH and can be classified into two

groups: serine proteases and matrix metalloproteinases (Palohati, Lauharanta et al.

1993). A previous study by Weckroth et al. (1996) showed that serine proteases of

polymorphonuclear neutrophil origin, e.g. cathepsin G and elastase, were both low in

activity in CWF (Weckroth, Vaheri et al. 1996). For this reason, the studies reported

in this chapter focussed on MMPs.

In terms of MMPs, there have been a number of studies measuring MMP activity in

CWF, but many of these studies have only used general techniques to identify a broad

range of MMPs e.g. the Azocoll assay (Tarnuzzer and Schultz 1996; Trengove, Stacey

et al. 1999), gelatin zymography (Wysocki, Staianocoico et al. 1993; Yager, Zhang et

al. 1996; Trengove, Stacey et al. 1999; Wysocki, Kusakabe et al. 1999), and antibody

detection (Valleala, Hanemaaijer et al. 2003). As most members of the MMP family

are structured into three essential, characteristic and highly conserved domains

(Massova, Kotra et al. 1998), it is often quite difficult to differentiate MMP species

based on molecular weight alone. In addition, antibody assays that are based on

specific MMPs often find it difficult to differentiate between closely related species,

as there is a very close evolutionary relationship within the family, as is reflected by

their highly conserved domains (Somerville, Oblander et al. 2003). Indeed, this high

degree of structural similarity is likely to be the main reason underlying the

controversy surrounding levels of specific MMPs in chronic wounds. In view of this,

the studies in this chapter used multiple techniques to identify specific MMPs species.

Together, the combination of an antibody-based identification assay with an activity

assay has allowed more conclusive and robust results. Indeed, the findings reported

herein indicate that there are minimal levels of MMP-1, -8 and -13 activity in CWF,

with MMP-9 being the most likely candidate responsible for Collagen Type I

degradation in CWF. This was further confirmed by using a different MMP-9

antibody for the ELISA as compared with immunoprecipitation.

57.

As reported in this chapter, all nine CWF samples exhibit higher levels of MMP

activity than the AWF and HS samples. CWF-6 appears to have less MMP activity

than the other CWF samples; however, it still contains more MMP activity than the

HS and AWF samples. These differences within the sample groups highlight the

difficulties of dealing with CWF as a diagnostic sample; due to the heterogeneity of

these samples, and the wounds from which they are obtained, it is often quite difficult

to achieve statistically conclusive results. However, in this study elevated levels of

Collagen Type I and IV degradation by CWF samples compared with those found

with HS and AWF samples has been reported. In addition, the collagen degradation

can be specifically attributed to MMPs as shown through the targeted inhibitor study

using GM6001, a specific MMP inhibitor (Levy, Lapierre et al. 1998). Thus, GM6001

was shown to inhibit the CWF-induced collagen degradation in a dose-dependent

manner. Together, these data confirm the hypothesis that elevated MMP levels in

CWF are indicative of a clinically worse chronic ulcer and furthermore, suggest that

inhibition of these proteases holds promise as a therapeutic treatment for these

debilitating chronic wounds.

More specifically, these experiments suggest that MMP-9 appears to be the major

protease responsible for matrix degradation in CWF. This is similar to previous

reports by Ladwig et al. (2002), which suggested that there was a statistically

significant correlation between poor healing and elevated levels of MMP-9 in patients,

regardless of the treatment regime (Ladwig, Robson et al. 2002). Moreover, Wysocki

et al. (1999) state that as healing proceeds, the levels of MMP-9 decrease in CWF and

reach those found in acute wounds (Wysocki, Kusakabe et al. 1999). Further evidence

suggests that the MMP-9 present in CWF is derived from macrophages infiltrating the

wound bed (Moses, Marikovsky et al. 1996), which considering the excessive levels

of MMP-9 in CWF compared with AWF, could further confirm the hypothesis that

chronic wounds are partly caused by disproportionate inflammation and the inability

to proceed into the normal remodelling stage of wound healing (Chen, Schultz et al.

1999). Therefore, the clinically worse ulcers are those that show prolonged

inflammation, presenting as increased levels of macrophage-derived MMP-9 in the

resultant CWF.

58.

In the results reported herein, the lower and higher PUSH score groups were

indistinguishable through immunoprecipitation, as both groups showed a significant

increase in MMP-9 activity compared with HS and AWF samples (p<0.01). Similar

results have been obtained in previous studies, with MMP-9 in CWF showing a

minimum of a 2-fold increase when compared with AWF and HS (Wysocki,

Kusakabe et al. 1999; Ladwig, Robson et al. 2002). When considering the MMP-9

depleted fractions, there were significant MMP-9 activity differences between the

lower PUSH score (PUSH ≤11) group and the higher PUSH score (PUSH ≥12) group

(p<0.05), with the higher PUSH score (PUSH ≥12) group displaying a significant

increase in Collagen Type I degrading activity as compared with both HS and AWF

(p<0.01). This could be due to the large levels of MMP-9 present in these samples,

which in turn suggests that the zymograms may well have been overloaded. If indeed

this did occur, this may explain why statistically significant differences between the

two clinical groupings were not obtained. Nevertheless, Tarlton et al. (1999) were

able to use gelatin zymography to show a statistical correlation between pro-MMP-9

and the increasing severity of the ulcer (p=0.006) (Tarlton, Bailey et al. 1999).

In comparison, the Tarlton et al. (1999) study used wound site specific collection

techniques, i.e. they collected CWF from the advancing wound margin in a

deteriorating ulcer and not the ulcer it its entirety; so the results are not directly

comparable (Tarlton, Bailey et al. 1999). In this study, we specifically chose to use a

standardised collection technique that recovered the wound fluid in its entirety without

any added protein selection processes, i.e. differential protein absorption and retrieval

from wound dressings. Furthermore, while the samples were not collected from

clinically infected wounds, it is important to recognise that there would still be a

bacterial presence in the wound, which may then contribute to the wound fluid’s

proteolytic activity. The extent to which bacterial proteases contribute to the overall

amount of MMP activity in CWF will be a focus of future studies. Another interesting

point to note is that while some MMPs have been known to degrade IgG proteins,

namely MMP-3 and MMP-7 (Gearing, Thorpe et al. 2002), this was not observed to

contribute to cleavage of the antibody from the resin in these experiments. This could

be due to lower levels of these specific MMPs, i.e. MMP-3 and -7, in the CWF

samples, or indeed, competitive binding of MMP-9 to the antibody, which could then

potentially block any further interactions.

59.

To further investigate any potential differences in CWF collected from wounds with

different clinical status, a direct ELISA was used with a different MMP-9 antibody to

show quantitative levels of MMP-9 in these samples. This technique showed clear

differences between the levels of MMP-9 present in the two separate groupings. The

lower PUSH score (PUSH ≤11) group displayed significantly higher levels of MMP-9

than AWF (p<0.01), as well as significantly lower levels of MMP-9 than the higher

PUSH score (PUSH ≥12) group (p<0.01). As a result, it appears that there is a strong

correlation between increased MMP-9 levels in CWF from wounds in the higher

PUSH score (PUSH ≥12) group compared to AWF samples (p<0.01). This therefore

suggests that MMP-9 is the predominant protease involved in degrading the

extracellular matrix in the chronic wound environment and healing could potentially

be promoted by its attenuation. In contrast, Fray et al. (2003) propose that MMP-2 and

-9 are both essential for wound healing, a process that would therefore be impaired if

these two proteases were inhibited in chronic ulcer treatments (Fray, Dickinson et al.

2003). However, their hypothesis is based on the fact that gelatinases are required for

tissue remodelling and for the onset on re-epithelialisation – stages of wound healing

that normally occur when inflammation has subsided (Trengove, Stacey et al. 1999).

Therefore, if significantly increased levels of MMP-2 and -9 are present before

inflammation has abated, an excessively proteolytic environment will continually

degrade key growth promoting agents and thus will not allow normal wound healing

to occur.

60.

2.5 CONCLUSION

In this study, the levels of MMP-9 present in the CWF samples showed a statistically

significant increase compared with both HS and AWF. When these CWF samples

were separated by their clinical score, the fluids from the clinically worse ulcers, i.e.

those with the higher PUSH scores (PUSH ≥12), displayed significantly higher levels

of MMP-9 than those from the lower PUSH score (PUSH ≤11) group (p<0.01). In

addition, it appears that high levels of MMP-9 activity in CWF are a better indicator

of a wound's chronicity than the total protease activity levels alone. Further, these data

suggest it is not surprising that current topical treatments of chronic ulcers with

bioactives, e.g. growth factors, have proven to be only slightly effective in treating

chronic wounds since high levels of proteases can rapidly degrade these growth

promoting agents (Cullen, Watt et al. 2002).

As demonstrated in this chapter, it is likely that these bioactives have been ineffective

as therapeutics in the clinical setting due to the high levels of proteases found in CWF.

Therefore, many authors have suggested that the addition of a protease inhibitor prior

to topical treatment of the wound would promote healing (Tarnuzzer and Schultz

1996; Wysocki, Kusakabe et al. 1999; Yager and Nwomeh 1999; Cullen, Smith et al.

2002). In view of these previous studies, along with the results reported herein, it is

suggested that a specific inhibitor of MMP-9 could potentially be more therapeutically

effective than general MMP inhibitors in modulating chronic ulcers towards a healing

state. This hypothesis is further explored in Chapter 4.

61.

CHAPTER 3

SYNTHESIS OF HYDROGELS FOR WOUND

DRESSING APPLICATIONS

62.

3.1 INTRODUCTION

As outlined in Chapter 1, the hydrogel is of particular interest in many human

therapeutic applications as it possesses a high water content, good biocompatibility,

and also often a texture similar to that of the body’s own soft tissue (Schmedlen,

Masters et al. 2002). In addition, many human biomedical applications have been

suggested as suitable for hydrogels, including: drug delivery, contact lenses, corneal

implants, as well as substitutes for skin, tendons, and cartilage (Schmedlen, Masters et

al. 2002). More recently, hydrogels have also been investigated for wound dressing

applications. As a large amount of importance needs to be placed on patient

compliance, pain management and quality of life; the hydrogel is often chosen as it is

cool, non-sticking and comfortable for the patient (Queen, Orsted et al. 2004; Akita,

Akino et al. 2006). Current commercially available hydrogel wound dressings include:

IntraSite™ gel (Smith & Nephew); Tegagel™ (3M); Curasol® gel (Healthpoint); Nu-

Gel® (Johnson & Johnson); as well as various others from a large range of wound

care companies. Therefore, hydrogels appear to be a logical choice for the basis of

further wound dressing developments and modifications.

The choice of starting materials in hydrogel preparation is extremely important when

considering specific tissue engineering and medical applications (Lee and Mooney

2001). PHEMA based hydrogels have been widely studied for human biomedical

applications and are one of the most commonly used materials in biomedical

hydrogels e.g. soft contact lenses (Carenza and Veronese 1994; George, Wentrup-

Byrne et al. 2004). Therefore, PHEMA was chosen for this study as it not only

possesses good biocompatibility and low thrombogenicity (Caliceti, Salmaso et al.

2001; George, Wentrup-Byrne et al. 2004), but also is resistant to enzymatic

degradation and extremes in pH (Prashantha, Rashmi et al. 2006). This last point is

critical to chronic wound dressing applications as there is a marked difference

between acute and chronic wounds in terms of proteolytic activity: thus chronic

wounds exhibit excessive protease activity and a wide variability in pH (Tarnuzzer

and Schultz 1996; Ladwig, Robson et al. 2002; Li and Li 2003). A scaffold that is able

to degrade in this toxic environment may potentially exacerbate the inflammation

burden, i.e. the excessive neutrophil response, already present in these slow healing

wounds (Yager and Nwomeh 1999). In addition, PHEMA has extensively-

63.

documented resistance to non-specific protein adsorption, which will then promote

low levels of cell adhesion – another critical factor for wound dressing changes

(Martins, Wang et al. 2003). Furthermore, PHEMA is extremely inert and has not

been shown to change the pH of solutions (Montheard, Chatzopoulos et al. 1992),

which indicates that it will not adversely interfere with the biological system that it

comes into contact with when applied topically.

Gamma irradiation has been shown to be an favourable tool for hydrogel

polymerisation (Park, Nho et al. 2004). Primarily, this is because it eliminates the

need for potentially toxic catalysts or additives, as is required with chemical initiation

methods (Park, Nho et al. 2004). In terms of crosslinking, irradiative methods are

again advantageous as they do not use potentially toxic crosslinking agents, which

then allows for a easier transition through the commercial development of a human

biomedical product (Hassan and Peppas 2000). Furthermore, crosslinked hydrogels

are preferred over their water-soluble polymers, as they are inert and insoluble, i.e.

they cannot be absorbed into the body (Zahedi and Lee 2007).

The HEMA monomer itself can also contain impurities that are active crosslinking

agents when placed under gamma irradiation (Montheard, Chatzopoulos et al. 1992).

For example, as HEMA is synthesised through the transesterification of ethylene

glycol, often the monomer can contain small amounts of ethylene glycol

dimethacrylate (EGDMA) – even after distillation (Montheard, Chatzopoulos et al.

1992). In addition, as EGDMA contain difunctional reactive groups, it is a common

crosslinking agent; making resulting copolymers that are able to form hydrogels, even

at very low percentages of the impurity (Montheard, Chatzopoulos et al. 1992).

Therefore, while Carenza et al. (1993) claimed to crosslink the HEMA monomer with

PEG at a total dose of 25 kGy to create a completely polymerised crosslinked

hydrogel (Carenza, Lora et al. 1993), it is also possible that small amounts of

EDGMA contributed to the polymerisation (Figure 1.6).

PEG itself has been extensively shown to act as a porogen in the preparation of porous

polymers (Xi, Wu et al. 2006). This therefore assumes that it does not actually

covalently react into the polymer, potentially conflicting with Carenza et al.’s (1993)

work (Carenza, Lora et al. 1993). However, in terms of physical properties, PEG is

64.

highly biocompatible and is also able to exhibit versatile physical properties, which

are highly dependent on its weight percentage, molecular chain length and

crosslinking density (Almany and Seliktar 2005). Therefore, the idea of a potential

copolymerisation of these two materials certainly provides scope for an intriguing

novel material. Indeed, the specific aims of the experiments reported in this chapter

examine different PHEMA hydrogels synthesised both with and without the presence

of PEG. This was assessed in terms of properties desirable in a wound dressing, i.e.

ease of application, comfort to the patient, as well as looking at skin irritation

potential. Furthermore, the differing characteristics of the hydrogels were examined in

terms of polymer conversion, water uptake, and protein release, while cell based

assays examined the dressing’s biocompatibility.

65.


3.2.1 Chemicals

2-Hydroxyethyl methacrylate (HEMA, assay ≥ 99% GC, Sigma-Aldrich, St Louis,

MO, USA) was distilled under reduced pressure immediately prior to use.

Polyethylene glycol 20,000 (PEG, Sigma-Aldrich), peroxidase from horseradish

(Sigma-Aldrich) and 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-

diammonium salt (ABTS, Pierce Biotechnology, Rockford, IL, USA) were used as

supplied under the manufacturers’ instructions. All H2O used was double deionised by

ion exchange (MilliQ, Millipore, Billerica, MA, USA).

3.2.2 Synthesis of hydrogels

Aqueous solutions of distilled HEMA were prepared in two separate ratios, both with

PEG 20,000 (50% water, 30% PEG and 20% HEMA) and without PEG 20,000

(71.4% water, 28.6% HEMA). Initial optimisation studies were performed to obtain

these ratios, as hydrogels synthesised with a lower HEMA ratio were not structurally

sound. In addition, the PHEMA alone ratio was obtained by simply preparing the

same solutions in the absence of PEG to ensure comparability. Solutions were placed

between two glass plates separated by a silicone gasket and purged with argon. Each

mould was approximately 75 mm x 50 mm x 3 mm and filled with 10 mL of monomer

solution. The moulds were then gamma irradiated by a Gamma-cell 220 (Atomic

Energy of Canada Ltd, Ottawa, Canada) using a Co60 source to give a total dose of 5

and 10 kGy, respectively (Table 3.1).

Sample ID H2O (g) HEMA (g) PEG 20,000 (g) Total Dose (kGy)

C (50:20:30 H2O:HEMA:PEG) 5.0 2.0 3.0 5

G (50:20:30 H2O:HEMA:PEG) 5.0 2.0 3.0 10

PH-5 (71:29 H2O:HEMA) 5.0 2.0 - 5

PH-10 (71:29 H2O:HEMA) 5.0 2.0 - 10

Table 3.1 Hydrogel formulations prepared for analysis as potential wound dressings

66.

Hydrogels were removed from their moulds and cut into 1 cm x 1 cm square pieces

for further analysis.

3.2.3 Analysis of polymerisation through NIR FT-Raman Spectroscopy

Un-irradiated and irradiated samples were analysed by a Perkin Elmer System 2000

NIR FT-Raman spectrophotometer (Perkin Elmer, Waltham, MA, USA). The

hydrogel samples were placed into glass vials for spectroscopic analysis and the

spectra were analysed using Grams/AI (Thermo Electron Corporation, Waltham, MA,

USA).

3.2.4 Swelling of hydrogels

Hydrogels were then placed into 12-well tissue culture plastic plates (Nunc A/S,

Roskilde, Denmark) with 3 mL of H2O and swollen at room temperature for a 2-week

period. The hydrogels were then dried in a vacuum desiccator and rehydrated under

similar conditions for one week. The hydrogel samples were both weighed and

measured at appropriate intervals to provide an indication of water uptake into the

system. Swelling ratios were then measured by the following equations:

St = Mt

Mp

where Mt is the mass of the swollen polymer at time t and Mp is the mass of the

original polymer (George, Wentrup-Byrne et al. 2004). Similarly,

Vt = Vt

Vp

where Vt is the volume of the swollen polymer at time t and Vp is the volume of the

original polymer. Furthermore, to gravimetrically analyse the loss of PEG in the

hydrogels, the theoretical amount of PEG was calculated from the weight of the

hydrogel and this was then compared to the percentage of the actual weight lost.

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These swelling experiments were performed to gain an appreciation of the hydrogel’s

ability to take up wound exudate as would be required in its final application.

3.2.5 Protein loading

Peroxidase from horseradish (HRP ~ 40 kDa) was prepared in solutions of ddH2O at 2

µg/mL. All solutions were filtered sterilised (0.22 μm) before use. The hydrogels were

placed in 24-well plates (Nunc) that had been siliconised with Sigmacote (Sigma-

Aldrich) as per the manufacturer’s directions, along with 1 mL of HRP solution and

then placed on a rotary shaker at room temperature for 24 hours.

3.2.6 Protein release

Hydrogels that had been loaded with HRP were first briefly dried for approximately 1

hour under vacuum at room temperature. Next, the hydrogels were placed in

siliconised 24-well plates (as per protein loading) and immersed in 1 mL of 50 mM 4-

(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES – Roche, Basel,

Switzerland) at pH 7.4 and placed on a rotary shaker at room temperature. Sub-

samples of the HEPES solutions were taken at intervals of 1, 2, 6, 24, 48, 72 and 168

hours and measured for HRP concentration. Briefly, this was achieved by adding the

HRP sample to 150 µl ABTS in a 96-well plate (Nunc) and incubating this at room

temperature for 30 minutes. The optical densities of the samples were then read at 410

nm in a Benchmark Plus Microplate Spectrophotometer (Bio-Rad Laboratories,

Hercules, CA, USA) and compared to a standard curve to measure HRP

concentrations. This concentration was then used to calculate the cumulative HRP

released from the hydrogels (ng).

3.2.7 Skin collection

Skin samples were collected from consenting patients undergoing breast or abdomen

reductions at the St. Andrews and Wesley Hospitals, Brisbane, QLD, Australia.

Human ethical approval was obtained from both the hospitals and the Queensland

University of Technology. The skin samples were collected in sterile jars containing

antibiotic/antimycotic solution containing 10,000 units of penicillin/mL, 10,000 µg of

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streptomycin/mL and 25 µg of amphotericin B/mL, with penicillin G, streptomycin

sulfate and amphotericin B as antimycotic in 0.85% saline (Invitrogen, Carlsbad, CA,

USA). These samples were processed within 12 hours following storage at 4 ºC in

Dulbecco’s Modified Eagle’s Medium (DMEM) containing antibiotics.

3.2.8 Primary keratinocyte cell cultures

Primary skin keratinocytes were isolated as previously described (Topping, Malda et

al. 2006) and expanded on a feeder layer of lethally irradiated 3T3 mouse fibroblast

feeder cells (i3T3s) in Green’s Media (Chakrabarty, Dawson et al. 1999) that

contained 10% foetal calf serum (FCS – Hyclone, Logan, UT, USA). Keratinocytes

were grown for 7 days with the medium replaced every 3-4 days.

3.2.9 Toxicity testing using a cell number assay

Polymers were synthesised as above and soaked in 3 mL Green’s media for 3 days in

12-well plates (Nunc) along with samples of Green’s media and serum-free media

(SFM) alone at 37 ºC/5% CO2. These solutions are subsequently referred to as

“treated media”. Primary keratinocytes at p0 were passaged and seeded into black 96-

well plates (5x103 cells/well) that had been preseeded 24 hours earlier with i3T3s at

the same concentration. The keratinocytes were allowed to adhere for 24 hours before

the treatments were applied. Briefly, the media was aspirated, the cells were washed

with a buffered salt solution and then the “treated” media replacements were added at

final “treated” media concentrations of 10, 50 and 100% respectively (Figure 3.1).

Cells were then incubated for 48 hours at 37 ºC/5% CO2. Cell number was determined

using the CyQUANT® NF Cell Proliferation Assay (Invitrogen) as per the

manufacturer’s instructions. Fluorescence was read at λex485 nm λem530 nm by a

POLARstar Optima Microplate Reader (BMG LABTECH GmbH, Offenburg,

Germany). Results were expressed as a % of the SFM negative control.

3.2.10 Preparation of a three-dimensional human skin equivalent

A de-epidermised dermis (DED) model was used as previously described

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Figure 3.1 Experimental design of the toxicity testing

70.

(Topping, Malda et al. 2006) with the following modifications. Following

keratinocyte addition to the sterile stainless steel rings on top of the DEDs, the

composites were incubated for 24 hours at 37 ºC/5% CO2. After this period, the rings

were removed and the composites were raised to the air-liquid interface by moving

them onto stainless steel grids in 6-well plates (Nunc). Cultures were maintained for 5

days at 37 ºC/5% CO2 before the hydrogel treatments were applied.

3.2.11 Biocompatibility testing of hydrogels using the DED model

The DED model was prepared as described above. At five days post air-liquid

interface culture, the hydrogels were placed on top of the composite, along with no

treatment and Allevyn™ dressing (Smith & Nephew, London, United Kingdom), a

commercially available wound dressing, as controls. These wound dressings were

exposed to the composite for 7 days at 37 ºC/ 5% CO2, with the media being replaced

at 3 days (Figure 3.2). At completion of the treatment, a 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide conversion (MTT) assay (Denizot and Lang 1986)

was used to analyse one of each treatment group. Briefly, the DED was submerged in

6 mL of 0.5 mg/mL MTT dye (Sigma-Aldrich) and incubated at 37 ºC for 2 hours.

Following incubation, the metabolically active cells could be visualised as a purple

colour and the DED were photographed. Next, both the MTT-stained and un-stained

samples were fixed and paraffin-embedded using standard protocols for haematoxylin

and eosin (H&E) histological analysis (Sharpey-Schafer 1954).

3.2.12 Immunohistochemistry

Paraffin sections were cut (3 μm sections) and then deparaffinised in ethanol and

xylene. Briefly, this involved sequential incubations with solutions of 100% xylene,

100% ethanol, 95% ethanol, 70% ethanol and distilled water. Sections were then

probed separately for: keratin 1/10/11 (K1/10/11), a marker for cornification and

squamous cell differentiation; p63 (RDI Research Diagnostics, Concord, MA, USA), a

p53 analogue that identifies normal basal cells as opposed to malignant tumours; and

cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), a critical

mediator of apoptosis in mammalian cells. For slides that were probed for cleaved

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Figure 3.2 Experimental timeline of the three-dimensional ex vivo human skin equivalent model

72.

caspase-3, antigen unmasking was required before the blocking step. Briefly, the

slides were incubated at 37 ºC in 10 mM citrate buffer (pH 3.0) for 30 min, before

proceeding as normal. After incubation with primary antibodies for K1/10/11 (1:400),

p63 (1:100) and cleaved caspase-3 (1:100), sections were stained using a Dako

Envision kit (Dako Denmark A/S, Glostrup, Denmark) as per the manufacturer’s

instructions, with the exception that phosphate buffered saline (PBS) was used instead

of TBS. After antibody development of the labelled secondary antibody with the 3,3’

diaminobenzidine (DAB) chromogen solution, all sections were counterstained with

haematoxylin for 30 seconds and analysed using light microscopy.

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3.3 RESULTS

3.3.1 Hydrogel preparation

Four sets of hydrogels were synthesised according to the conditions outlined earlier

(Table 3.1). There were clear differences in the PHEMA hydrogels polymerised in the

presence of PEG compared to those without PEG, namely in terms of transparency

and the apparent ease of handling and topical application. The PHEMA alone

hydrogels appeared as opaque sheets for both doses of gamma irradiation. The sheets

were then characterised using a number of techniques, including NIR FT-Raman

Spectroscopy, ability to swell with water, protein uptake and release, and two- and

three-dimensional cell culture biocompatibility studies.


NIR FT-Raman Spectroscopy demonstrated that a minimal dose of gamma irradiation

induced almost compete polymerisation of the HEMA monomer to its polymer form.

This was displayed by the characteristic C=C peak at 1639 cm-1 becoming reduced in

the non-irradiated samples as compared to the irradiated samples at a total dose of 5

kGy. This peak completely disappeared in the samples irradiated to a dose of 10 kGy

(Figure 3.3B). Polymerisation was observed in samples synthesised both with and

without PEG. However, for the 5 kGy samples containing PEG, it appears that there is

a small amount of residual monomer still present, as shown from the slight peak

present at 1639 cm-1 (Figure 3.3B). Therefore, it appears that the PEG interferes to a

degree with the HEMA polymerisation, but this is overcome by the increased gamma

irradiation dose as demonstrated with the 10 kGy sample.

3.3.3 Hydrogel swelling ratios in water

To further characterise the hydrogel sheets, swelling studies were performed in water.

Small squares of each hydrogel (1 cm x 1 cm) were immersed in water for a period of

time and analysed according to certain variables. The weight and volume of the

hydrogel at various time-points were compared with the original weight and volume

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Figure 3.3 Hydrogel sample analysis using NIR FT-Raman Spectroscopy. Spectra indicate intensity/ Raman shift in cm-1.(A) 1 – Unirradiated H2O:HEMA, 2 – Irradiated H2O:HEMA (5 kGy) (B) 1 – Unirradiated H2O:HEMA:PEG, 2, 3 – Irradiated H2O:HEMA:PEG (5, 10 kGy)

75.

of the hydrogel and expressed as this ratio ± SEM. Furthermore, after the initial

swelling, samples were dried and then rehydrated to gain information on the capacity

of the “washed” hydrogels to swell. The swelling ratios, as determined from the initial

swelling profile compared to the rehydration profiles, varied greatly (Figure 3.4). In

addition, the hydrogels that were polymerised without the presence of PEG showed

very different initial swelling profiles to those polymerised in the presence of PEG.

However, both groups of samples appeared quite similar in the rehydration study.

In the initial swelling study, the hydrogels polymerised in the presence of PEG

displayed a quick burst of swelling in the period up to six hours, and then decreased

down to below their initial weight and volume from approximately 48 hours onwards

(Figure 3.4A-B). In contrast, those hydrogels polymerised without the presence of

PEG displayed a continuous steady increase in swelling until the conclusion of the

experiment at 336 hours (2 weeks). The ratios were quite similar for both the 5 and 10

kGy samples in each group. Overall, there were minimal differences between

polymers PH-5 and PH-10 as shown by initial and rehydration swelling in terms of

both weight and volume (Figure 3.4). For the hydrogels polymerised in the presence

of PEG, polymer G consistently appeared to be capable of swelling with a higher ratio

than polymer C. Furthermore, the percentage of PEG released into the water compared

to the amount of PEG initially in the monomer solution was calculated to be

approximately 80% (Figure 3.5), indicating a large amount of PEG is not covalently

linked to the hydrogel.

3.3.4 HRP release from the hydrogel sheets following diffusion loading

Release studies were performed with HRP as a model protein (40 kDa) to analyse the

capacity of the hydrogel to release protein in a controlled manner, while still

maintaining protein function. In all cases, the release profiles were quite similar,

averaging between approximately 400 ng and 500 ng of enzymatically active HRP

being released over the one-week period (Figure 3.6a).

The HRP release profiles for all hydrogels correlated well with the Fickian diffusion

model (Figure 3.6b), which was described in more detail in Section 1.3.1. Briefly, in

this type of diffusion the rate of polymer chain relaxation is increased compared with

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Figure 3.4 Swelling studies of hydrogels in water. Levels are shown as a ratio of the measurement at a specific time-point compared with the initial measurement ± SEM (n=3). (A) Initial weight (B) Initial volume (C) Rehydration weight (D) Rehydration volume

77.

Figure 3.5 Gravimetric study of the loss of PEG from the HEMA:PEG hydrogels. Level is calculated from the initial swelling studies in water and is shown as the percentage of PEG released from the hydrogel compared with the amount of PEG in the monomer solution ± SEM (n=3).

78.

the rate of solute diffusion (Singh, Sharma et al. 2007). This means that as the

hydrogel swells, more protein is then able to be released from the hydrogel into the

surrounding buffer solution. All of the R2 values are approximately 1, indicating

Fickian diffusion. In the hydrogels that had been synthesised in the presence of PEG,

the cumulative amount of HRP released appeared slightly lower than that obtained

with the samples that had been synthesised without the presence of PEG (Figure 3.6).

However, the total dose of gamma irradiation did not appear to affect the amount of

HRP released over the one-week period in both the hydrogels synthesised with and

without the presence of PEG at 5 and 10 kGy. Finally, it is important to note that these

HRP measurements are based on the enzymatic conversion of a substrate to its

coloured product and, therefore, will only detect levels of functional protein.

3.3.5 Toxicity testing using a cell number assay

Primary skin keratinocytes that had been isolated from patient skin samples were used

to identify any potentially toxic effects of leached components from the hydrogel

sheets. Hydrogels were soaked in cell culture media for 3 days. This media was then

applied to cultured cells at various concentrations and cell numbers determined using

the CyQUANT® NF Cell Proliferation Assay. Toxicity of the hydrogel-conditioned

media was expressed as the relative % of the negative (SFM) control ± SEM. In this

particular case, treatments were compared to Green’s media + serum (FG), the current

“gold standard” media for culture of skin keratinocytes, that had also spent 3 days at

37 ºC/5% CO2, but in the absence of hydrogels.

There were no significant differences between the control (FG) and all three

concentrations of polymer G “conditioned” media (Figure 3.7). In contrast, and as

expected due to the lack of serum proteins that are essential for keratinocytes to

survive in culture, the highest concentration of SFM (100%) displayed significant

toxicity to the primary keratinocytes (p<0.05). Further, the highest concentration of

PH-10 “conditioned” media appeared to be the most toxic of the samples applied to

these primary skin cells, with a significant decrease in cell numbers (p<0.01). Indeed,

there appears to be a trend in the PH-10 “conditioned” media samples with a decrease

in cell number as higher concentrations of the “conditioned” media were exposed to

the cells. However, this effect is not apparent when PEG is present in the synthesis of

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Figure 3.6 Cumulative release of HRP from the hydrogel sheets into a HEPES buffer at pH 7.4. Hydrogels were first immersed in an aqueous HRP solution for 24 hours and then dried under vacuum. (A) Levels are shown as cumulative HRP released (ng) as determined by ABTS conversion to a coloured product at 410 nm ± SEM (n=3). (B) HRP diffusion is shown through plotting Mt/M∞ versus time1/2. All of the R2

values are between 0.9890 and 1.000 indicating Fickian diffusion.

80.

Figure 3.7 Toxicity testing of components leached from the hydrogels in primary skin keratinocytes using the CyQUANT® NF Cell Proliferation Assay to determine cell number. Hydrogels were immersed in FG media for 3 days and then this “conditioned” media was added at a final “conditioned” media concentration of 10, 50 and 100% respectively (shown from left to right). FG control and SFM control were fresh media samples; compared with FG and SFM replacements, which were media samples that were stored at 37 ºC/5% CO2 for the same 3-day period. Levels are shown as % of the SFM control ± SEM (n=3).

81.

the polymer G hydrogels, as shown by the minimal differences in cell numbers

observed with keratinocytes exposed to polymer G “conditioned” media as compared

to the positive (FG) control.

3.3.6 Biocompatibility testing using a three-dimensional human skin equivalent

A three-dimensional ex vivo human skin model was used to compare potential toxicity

effects from the synthesised hydrogels with a commercially available wound dressing,

namely Allevyn® (Smith & Nephew). The wound dressings were applied to the skin

models for a seven-day period, followed by histological and immunochemical

staining. As shown through the H&E staining, all treatments consist of a

haematoxylin-stained basal layer, with an eosin-stained cornified layer of relatively

the same thickness throughout (Figure 3.8). The Allevyn® treatment displayed

minimal basal cells, along with large numbers of nucleated cells in the cornified layer.

The other two hydrogel treatments also show some nucleated cells in the cornified

layer, but these appear to be present at a reduced level to the Allevyn® treatment. In

addition, the three wound dressing treatments all appear to result in the generation of a

“looser” weave in the cornified cells. However, this could potentially also be due to

artefacts from the paraffin embedding process. Artefacts can be introduced by several

common histological processes, including: the initial fixing in formalin; the presence

of methanol, formic acid, as well as the lack of control over osmolarity and pH;

mechanically induced artefacts involved in handling the tissue; and heating induced

tissue damage during the paraffin embedding process (Melo, Rosa et al. 2007).

In terms of specific skin markers, both the keratin 1/10/11 and p63 appear to be

relatively uniform in all samples, with the main difference being that the control

treatment shows more intense immunoreactivity in both cases (Figure 3.8). In terms of

the apoptotic marker, cleaved caspase-3, there are strong differences between the

Allevyn® treatment and all other samples. In the control treatment, there is no visible

immunoreactivity with apoptotic cells identified and defined by immunoreactivity to

the cleaved caspase-3 antibody, which is quite similar to the two other synthetic

hydrogel treatments. However, in the commercially available dressing Allevyn®,

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Figure 3.8 Histological and immunohistochemical analysis of an ex vivo human skin model following exposure to both a commercially available wound dressing and synthesised hydrogels. From left to right the staining or immunoreactivity is: haematoxylin and eosin (H&E), keratin 1/10/11 (K1/10/11), p63 and cleaved caspase-3 (CC-3). The arrow heads point to positively stained cells and/or regions. Scale bar = 20 µm.

83.

there is strong immunoreactivity in the cornified layer, thus revealing increased

apoptosis in cells in this layer. In terms of the specific markers tested, this is the most

marked difference between treatments (Figure 3.8).

84.

3.4 DISCUSSION

Hydrogels have been widely used for medical and tissue engineering applications as

they possess a number of advantageous properties. These include, but are not

restricted to: a high water content; good biocompatibility; patient compliance in

wound dressing applications as they are cool and comfortable for the wearer

(Schmedlen, Masters et al. 2002; Queen, Orsted et al. 2004). In addition, when

hydrogels are used as dressings they generally discourage the formation of capillary

loops in the dressing structure, i.e. the dressing is able to prevent the inward migration

of endothelial cells and subsequent division-forming capillaries, which then allows for

easy dressing changes (Queen, Orsted et al. 2004). Furthermore, as there are already a

number of non-interactive hydrogel wound dressings currently on the market, this

polymer category appears to be favourable for further wound dressing development.

In this study, synthesis of a hydrogel comprised of aqueous PHEMA has been

successfully demonstrated through gamma irradiation in the presence of PEG. Most

importantly, this dressing appears to have the ability to take up water, shows

controlled release of a model protein, and does not demonstrate toxicity effects when

assessed in both two-dimensional and three-dimensional primary skin cell culture

systems.

All four sets of hydrogel solutions effectively created crosslinked hydrogels that

possessed the ability to swell with water. In terms of appearance, the hydrogels

synthesised in the presence of PEG were both transparent and easier to manoeuvre, i.e.

would be easily applied to an uneven surface, than those synthesised as a simple

aqueous HEMA solution. Furthermore, the PHEMA alone hydrogels appeared to

show some phase separation during irradiation as they emerged opaque following

polymerisation. This could be due to the fact that the presence of PEG in the

water:HEMA solution increased the solubility of the PHEMA as it forms under

gamma irradiation. Furthermore, while the FT-Raman results suggest close to a

complete conversion, in the PHEMA alone hydrogels, it is possible that with the phase

separation some local gel particles may be created with less than 100% conversion. In

terms of the instrument, FT-Raman spectroscopy suffers from quite low sensitivity

compared with other characterisation methods (Tian, Zhang et al. 2007) and generally

cannot detect <1% impurity. In addition, while the PEG does not appear to be

85.

covalently linked to the hydrogel, it does interfere slightly with the conversion

process. However, this phenomenon was overcome when the total dose of irradiation

exceeded 10 kGy. This could primarily be due to the increase in energy required for

the HEMA polymerisation to occur when in the presence of an interfering molecule,

even though the dose does not appear to be sufficient for the PEG to react into the

polymer.

Previous studies have shown that PEG is highly biocompatible and has also been

investigated in terms of minimising biofouling (Courtois, Bystrom et al. 2006). Of

importance to this work, PEG has been demonstrated to act as a porogen in the

preparation of various porous polymers (Courtois, Bystrom et al. 2006; Xi, Wu et al.

2006). From the initial swelling studies examined here, it was apparent that the PEG

present in the gamma irradiated hydrogels C and G allowed for an initial burst of

swelling, followed by a steady decrease at approximately 48 hours to their initial

weight and volume. This can be explained by the high solubility of PEG in water, thus

the PEG started to dissolve rapidly in the first 6 hours, and thereby reduced the weight

and volume of the sheet once the hydrogels had reached their saturation point. Further

confirmation of this was obtained from the gravimetric study of PEG’s removal from

the polymer during water uptake. While it was initially hypothesised by Carenza et al.

(1993) that the PEG formed physical crosslinks between the HEMA backbone

(Carenza, Lora et al. 1993), it appears from this work, along with the proposed

gamma-induced reaction schemes (Figures 1.4-1.6) that the crosslinking occurs

through small amounts of EDGMA still present in the monomer after distillation, and

not through PEG acting as a crosslinking agent. In addition, the doses used in this

study, 5 and 10 kGy respectively, do not appear to be sufficient to create the alkoxy

radical on the end of the PEG chain. Previous studies that have used gamma

irradiation to form PEG-copolymers have ranged in total irradiation doses of 50-200

kGy (Abd Alla, Said et al. 2004). In terms of rehydration, there were minimal

differences between the PHEMA alone hydrogels. However, for the PEG-containing

hydrogels, polymer G showed a higher swelling ratio than its 5 kGy counterpart,

Polymer C. This is possibly due to a more complete polymerisation as shown by the

FT-Raman spectroscopy, thereby ensuring that no weight was lost due to possible

small percentages of unreacted HEMA monomer being soluble in the water.

86.

Both sets of hydrogels appeared to have the ability to release physiologically relevant,

i.e. similar amounts of protein as would be found in an in vivo environment, amounts

of protein, in this case HRP, over a seven-day period. Recent studies in our laboratory

have shown that growth factors present in nanogram levels are required to elicit a

beneficial wound healing response in vivo. Therefore, while many studies describe

protein release using milligram amounts (Caliceti, Veronese et al. 1992), it is

hypothesised that these supra-physiological levels of growth factors may contribute to

the down-regulation of growth factor receptors, and in turn down-regulation of

intracellular signalling – well documented outcomes of prolonged exposure to growth

factors (Rubin, Gur et al. 2005). Furthermore, HRP was used as a model protein as it

is detected using the functional conversion of a substrate, namely ABTS, to its

coloured product. Therefore, if the hydrogels themselves were to interact in a negative

manner with the protein, e.g. cause detrimental conformational changes, only the

functionally active proteins would be measured. This is extremely important in

biological applications as minor conformational changes in proteins caused by

scaffold interactions may produce major effects on the protein’s functional properties

in vivo. Furthermore, this protein release data is a good indicator that proteins are able

to access immobilised compounds on the polymer backbone.

Next, the toxicity of extracts from both sets of hydrogels was tested. As indicated

previously, the addition of PEG in the water:HEMA solution may allow for increased

solubility of the PHEMA upon synthesis. This increased solubility should also aid in

limiting phase separation between the water and HEMA, thereby increasing the

conversion from monomer to polymer and preventing monomer “pockets” from

forming. This is extremely important in terms of cell biocompatibility as the HEMA

monomer itself has demonstrated cytotoxicity in several studies, primarily through

inducing apoptosis in vitro by the differential activation of mitogen-activated protein

(MAP) kinases leading to initiation of the apoptotic cascade (Samuelsen, Dahl et al.

2007). In addition, many of the leachable components of polymers have been shown

to activate caspases and produce reactive oxygen species (ROS) – two processes that

can initiate progression into an apoptotic cascade and cause cell apoptosis and

necrosis (Samuelsen, Dahl et al. 2007). From our two-dimensional in vitro cell culture

results, it appears that the presence of PEG during aqueous PHEMA synthesis

minimises the presence of toxic leachable components. In contrast, the highest

87.

concentration of PH-10 “conditioned” media had a highly toxic effect on the

keratinocytes as shown by the strong decrease in cell numbers (p<0.01). As the PH-10

polymer is more likely to have small pockets of residual HEMA monomer, which

were undetectable by the 1% impurity limit of FT-Raman spectroscopy, leaching into

the model; and since HEMA has been associated with increasing levels of active

caspase-9-inducing apoptosis (Samuelsen, Dahl et al. 2007), this provides an

explanation for the differences between the polymer G and PH-10 treatments. Another

important point to note in this experiment is that the observed cytotoxicity can be

attributed to components leaching out from the polymers, and not the polymers

binding proteins from the media and thus depleting the media of essential

components. This conclusion can be reached since the low protein-fouling properties

of PHEMA itself is extensively documented (Martins, Wang et al. 2003). Therefore,

while the PEG itself does not polymerise into the polymer system, it is still very

important in terms of in vitro cell biocompatibility.

Three-dimensional ex vivo skin models are a useful tool for evaluating topical

products, as well as overcoming a number of ethical and practical issues associated

with animal studies (Tornier, Rosdy et al. 2006). It is important to note that this

composite does not contain inflammatory cells or a blood supply, which means that

there are some limitations in correlating this data to an in vivo system. However, 3D-

stratified layers of human epidermal cells grown in culture systems in vitro have been

shown to be useful in assessing skin irritation potential (Lawrence 1997). In this

study, the DED model showed that there were minimal differences between the

commercially available wound dressing, Allevyn®, and the two chemically

synthesised hydrogels in terms of visible structure and experimentally tested skin cell

markers. Allevyn® was selected as the commercial wound dressing as it is commonly

used in clinical practice, and is recognised as a non-interactive dressing. An

interesting point to note is that with all three wound dressing treatments, nucleated

cells became visible in the previously anuclear cornified layer. Furthermore, the

Allevyn® treatment showed a marked reduction in the number of cells in the basal

layer. These differences, compared with the control of no wound dressing treatment,

could be due to a number of causes. Primarily, the application of a topical dressing

results in culture medium being absorbed into the dressing, and therefore converts the

air-liquid interface of the 3D skin model to a liquid-liquid interface. This may then

88.

stimulate the keratinocytes to migrate faster through the cornified layers and produce a

more immature phenotype. This particular behaviour has also been described in

previous reports, where cells in a similar three-dimensional skin model did not

completely differentiate when still covered with culture medium (Ohsawa, Maruyama

et al. 1999).

In terms of skin markers, the four treatments appeared relatively similar when probed

with the skin differentiation marker keratins 1, 10 and 11 and the basal cell marker,

p63. Interestingly, there were clear differences between the treatments when the

sections where analysed in regards to their levels of apoptotic cells. Apoptosis in the

keratinocytes was analysed by staining with a marker for cleaved caspase-3 – a major

effector caspase that is specific for keratinocytes undergoing apoptosis and not just

those undergoing normal terminal differentiation (Xue, Campbell et al. 2007). The

Allevyn® treatment showed strong immunoreactivity for cleaved caspase-3

throughout the cornified layer of the skin model, thus indicating consistent apoptosis

in the cells in this stratified layer. This was not apparent in the other three treatments,

with only minimal immunoreactivity present in the skin models with the two

synthesised hydrogels. In the polymer G treated three-dimensional ex vivo skin model

there was some immunoreactivity in the outermost section of the cornified layer, and

with the PH-10 treatment there was minimal staining below the basal layer.

Concerning Allevyn®, Shuster et al. (2001) reported induction of a CD-95-dependent

apoptosis pathway in human T cells following exposure to polyurethane based

biomaterials (Schuster, Ankersmit et al. 2001). As Allevyn® is composed of a

polyurethane foam core, it appears likely that this material, or a leachable by-product

of its processing, is responsible for the apoptosis seen in this skin model. This is a

particularly interesting finding, as this product is regularly used in a clinical setting

without apparent adverse effects. This may indicate that the HSE model used in this

study is more sensitive to subtle variations that may not be visible in an in vivo clinical

situation.

89.

3.5 CONCLUSION

From the results reported above, these hydrogels, in particular polymer G, appear

promising for potential wound dressing applications. This is due to a number of

experimentally tested properties, including: the ability to swell with water; uptake and

controlled release of a biologically active protein; primary keratinocyte compatibility

in a two-dimensional in vitro cell culture system; and, biocompatibility in a three-

dimensional ex vivo skin model. Importantly, polymer G appears to demonstrate

similar, if not improved, properties to that of the commercially available wound

dressing, Allevyn®, which was tested in this study. In conclusion, this hydrogel will

be further explored in Chapter 4 with the possibility of improving it from a passive

polymer simply placed on the wound bed to that of an interactive dressing that is able

to aid in modulating the chronic wound environment such that it is conducive to

healing.

90.

CHAPTER 4

SYNTHESIS AND EVALUATION OF

FUNCTIONALISED-ALENDRONATE HYDROGELS

FOR TREATMENT OF EXCESSIVE PROTEASE

ACTIVITY IN CHRONIC WOUND FLUID

92.

4.1 INTRODUCTION

Many current topical treatments for chronic ulcers have been designed to rectify the

decreased levels of growth-factors in these wounds, e.g. Regranex® (Johnson &

Johnson), but have only been minimally effective (Cullen, Watt et al. 2002). Many

authors have postulated that this is due to the excessively high levels of proteolytic

activity found in CWF (Tarnuzzer and Schultz 1996; Stadelmann, Digenis et al. 1998;

Trengove, Stacey et al. 1999; Cullen, Watt et al. 2002), as described in Chapter 2. As

outlined in the literature review, there are a number of specific synthetic inhibitors of

MMP, namely tetracyclines and their chemically modified derivatives, e.g.

doxycycline (Ramamurthy, Kucine et al. 1998; Ramamurthy, McClain et al. 1999;

Yager and Nwomeh 1999); hydroxamic acids, e.g. GM6001 (Lund, Romer et al.

1999); and bisphosphonates, e.g. clodronate (Valleala, Hanemaaijer et al. 2003).

However, recent reports have suggested that the MMPs present in the wound bed may

also be involved in other biological processes important to wound healing, such as

growth factor activation and immune system regulation, rather than simply degrading

the wound bed matrix and growth factors (Levi, Fridman et al. 1996; Suzuki, Raab et

al. 1997; McQuibban, Butler et al. 2001; Gearing, Thorpe et al. 2002). Therefore,

while many researchers have suggested that the addition of a protease inhibitor prior

to topical treatment of the wound with growth factors would promote healing

(Tarnuzzer and Schultz 1996; Wysocki, Kusakabe et al. 1999; Yager and Nwomeh

1999; Cullen, Smith et al. 2002), other MMP-interactions in the wound healing

process also need to be considered (Somerville, Oblander et al. 2003). The goal of this

approach, which is significantly different from other proteases-inhibitory strategies

proposed by others, is to inactivate the proteases in the wound fluid after the fluid is

absorbed away from the actual wound bed. In this situation, the proteases required for

healing-associated functions within the upper cellular layers of the wound bed remain

available.

The bisphosphonates are particularly appealing for use in chronic ulcers as they have

been used clinically for a number of years to treat MMP-related disorders.

Furthermore, they exhibit low toxicity and are therefore generally well-tolerated

(Teronen, Heikkila et al. 1999). Bisphosphonates are small molecules, characterised

93.

by two C—P bonds (Heymann, Ory et al. 2004). They are also more specifically

referred to as geminal bisphosphonates when the two bonds are found on the same

carbon atom (Fleisch 1997). Quite similar to endogenous pyrophosphates,

bisphosphonates replace the O—P with a C—P, thereby allowing two additional

functional groups (Heymann, Ory et al. 2004), as well as creating a hydrolysis-

resistant P—C—P bond (Vasikaran 2001) (Figure 1.2). In terms of MMP-inhibition,

they are capable of binding to divalent metal ions, e.g. Zn2+, Ca2+ (Heikkila, Teronen

et al. 2002), through a three-dimensional structure that allows the coordination of one

oxygen from the phosphate group with the cation (Heymann, Ory et al. 2004). This

affinity for divalent cations can be increased even further if one of the functional

groups is either a hydroxyl (OH) or a primary amine (NH2), as this then facilitates the

formation of a tridentate conformation with the cation (Heymann, Ory et al. 2004). In

addition, the nitrogen atom in the side chain must be in a particular spatial

arrangement, as well as being a critical distance from the cation, to be the most potent

(Russell and Rogers 1999). Therefore, there has been a lot of work aimed at designing

new and improved bisphosphonates in recent years, with current numbers being

synthesised in the hundreds (Russell and Rogers 1999).

As mentioned previously, bisphosphonates have been shown to inhibit MMP-1, -2, -3,

-8, -9, -12, -13 and -20 at both therapeutically obtainable and, most importantly, non-

cytotoxic concentrations (Heikkila, Teronen et al. 2002). Current clinical applications

generally include the management of calcium and bone metabolism disorders, e.g.

osteoporosis, Paget’s disease, hypercalcaemia and metastatic cancer (Vasikaran 2001).

For a number of years now, these diseases have been effectively treated with

bisphosphonates. Indeed, Fosamax® (sodium alendronate, Merck & Co Inc.) has

recently been listed on the Australian Pharmaceutical Benefits Scheme (Australian

Government 2007). Therefore, it appears that for future indications, including those

primarily caused by significant soft tissue destruction, e.g. chronic ulcers,

bisphosphonates may be a promising treatment (Teronen, Heikkila et al. 1999). In

terms of chronic ulcer treatments, the delivery of specific bisphosphonates, or indeed

simply the presentation of this particular chemical to the site of excessive protease

activity, has to be carefully contemplated.

94.

Clinically, bisphosphonates can be classified into at least two groups with different

modes of action. Firstly, there are those that closely resemble pyrophosphate, e.g.

clodronate, which can be metabolically incorporated into non-hydrolysable analogues

of ATP, thereby altering the structure and potentially inhibiting ATP-dependent

intracellular enzymes. Next, there are the nitrogen-containing bisphosphonates, e.g.

alendronate, which are able to interfere with the mevalonate pathway, thus prevent the

synthesis of isoprenoid compounds, which can then lead to cellular apoptosis (Russell

and Rogers 1999). In addition, bisphosphonates also have the ability to chelate

divalent cations, which can also inhibit MMP activity by removing the zinc ions

required for catalytic activity and the calcium ions required for protein stability

(Boissier, Ferreras et al. 2000; Heikkila, Teronen et al. 2002; Neville-Webbe, Holen et

al. 2002). In this particular application, it is the bisphosphonate’s chelating ability that

is thought to decrease MMP activity in CWF.

Bisphosphonates have previously been modified for clinical delivery, especially in

terms of bone-specific applications (Uludag and Yang 2002; Wang, Miller et al. 2003;

Balas, Manzano et al. 2006). In addition, alendronate is a prime candidate

bisphosphonate for functionalisation as it contains a primary amine – a convenient

chemical bond that can be easily manipulated for conjugation into a polymer using

already-established methods (Wang, Miller et al. 2003). Through a nucleophilic acyl

substitution reaction, an amine, present in the alendronate sodium salt form, can be

reacted with an acid halide (Otera 1993) to produce an alendronate tethered to an

unsaturated carbon group. This alkene bond then allows for further polymerisation

into an established hydrogel system, such as that reported in Chapter 3. The

experiments in this chapter focus on the details surrounding this preliminary

alendronate condensation reaction, and then describe how this methacrylated-

alendronate can be copolymerised into the PHEMA/PEG hydrogels described in

Chapter 3. Furthermore, this bioactive wound dressing is tested for efficacy against

protease activity in CWF, and is assessed in further cell-based assays to confirm

biocompatibility with a human ex vivo skin model. Therefore, the specific aim of the

experiments in this chapter were to analyse whether a functionalised alendronate

hydrogel can be successfully synthesised, and in turn whether it can still inhibit MMPs

in CWF.

95.


4.2.1 Chemicals

2-Hydroxyethyl methacrylate (HEMA, assay ≥ 99% GC, Sigma-Aldrich, St Louis,

MO, USA) and methacryloyl chloride (Sigma-Aldrich) were distilled under reduced

pressure immediately prior to use. Polyethylene glycol 20,000 (PEG, Sigma-Aldrich),

alendronate sodium salt (Merck, San Diego, CA, USA) and 4-methoxy phenol

(Sigma-Aldrich) were used as supplied under the manufacturers’ instructions. All H2O

used was double deionised by ion exchange (MilliQ, Millipore, Billerica, MA, USA).

4.2.2 Functionalisation of alendronate

In a round-bottom flask, alendronate sodium salt (300 mg, 0.92 mmol) was dissolved

in aqueous NaOH (148 mg, 3.70 mmol in 7.4 mL H2O) with 4-methoxy phenol to

inhibit polymerisation. The solution was cooled in an ice-salt bath then freshly

distilled methacryloyl chloride (120 mg, 1.15 mmol) and NaOH (111 mg, 2.768 mmol

in 5.5 mL H2O) was added step-wise, making sure that the pH remained above 11.

The reaction was stirred vigorously for 20 hours, then acidified with HCl to pH 7. The

resulting product was evaporated to dryness using a rotary evaporator and extracted

with chloroform (CHCl3) to remove any impurities. The water layer was then

precipitated with N,N-Dimethylformamide [DMF or (CH3)2NC(O)H] to obtain

methacrylated-alendronate.

4.2.3 Characterisation of methacrylated-alendronate through Fourier

Transform Nuclear Magnetic Resonance (FT-NMR) spectroscopy

1H and 31P spectra were recorded using a 400 MHz NMR Spectrometer (Bruker,

Ettlingen, Germany) at room temperature in deuterated water (D2O). Spectra were

analysed using MestReC Version 4.9.9.6 software (Mestrelab Research, Santiago de

Compostela, Spain).

96.

4.2.4 Analysis of MMP-inhibition through incubation with the methacrylated-

alendronate

A pooled sample of CWF was run on Collagen Type I zymograms as described

previously in Section 3.2.2, with the following exception. After the electrophoresis

had been performed, the zymograms were washed in triton X-100. Next, alendronate

(2 mM) and methacrylated-alendronate (2 mM of the equivalent bisphosphonate

segment of methacrylated-alendronate) were included separately in the zymogram

incubation buffer and the zymograms were incubated at 37 ºC for 24 hours. Collagen

Type I zymograms were then analysed as described previously in Section 2.2.3.

4.2.5 Synthesis of hydrogels

Aqueous solutions of distilled HEMA were prepared as previously described (50%

water, 30% PEG and 20% HEMA) with a vehicle control (no methacrylated-

alendronate) and two concentrations of methacrylated-alendronate (2 mM and 20 mM

of the equivalent bisphosphonate segment of methacrylated-alendronate in the

monomer solution). Solutions were then placed between two glass plates separated by

a silicone gasket and purged with argon. Each mould was approximately 75 mm x 50

mm x 3 mm and filled with 10 mL of monomer solution. The moulds were then

gamma-irradiated in a Gamma-cell 220 (Atomic Energy of Canada Ltd, Ottawa,

Canada) using a Co60 source at a rate of 3.25 kGy/h to give a total dose of 10 kGy

(Table 4.1). Hydrogels were removed from their moulds and cut into 1 cm x 1 cm

square pieces for further analysis.

Sample ID H2O (g) HEMA (g) PEG 20,000

(g)

Methacrylated-

alendronate (mg)

G 5.0 2.0 3.0 -

GA1X 5.0 2.0 3.0 7.68

GA10X 5.0 2.0 3.0 76.80

Table 4.1 Functionalised hydrogel formulations prepared for analysis as potential wound dressings


97.

Irradiated samples were analysed by a Perkin Elmer System 2000 NIR FT-Raman

spectrophotometer (Perkin Elmer, Waltham, MA, USA). The hydrogel samples were

placed into glass vials for spectroscopic analysis and the spectra were analysed using

Grams/AI (Thermo Electron Corporation, Waltham, MA, USA).

4.2.7 Analysis of MMP-inhibition through incubation with the alendronate-

functionalised hydrogels

Similar to that described in Section 3.2.2, a pooled sample of CWF was analysed

through Collagen Type I zymography with the following alteration. Alendronate-

functionalised hydrogels (as prepared in Section 4.2.5) were cut into small pieces and

included in the incubation buffer, along with a blank and a non-functionalised

hydrogel to act as a vehicle control, and then incubated at 37 ºC for 24 hours.

Collagen Type I zymograms were then analysed as described previously in Section

3.2.3.

4.2.8 Biocompatibility testing of hydrogels using the DED model

The DED model was prepared as described previously in Section 2.2.10 – 2.2.11. At

five days post air-liquid interface culture, the alendronate-functionalised hydrogels

were placed on top of the composite, along with “no treatment” and “non-

functionalised” hydrogels as controls. These wound dressings were exposed to the

composite for 7 days at 37 ºC/5% CO2, with the media being replaced at 3 days. At

completion of the treatment, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide conversion (MTT) assay (Denizot and Lang 1986) was used to analyse one of

each treatment group. Briefly, the DED was submerged in 6 mL of 0.5 mg/mL MTT

dye (Sigma-Aldrich) and incubated at 37 ºC for 2 hours. Following incubation, the

metabolically active cells could be visualised by a purple colour and the DED were

photographed. Next, both the MTT-stained and un-stained samples were fixed and

paraffin-embedded using standard protocols for haematoxylin and eosin (H&E)

histological analysis.

4.2.9 Immunohistochemistry

98.

As described previously in Section 2.2.12, paraffin sections were cut and

deparaffinised in ethanol and xylene. Sections were probed separately for keratin

1/10/11 (K1/10/11), a marker for cornification and squamous cell differentiation; p63

(RDI Research Diagnostics, Concord, MA, USA), a p63 analogue that identifies

normal basal cells as opposed to malignant tumours; and cleaved caspase-3 (Cell

Signaling Technology, Danvers, MA, USA), a critical mediator of apoptosis in

mammalian cells. For slides that were probed for cleaved caspase-3, antigen

unmasking was required before the blocking step. Briefly, the slides were incubated at

37 ºC in 10 mM citrate buffer (pH 3.0) for 30 min, before proceeding as normal. After

incubation with primary antibodies for K1/10/11 (1:400), p63 (1:100) and cleaved

caspase-3 (1:100), sections were stained using a Dako Envision kit (Dako Denmark

A/S, Glostrup, Denmark) as per the manufacturer’s instructions, with the exception

that phosphate buffered saline (PBS) was used instead of Tris-buffered saline (TBS).

After antibody development, all sections were counterstained with haematoxylin and

analysed using light microscopy.

99.

4.3 RESULTS

4.3.1 Synthesis and characterisation of methacrylated-alendronate

Following the identification of excessive MMP levels in CWF as described in Chapter

3, potential MMP inhibitors were considered for incorporation into a wound dressing.

The bisphosphonate alendronate was chosen as it has been used for a number of years

in clinical practice (Vasikaran 2001) and possessed an easily modified primary amine.

This was important as the approach to be followed differed significantly from

previous approaches in that it was proposed to inactivate the proteases in the wound

fluid after the fluid is absorbed away from the actual wound bed. This strategy was

chosen so that proteases required for functions within the upper cellular layers of the

wound bed remain available. In view of this, the alendronate had to be modified to

allow eventual polymerisation into a hydrogel system. The primary amine in

alendronate appeared a logical place to perform the modification, and was reacted

with methacryloyl chloride to form the amide along with a pendant alkene bond

(Figure 4.1A). This unsaturated carbon then allowed for further polymerisation

(Figure 4.1A) into the already established aqueous PHEMA:PEG hydrogel system

outlined in Chapter 3.

Following the reaction between alendronate and methacryloyl chloride, the resulting

product required structural analysis. The two 1H NMR spectra reveal the original

alendronate, along with its methacrylated counterpart, which also contains some

contaminants from the synthesis protocol (Figure 4.2). The peaks have been assigned

and further details of the spectrum are outlined in Table 4.2. The four equivalent

protons on the phenol group of the 4-methoxy phenol used in the reaction as a

polymerisation inhibitor appear as a single peak at 7.84 ppm. In addition, the spectrum

reveals that there some reaction impurities, namely, methacrylic acid and

poly(methacrylic acid), which are still present even after multiple chloroform washes

and DMF precipitation. Two separate 31P spectra of the methacrylated products, along

with the original alendronate, are shown in Figure 4.3 to demonstrate the differences

following reaction work up and medium term storage. Again, the peaks have been

assigned and further details of the spectrum are outlined in Table 4.3. In Figure 4.3C a

second triplet is seen upfield of the first, which can be assigned to a dimer of the

100.

(A)

O

NH PO

OH OH

OHP

O OHONa

O

Cl NH2PO

OH OH

OHP

O OHONa . 3 H2O

NaOHIce-salt bathStir vigorously20 h

+ HCl . 3 H2O

+

(B)

O

O OH

O

NH PO

OH OH

OHP

O OHONa O

O

O

O OH

O

NH

OOH

O OH

O

OOH

P

PO OH

ONa

OOH OH

OHgamma-irradiation

+

Figure 4.1 Reaction schemes. (A) Condensation reaction of methacryloyl chloride and alendronate sodium salt. (B) Gamma induced polymerisation of HEMA and methacrylated-alendronate.

101.

(A)

(B)

(C)

Figure 4.2 FT-NMR 1H spectrum of methacrylated-alendronate. (A) Labelled schematic of the methacrylated-alendronate. (B) 1H spectrum of alendronate. (C) 1H spectrum of methacrylated-alendronate.

102.

Peak ppm Multiplicity, coupling constant (Hz)

Norm. Int Identity

1 7.84 s 0.15 CH on 4-methoxy phenol

c 5.60 m, * 1.00 Alkenyl H-trans to CH3 on product

5.562

5.56m, * 0.83

Alkenyl H-trans to CH3 on methacrylic acid

b 5.34 m, * 1.01 Alkenyl H-cis to CH3

on product

3 5.25 m, * 0.88 Alkenyl H-cis to CH3

on methacrylic acid

# 4.70 s 18.04 Water 3.213.20d,e 3.18

t, 6.8 2.01 CH2 on product adjacent to N

4,5 2.93 s 0.73 CH2 on polymer

4,5 2.77 s 0.56 CH2 on polymer 1.901.881.871.841.781.771.761.751.74

a 6

f,g h,i

1.73

m, * 10.41

Various CH3 on product (a), methacrylic acid, and other polymer forms

CH2 on product

* Coupling constants were unresolved

Table 4.2 Features of methacrylated-alendronate FT-NMR 1H spectrum. Columns are from left to right: peak number; shift (ppm); multiplicity (s, d, t, m) and coupling constant (Hz); normalised integral; and identity.

103.

(A)

(B)

(C)

(D)

Figure 4.3 FT-NMR 31P spectrum of methacrylated-alendronate. (A) Labelled schematic of the methacrylated-alendronate. (B) 31P spectrum of alendronate (C) 31P spectrum of product (D) 31P spectrum of worked up product with longer sample storage

104.

NH2PO

OH OH

OHP

O OHO

NH2P O

OHOH

OHP

OOHO

Figure 4.4 Potential structure of methacrylated-alendronate dimer

Peak ppm Multiplicity,

coupling constant (Hz)

Norm. Int Identity

21.5121.43a,b

21.35

t, 12.7 1.00 P on methacrylated- alendronate, split by CH2

21.1621.091 21.01

t, 11.8 0.35 P on methacrylated-alendronate dimer (Figure 4.4), split by CH2

Table 4.3 Features of methacrylated-alendronate FT-NMR 31P spectrum. Columns are from left to right: peak number; shift (ppm); multiplicity (s, d, t, m) and coupling constant (Hz); normalised integral; and identity.

105.

original methacrylated-alendronate formed through P—O—P bonds of the

bisphosphonate groups (Figure 4.4), and not the starting product as shown by the shift

upfield. This becomes more apparent after the work-up of the reaction, teamed with

longer term storage of the product (Figure 4.3B-C).

4.3.2 Analysis of MMP-inhibitory action of methacrylated-alendronate

To analyse the methacrylated-alendronate, an activity assay was used to compare its

MMP-inhibitory activity to that of the original alendronate. Through incubation of

CWF with increasing concentrations of either the original or the methacrylated-

alendronate, the proteolytic activity was revealed by the use of Collagen Type I

zymography (Figure 4.5A-B). This demonstrated that both forms of the alendronate

are able to inhibit CWF samples 1-6 to varying degrees. Densitometry was used to

quantify the reduction in proteolytic activity revealed by zymography and are

represented graphically in Figure 4.5C. Quantitatively, both inhibitors were able to

decrease the amount of Collagen Type I degradation, as compared to the untreated

CWF samples (p<0.01). From this functional assay, it appears that the addition of the

methacrylate group still allows for inhibition of MMPs at a physiological temperature

over 24 hours, although at a reduced level of function.

4.3.3 Preparation and characterisation of alendronate-functionalised hydrogels

Following successful inhibition of MMPs in CWF with the methacrylated-

alendronate, further polymerisation was required to form the functionalised wound

dressing. Three sets of hydrogels were successfully synthesised according to the

conditions outlined earlier (Table 4.1, Figure 4.1B). NIR FT-Raman Spectroscopy

demonstrated that a total dose of 10 kGy of gamma irradiation induced almost

compete polymerisation of the methacrylated-alendronate and HEMA monomer to its

copolymer form (Figure 4.6). This was evident by the absence of a characteristic C=C

band at 1639 cm-1 in the irradiated samples when compared with the monomer

mixture as described in Figure 3.1B. The hydrogel sheets were then characterised

using both a functional protease assay, along with exposure to a three-dimensional ex

vivo skin model to determine biocompatibility.

106.

(A) (C)

(B)

Figure 4.5 Collagen Type I zymography demonstrating inhibition of protease activity present in wound fluid samples by alendronate sodium salt and its methacrylated-counterpart. (A) Lanes 1-6 are CWF samples 1-6 (500 ng); Lanes 7-12 are CWF samples 1-6 (500 ng) and 2 mM of alendronate present in the incubation buffer at 37 ºC for 24 hours. (B) Lanes 1-6 are CWF samples 1-6 (500 ng); Lanes 7-12 are CWF samples 1-6 (500 ng) and 2 mM of methacrylated-alendronate present in the incubation buffer at 37 ºC for 24 hours. (C) Relative levels of protease activity in pooled CWF samples treated with respective inhibitors. The MMP-specific inhibition of collagen degrading activity was represented quantitatively through densitometric analysis. Levels are shown as the % collagen degrading activity as compared to the untreated samples, ± SEM (n=3). Statistical significance is relative to the untreated samples and shown as # (p<0.01) as determined by Tukey’s test.

107.

Figure 4.6 Analysis of alendronate-functionalised hydrogels using NIR FT-Raman Spectroscopy. Spectra indicate intensity/ Raman shift in cm-1. (A) Unirradiated H2O:HEMA:PEG (B) GA1X Irradiated H2O:HEMA:PEG:1X methacrylated-alendronate (10 kGy) (C) GA10X Irradiated H2O:HEMA:PEG:10X methacrylated-alendronate (10 kGy)

108.

4.3.4 Analysis of MMP-inhibitory action of alendronate-functionalised hydrogels

Following the demonstrated MMP-inhibitory activity of the methacrylated-

alendronate, this molecule was then co-polymerised into a hydrogel-based support to

examine whether the tethered form had similar properties. The alendronate-

functionalised hydrogels were analysed using Type I Collagen zymography to

determine if the tethered alendronate still demonstrated MMP-inhibitory action. The

hydrogels were cut into small pieces and incubated with the zymograms containing

the CWF, along with an untreated control. Collagen Type I zymography of the pooled

CWF samples showed a large amount of protease activity in the untreated control

(Figure 4.7A). However, when the hydrogel pieces were present, all three treatments

showed a visible decrease in the amount of Collagen Type I degradation (Figure

4.7A). When the treatments were analysed using densitometry to quantitate the

relative decrease in protease activity, the GA10X hydrogel, i.e. the hydrogel

containing 20 mM of methacrylated-alendronate, displayed the ability to significantly

reduce Collagen Type I degradation as compared with the non-hydrogel treated

control (p<0.01) (Figure 4.7B). These results suggest the tethered alendronate is still

able to inhibit MMPs as shown through a functional assay.

4.3.5 Biocompatibility testing using a three-dimensional human skin equivalent

A three-dimensional de-epidermised skin model was used to assess potential toxicity

effects from the alendronate-functionalised hydrogels as compared to the vehicle

hydrogel. The various hydrogels were applied to the stratified skin models for a seven-

day period, with histological and immunochemical staining carried out upon

completion of the experiment. Treatments included the vehicle control of hydrogel G,

along with the two alendronate-functionalised hydrogels, GA1X and GA10X.

Histological analysis showed a haematoxylin-stained basal layer, with an eosin-

stained cornified layer of relatively the same thickness throughout for all treatments

(Figure 4.8). For the GA10X hydrogel there appears to be minimal basal cells, along

with increased numbers of nucleated cells in the cornified layer. Hydrogel G and

GA1X also show some nucleated cells in the cornified layer, however, they do not

appear to be as numerous. When the sections were probed for specific skin markers,

keratin 1/10/11 and p63, and the apoptotic marker, cleaved caspase-3,

109.

(A) (B)

Figure 4.7 Collagen Type I zymography demonstrating inhibition of protease activity present in wound fluid samples by alendronate-functionalised hydrogels.(A) Lane 1 is the pooled CWF sample with no treatment in the incubation buffer. Lanes 2-4 are the same pooled CWF sample with polymer G, GA1X and GA10X respectively cut into small pieces and present in the incubation buffer at 37 ºC for 24 hours. (B) Relative levels of protease activity in pooled CWF samples treated with respective polymers. The MMP-specific inhibition of collagen degrading activity was represented quantitatively through densitometric analysis. Levels are shown as the % collagen degrading activity as compared to the control sample, ± SEM (n=3). Statistical significance is relative to the control sample and shown as # (p<0.01) as determined by Tukey’s test.

110.

Figure 4.8 Histological and immunohistochemical analysis of an ex vivo human skin model following exposure to the alendronate-functionalised hydrogel. From left to right the staining is: haematoxylin and eosin (H&E), keratin 1/10/11 (K1/10/11), p63 and cleaved caspase-3 (CC-3). The scale bar measures 20 µm.

111.

hydrogel revealed a minimal amount of p63 immunoreactive basal cells, confirming

the histological analysis. In the control and hydrogel G treatments, there is no visible

immunoreactivity of apoptotic cells, with minimal cell surface marker identification in

its functionalised counterparts, GA1X and GA10X. This indicates that the synthesised

hydrogels appear promising for potential wound dressing treatments, especially when

compared with the commercial wound dressing tested in Chapter 3 (Figure 3.8).

112.

4.4 DISCUSSION

From the results reported in Chapter 2, MMP-9 was identified as the predominant

protease present in CWF. Hence it is likely to have a key role in degrading the

extracellular matrix in the chronic wound environment. In addition, it appears that

high levels of MMP-9 activity in CWF are a stronger indicator of a wound's chronicity

than the total protease activity levels alone. Hence, it is not surprising that current

topical treatments of chronic ulcers with bioactives, i.e. growth factors, have proven to

be only slightly effective in treating this condition (Cullen, Watt et al. 2002). In view

of this, a specific MMP inhibitor, namely the bisphosphonate alendronate, was chosen

as a clinical treatment to aid in neutralising the aggressive proteolytic chronic wound

environment; the ultimate goal of this strategy being to modulate the ulcer towards a

healing state. The bisphosphonate family, which exhibits low toxicity and has been

well tolerated for several years of human use, seems to be the ideal candidate for

MMP-related diseases (Teronen, Heikkila et al. 1999). The wound treatment explored

in this study examined the use of bisphosphonates to inhibit MMP activity in CWF.

Importantly, the approach described differs significantly from previous approaches in

that the proteases are inactivated in the wound fluid, after the fluid is absorbed away

from the actual wound bed, so that proteases required for functions within the upper

cellular layers of the wound bed remain available.

Alendronate was chosen as the MMP-inhibitor for inclusion into the hydrogel,

primarily due to the fact that it contains both amine and hydroxyl functional groups,

which together can enhance the bisphosphonate’s affinity for divalent cations

(Heymann, Ory et al. 2004). However, it first has to be modified to allow further

polymerisation into the synthetic wound dressing. Because alendronate is insoluble in

common organic solvents, a two phase reaction called the Shotten-Baumann reaction

was chosen to react an acid chloride with the amine of alendronate. This reaction is

well characterised and allows high yields of amines, mainly due to the fact that they

are able to successfully compete with other reactive species present (Kemp and

Vellaccis 1980). Briefly, this is a two phase reaction with the acid chloride suspended

in the water phase containing the amine compound. At the boundary of the two

phases, the amine reacts with the acid chloride, thereby generating the desired

product. The acid (HCl) generated from the reaction of the acid chloride must be

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neutralised by a base (in this case, NaOH) in order to prevent the conversion of the

amine to the non-nucleophillic ammonium salt. Unfortunately, one of the unavoidable

by-products of the Schotten Baumann reaction is the hydrolysis of the acid chloride to

generate the corresponding acid, in this case, methacrylic acid. Typically, this can be

removed during work-up. However, due to the small scale of the reaction, not all of

this impurity could be removed in the reaction reported here. In addition, the 31P

spectra showed an additional phosphorus-containing species in the resulting product,

possibly due to the dimerisation of two functionalised alendronate molecules through

the –OH group on the phosphate. Similar dimerisation has been shown with a similar

phosphate-containing molecule, i.e. ethylene glycol methacrylate phosphate (MOEP)

(Suzuki, Whittaker et al. 2007). Following the reaction to modify alendronate, the

resulting product was then copolymerised into the PHEMA hydrogel system

previously outlined in Chapter 3. FT-Raman spectroscopy revealed a complete

polymerisation, thereby ensuring that no functionalised alendronate could be released

from the hydrogel.

Critically, it is demonstrated that the alendronate tethered to a hydrogel, along with its

methacrylated-counterpart, were able to significantly decrease the levels of Type I

Collagen degradation in CWF as compared to the untreated control (p<0.01).

Interestingly, the vehicle hydrogel, i.e. the hydrogel without the methacrylated-

alendronate, was also able to inhibit MMP activity as revealed through the Collagen

Type I zymography, but to a lesser degree than the most concentrated alendronate-

functionalised hydrogel, GA10X. A possible explanation for this is the chemical

composition of PHEMA itself, as it contains 3 oxygen atoms in its unit structure that

are all available for chelation (Zainuddin, Hill et al. 2006). Further evidence for this

hypothesis is that Ca2+ itself has a strong tendency to chelate to oxygen atoms (Levine

and Williams 1982). Therefore, through the use of a “self-chelating” hydrogel support,

the vehicle treatment (hydrogel G) has the ability to chelate the cations required by

MMPs for their stability and catalytic activity, e.g. Ca2+, Zn2+. This chelating ability is

then disrupted by copolymerisation with the methacrylated-alendronate at the lowest

concentration (hydrogel GA1X).

Zainuddin et al. (2006) previously postulated that by reducing the number of available

oxygen atoms, the chelating mechanism is thereby reduced, and may then prevent the

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chelation of metal ions with PHEMA. Furthermore, Chirila et al. (2007) report

massive calcification of PHEMA, both in vitro in simulated body fluid and in vivo as

subcutaneous implants (Chirila, Zainuddin et al. 2007). However, when the highest

concentration of methacrylated-alendronate is copolymerised into the system

(GA10X), the chelating ability of alendronate alone is stronger than the PHEMA

support and overcomes the less MMP-inhibitory response seen previously. Therefore,

taken together, these results indicate that the alendronate is still available to inhibit the

MMPs in the CWF, while not being released into the wound bed. This is a significant

advance over topically applied inhibitors as it allows MMPs to remain active in the

wound bed where they perform vital roles in growth factor activation and immune

system regulation (Levi, Fridman et al. 1996; Suzuki, Raab et al. 1997; McQuibban,

Butler et al. 2001; Gearing, Thorpe et al. 2002), yet at the same time the unwanted

MMPs, those in the CWF, are absorbed into the hydrogel where they are inactivated.

Furthermore, in this chapter, the experiments with the DED model showed that there

were minimal differences between the three treatments, i.e. the vehicle and two

alendronate-functionalised hydrogels, in terms of impact on visible structure and

effect on a range of skin cell surface expression markers. An interesting point to note

is that with both wound dressing treatments, nucleated cells became visible in the

previously anuclear cornified layer. As discussed previously, this is due to the

application of a topical dressing inducing an increased amount of culture medium into

the dressing, and therefore converting the air-liquid interface to a liquid-liquid

interface. Indeed, this may well underlie the well accepted evidence that a moist

wound environment is essential for optimal wound healing (Winter 1962). In terms of

skin cell surface expression markers, the three treatments appeared relatively similar

when probed with the skin differentiation marker keratins 1, 10 and 11 and the basal

cell marker, p63. The main difference observed between the three treatments being the

minimal immunoreactivity seen with p63 in the GA10X hydrogel. This is likely to be

alleviated by first washing the alendronate-functionalised hydrogels, thereby

minimising any potential “stickiness” of the phosphonate polymer component.

Apoptosis in the keratinocytes was also analysed by immunoprobing for cleaved

caspase-3 – a major effector caspase that is specific for keratinocytes undergoing

apoptosis and not just those undergoing terminal differentiation (Xue, Campbell et al.

2007). None of the three treatments showed obvious levels of immunoreactivity,

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which appears promising for future clinical treatments, especially in terms of

developing MMP-inhibiting, CWF-absorbing functionalised hydrogels. In the future,

efficacy of the hydrogel wound dressing in promoting wound healing will be assessed

using a HSE model that has artificially created wounds, e.g. punch biopsies, and that

exhibits impaired healing in the presence of exogenous CWF. This would provide

further information to confirm both the safety and efficacy of the dressings before

proceeding to an initial human clinical trial.

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4.5 CONCLUSION

Taken together, the results reported herein suggest this novel alendronate-

functionalised hydrogel holds promise as a dressing treatment for chronic wounds.

This is due to its ability to inhibit the excessive levels of MMPs in CWF, as well as its

biocompatibility when exposed to a human ex vivo skin model. Of importance to the

development of this wound dressing was the ability to attach a functional group to the

alendronate sodium salt, which then allowed polymerisation into the already-

established hydrogel system. This was achieved through a Shotten-Baumann reaction,

which permitted high yields of the amide in a single-step reaction. Furthermore, it has

been demonstrated that the MMPs in CWF can be inhibited by the original

bisphosphonate alendronate, a methacrylated-analogue, and in a tethered state, i.e.

attached to a hydrogel support. This last point is critical to the development of a

successful chronic ulcer wound dressing as it inhibits the MMPs in CWF, while still

allowing those on the wound bed itself to perform their essential functions in wound

healing, namely, activation of growth-promoting agents and immune system

modulation.

CHAPTER 5

GENERAL DISCUSSION

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Chronic ulcers are a very controversial topic in terms of both pathophysiology and

optimal treatment practices. Considering the significant burden that they place on

many healthcare systems around the world, it is surprising that more is not known

about this costly medical challenge. Current prevalence rates of chronic wounds in

Australia are estimated to be between 13-3% of the population/year and cost the

Australian community upwards of AU$500 million per annum to treat (Baker and

Stacey 1994; Gruen, Chang et al. 1996). These wounds are a major cause of grief and

concern for affected individuals and also contribute significantly to their overall

diminished quality of life (MacLellan 2000). However, at the commencement of the

studies described herein, very little research was occurring in Australia to investigate

new treatments, or even at a more general level, few groups were interested in

identifying potential causes of these ulcers themselves.

As a result of this situation, the aim of this thesis was to identify potential proteolytic

markers of chronic ulcers and, from this information, design a bioactive wound

dressing to modulate these ulcers towards a healing state. This thesis focussed on

proteases as their excessive levels have been identified by a number of studies as

being the main difference between chronic and acute wounds (Tarnuzzer and Schultz

1996; Ladwig, Robson et al. 2002; Li and Li 2003). Therefore, this dissertation

involved the synthesis and development of a novel hydrogel wound dressing that

would interact with the chronic wound environment to promote healing.

Characterisation of this hydrogel employed numerous techniques including, FT-

Raman spectroscopy, swelling studies, protein uptake and release, along with cell

based assays in both two- and three-dimensional environments. The main objective

was to also identify specific protease inhibitors that may be applied to chronic wounds

in order to attenuate excessive proteolytic activity and restore normal healing. As

reported herein, it has been ascertained that MMP-9 is indeed the most abundant

protease activity in non-healing wounds, revealed through Collagen Type I

zymography, immunoprecipitation and a direct ELISA. In addition, after identifying

bisphosphonates, namely alendronate, as MMP inhibitors, an alendronate-containing

functionalised wound dressing was created that holds significant promise as a novel

strategy to modulate and enhance the healing of chronic wounds. Therefore, this thesis

contains a number of novel results that confirm the hypotheses that were outlined in

Chapter 1.

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Chronic wound healing is an extremely complex issue due to the lack of knowledge

surrounding the cascade of events leading to a wound’s nonhealing status. Venous

hypertension itself has been indicated to lead to capillary bed distension, resulting in

the leakage of large molecules, namely fibrinogen, from the blood into the dermis

(Falanga 1993). This can then lead to the formation of fibrin cuffs in the surrounding

dermis, which in turn cause ulceration due to the impaired delivery of oxygen and

other nutrients to the surrounding tissue (Falanga 1993). In addition, more recent

studies have shown that the in vivo capillary bed distension is extremely complicated,

with some fibrin cuffs being shown to contain highly organised ECM molecules.

These are also often found in other inflammatory diseases, e.g. ulcers of the oral

cavity (Chen and Rogers 2007). Therefore, while this thesis focuses on proteolytic

causes of an ulcer’s chronicity, it is important to note that there are many processes

involved in chronic wounds and proteases are simply one of the major contributors.

That being said, chronic wounds are commonly characterised by elevated levels of

pro-inflammatory cytokines and proteases (Chen, Schultz et al. 1999). These wounds

also contain reduced levels of TIMPs (Chen, Schultz et al. 1999; Baker and Leaper

2000). The majority of previous studies involved with analysing the chronic wound

environment use CWF as their clinical sample, due to the difficulties in obtaining a

wound bed biopsy. However, the one study that did use cells isolated from a wound

bed biopsy, instead of the fluid itself, reported significantly different results. Cook et

al. (2000) reported that significantly decreased levels of active MMPs, along with

marked increases in expression of TIMPs, led to the chronicity of non-healing wounds

(Cook, Stephens et al. 2000). This is in direct contrast with all other previously

reported findings, and indeed with the results reported in Chapter 2. Therefore, while

these differences were taken into account, for the study reported herein, CWF was

chosen for exploration as a possible indicator of the chronic wound environment. This

was due to the goal of this thesis project being to explore non-invasive diagnostic

approaches and also, develop potential future wound dressings that could modulate

this proteolytic environment.

There is much controversy surrounding MMP activity in CWF. A number of studies

suggest that elevated MMP-2 and -9 levels are detrimental to the wound healing

process (Wysocki, Kusakabe et al. 1999; Ladwig, Robson et al. 2002), while others

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recommend that they are essential for normal wound healing to occur (Cook, Stephens

et al. 2000). In particular, Ladwig et al. (2002) reported that levels of activated MMP-

9 tended to decrease as healing proceeded (Ladwig, Robson et al. 2002). Furthermore,

Wysocki et al. (1999) demonstrated that expression of MMP-9 in CWF was 32-fold

higher than in blood-derived serum, and activity was also increased in early collection

times (Wysocki, Kusakabe et al. 1999). As most members of the MMP family are

structured into three essential, characteristic and highly conserved domains (Massova,

Kotra et al. 1998), it is often quite difficult to differentiate MMP species based on

molecular weight alone. In addition, antibodies that are based on specific MMPs often

find it difficult to differentiate between closely related species, as there is a very close

evolutionary relationship within the family further reflected by their highly conserved

domains (Somerville, Oblander et al. 2003). Indeed, this is likely to be the main

reason for the controversy surrounding levels of specific MMPs in chronic wounds.

In view of these previously conflicting results, the aim of Chapter 2 was to use

multiple techniques to identify specific MMPs species in CWF across a number of

samples. Initially, total protease levels were analysed using both Collagen Type I and

Collagen Type IV zymography. All CWF samples revealed increased levels of

collagen-degrading activity compared to that found in both HS and AWF samples. In

addition, this activity could be attenuated through the use of a MMP-specific inhibitor,

namely GM6001. Having obtained these results, specific MMPs were

immunoprecipitated from the wound fluid samples and then analysed using a

functional assay to minimise any non-specific immunoreactivity. MMP-9 was

subsequently identified as the major protease responsible for collagen degradation by

CWF. Next, the clinical status of the ulcer was considered; with the CWF samples

separated into both higher and lower PUSH score groups. Through

immunoprecipitation, CWF samples in the lower and higher PUSH score groups were

indistinguishable, as both groups showed a significant increase in MMP-9 activity

compared with HS and AWF samples (p<0.01).

To discern if there were potential differences in clinical groupings, a direct ELISA

was used with a different MMP-9 antibody to determine quantitative levels of MMP-9

in these samples. This technique revealed clear difference between the levels of

MMP-9 present in the two separate PUSH score groupings. The lower PUSH score

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(PUSH ≤11) group displayed significantly higher levels of MMP-9 than AWF

(p<0.01), as well as significantly lower levels of MMP-9 than the higher PUSH score

(PUSH ≥12) group (p<0.01). As a result, it appears that there is a strong correlation

between increased MMP-9 levels in CWF from wounds in the higher PUSH score

(PUSH ≥12) group compared to AWF samples (p<0.01). Therefore, high levels of

MMP-9 activity in CWF may be a good indicator of a wound's chronicity.

A further characteristic of chronic wounds is reduced expression of growth-factors

(Stadelmann, Digenis et al. 1998). Many current topical treatments for chronic ulcers

attempt to rectify the decreased levels of growth-factors, but are only minimally

effective (Cullen, Watt et al. 2002). Evidence from previous reports, combined with

data from Chapter 2, indicates that this is due to the excessive levels of protease

activity, i.e. MMP-9, found in CWF (Tarnuzzer and Schultz 1996; Stadelmann,

Digenis et al. 1998; Trengove, Stacey et al. 1999; Cullen, Watt et al. 2002). There are

a number of specific synthetic inhibitors of MMP, namely tetracyclines and their

chemically modified derivatives (Ramamurthy, Kucine et al. 1998; Ramamurthy,

McClain et al. 1999; Yager and Nwomeh 1999); hydroxamic acids (Lund, Romer et

al. 1999); and bisphosphonates (Valleala, Hanemaaijer et al. 2003). The

bisphosphonates were chosen for potential use in modulating the wound environment

as they have been used in clinical practice for a number of years in conditions such as

Paget’s disease and hypercalcaemia, both of which are linked to excessive MMP

activity (Vasikaran 2001). Due to their low toxicity, bisphosphonates are generally

well-tolerated (Teronen, Heikkila et al. 1999), and have been shown to inhibit various

MMPs at therapeutically obtainable and non-cytotoxic concentrations (Heikkila,

Teronen et al. 2002).

The second aim investigated in this project was that a synthetic hydrogel, in particular

one created using PHEMA, represented an ideal wound dressing for chronic ulcers.

This hypothesis was supported by the findings in Chapter 3 revealing that an aqueous

PHEMA hydrogel synthesised in the presence of PEG was able to demonstrate

similar, if not improved, properties compared with a commercially available wound

dressing in both two- and three-dimensional cell culture systems. Furthermore, this

synthetic hydrogel showed complete polymerisation at a total gamma irradiative dose

of 10 kGy, displayed the ability to swell with water, and, could take up and release

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physiologically relevant amounts of a biologically active protein. Interestingly, initial

reports from Carenza et al. (1993) proved to be inaccurate in their claims that PEG

acted as a crosslinker between the PHEMA backbone chains when synthesised using

gamma irradiation (Carenza, Lora et al. 1993). Instead, it appears that the crosslinked

hydrogels are formed from small amounts of residual EDGMA present in the HEMA

monomer. However, at higher total doses of irradiation, e.g. 100 kGy, there is the

potential for the alkoxy radical to form on the end of the PEG molecule to allow

copolymerisation. Indeed, Abd Alla et al. (2004) were able to form copolymers of

PVA and PEG with doses ranging up to 200 kGy (Abd Alla, Said et al. 2004).

A three-dimensional ex vivo skin equivalent model was also used to evaluate the

synthetic hydrogels against a readily available commercial wound dressing. An

interesting point to note is that with all three wound dressing treatments, nucleated

cells became visible in the previously anuclear cornified layer. A potential explanation

for this is that the application of a topical dressing converts the air-liquid interface of

the three-dimensional skin model to a liquid-liquid interface caused by culture

medium being absorbed through the dermis and into the wound dressing. This has

previously been shown by others in a similar model in which the cells did not

completely differentiate when the model remained covered with culture medium

(Ohsawa, Maruyama et al. 1999).

Commercially, there are several hydrogel wound dressings on the market. Some

examples of these include: IntraSite™ gel (Smith & Nephew, Hull, UK); Nu-Gel®

(Johnson & Johnson, New Brunswick, NJ, USA); Curasol® (Healthpoint, Fort Worth,

TX, USA); and Tegaderm™ (3M, St Paul, MN, USA). Hydrogels are often selected as

an ideal wound dressing as they are quite cooling on the skin and do not stick to the

wound itself – factors that aid in minimising pain to the patient (Queen, Orsted et al.

2004; Akita, Akino et al. 2006). Therefore, it appears that hydrogels address the

important issues of patient compliance, pain management and the individual’s quality

of life (Queen, Orsted et al. 2004).

More recently, First Water (Wiltshire, UK) has developed a novel range of hydrogels

to overcome initial performance issues with hydrogels in a clinical setting (First-

Water 2004). In terms of chronic leg ulcers, their product, Hydroform Cool, aims to

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have finely tuneable properties to allow optimal water transport properties (First-

Water 2004). This means that they are able to create a favourable chronic wound

environment by maintaining a fine balance between absorbing the wound exudate, and

donating moisture to ensure the wound bed itself does not dry out and delay wound

healing. When considering this together with the results reported in Chapter 2, the

application of a topical hydrogel may even stimulate the keratinocytes to migrate

faster through the cornified layers. Indeed, this may well underlie the well accepted

evidence that a moist wound environment is essential for optimal wound healing

(Winter 1962) and explain why hydrogels appear beneficial as wound dressings.

First Water have also developed a proprietary hydrogel technology that claims to

provide antimicrobial properties against a range of microbes, namely, Candida

albicans, Aspergillus niger, Pseudomonas aeruginosa, Staphylococcus aureus and

Escherichia coli (Sparrow 2005). Furthermore, they assert that their patented

technology, i.e. a copolymer of 2-acrylamido-2-methyl- propanesulphonic acid

sodium salt (NaAMPS) and acrylic acid (3-sulphopropyl)ester potassium salt (SPA)

allows for improved overall patient outcomes. This is mainly due to their claims that it

is able to selectively adhere to the patient’s skin, i.e. it is able stick to the wound’s

peri-skin, but not to the wound bed itself (Munro and Yasin 2002). Such advances in

patient-focussed wound dressing treatments have not only enhanced wound dressing

compliance, but have also improved the patient’s overall quality of life.

More sophisticated ulcer therapies include the recently developed active wound

management treatments, e.g. growth factors (Regranex, Johnson & Johnson) and

dermal substitutes (Dermagraft, Smith & Nephew). However, the major obstacle for

patients obtaining effective growth factor treatments is price – Regranex® costs

upwards of US$375/15 g tube (Ugarte, Roberts et al. 2002), which considering the

daily treatment regime, can equate to thousands of dollars per wound. Moreover, even

though the cost of these treatments is extremely prohibitive, they have only proven to

be slightly effective in treating chronic wounds since high levels of proteases in CWF

can rapidly degrade these topically-applied growth-promoting agents (Cullen, Watt et

al. 2002). Therefore, the toxic chronic wound environment has to be analysed further

before designing an effective and affordable interactive dressing to modulate the

chronic wound environment to that of a healing ulcer.

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In view of previous reports, as well as the evidence displayed in Chapter 2, a specific

MMP inhibitor, namely the bisphosphonate alendronate, was chosen as a treatment to

aid in neutralising the aggressive proteolytic chronic wound environment. Of note, the

wound treatment explored in the study described herein differs significantly from

previous approaches in that the goal was to inactivate proteases in the wound fluid,

after the fluid is absorbed away from the actual wound bed, so that proteases required

for functions within the upper cellular layers of the wound bed remain available.

Indeed, it is demonstrated in Chapter 4 that alendronate tethered to a hydrogel, along

with its methacrylated counterpart, were able to significantly decrease the levels of

Type I Collagen degrading activity in CWF as compared to the untreated control

(p<0.01). This indicates that the alendronate is still available to inhibit the MMPs in

the CWF, while not being released into the wound bed. This approach represents a

significant advance over topically applied inhibitors as it allows MMPs to remain

active in the wound bed where they perform vital roles such as activation of select

growth promoting factors and in immune system regulation (Levi, Fridman et al.

1996; Suzuki, Raab et al. 1997; McQuibban, Butler et al. 2001; Gearing, Thorpe et al.

2002), yet inactivates the abundant proteases in the wound fluid.

Pharmacologically, bisphosphonates act in a number of ways to inhibit MMP activity.

When used in vivo as an injectable or through oral administration, MMPs are able to

interact with two separate metabolic pathways depending on their structure (Russell

and Rogers 1999). Nitrogen-containing bisphosphonates, e.g. alendronate, are able to

inhibit enzymes of the mevalonate pathway, which can then affect further downstream

processing events, e.g. post-translational modifications of small GTPases (Russell and

Rogers 1999). Mevalonate is a precursor for isoprenoid intermediates and therefore is

responsible for a number of end-products, namely, cholesterol, farnesylated proteins

and geranylgeranylated proteins. Alterations in the end products, e.g. mutations in

GTP that make it resistance to hydrolysis by GTPases, can then constitutively activate

both the PI3K/AKT and Raf/MEK/MAPK/ERK pathways and cause disruption in

both apoptosis and cell cycle progression, both of which are commonly seen in a

variety of cancers (Swanson and Hohl 2006). In terms of this particular application,

due to the fact that the bisphosphonate is not intended to be released into the wound

bed, the main mode of action for MMP-inhibition will be through cation chelation. As

mentioned previously, MMPs are zinc-dependent endopeptidases, which also contain

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a number of calcium ions for stability (Parks 1999). Bisphosphonates are able to bind

to divalent metal ions, e.g. Zn2+, Ca2+ (Heikkila, Teronen et al. 2002), through the two

phosphate groups to form a three-dimensional structure between one oxygen from the

phosphate group and the cation itself (Heymann, Ory et al. 2004). Furthermore,

previous studies have also shown that the addition of high levels of either Zn2+or Ca2+

to in vitro cell culture systems can reduce the inhibitory activity of bisphosphonates

towards MMPs (Boissier, Ferreras et al. 2000; Heikkila, Teronen et al. 2002; Neville-

Webbe, Holen et al. 2002). Moreover, when bisphosphonates are administered orally,

taking EDTA, a cation chelator, at the same time can increase bisphosphonate

absorption (Vasikaran 2001).

As mentioned previously, one of the major obstacles to the average patient receiving

the optimal level of wound care is cost (Eldor, Raz et al. 2004). Even though many

studies have shown that these advanced treatments are cost-effective in the longer-

term (Kantor and Margolis 2001), governments are still slow to subsidise these

treatment regimes. Therefore, for a wound dressing to be effective in the individual

patient, as well as the community, its development has to be focussed on minimising

expensive costs to the user. The wound dressing outlined in this thesis has paid close

attention to this important issue. Alendronate itself has been used for a number of

years in clinical settings to attenuate MMP activity (Russell and Rogers 1999;

Teronen, Heikkila et al. 1999; Vasikaran 2001), as well as recently being added to the

Australian Pharmaceutical Benefits Scheme (Australian Government 2007) in the

form of its sodium salt, Fosamax® (Merck & Co, Inc, Whitehouse Station, NJ, USA).

Furthermore, this compound reaches the end of its life of patent protection in 2008,

which will then encourage an increased number of generic products. This recent

development will ensure that price and availability of this compound is within the

reach of the ordinary chronic ulcer sufferer. In addition, PHEMA itself has been used

for specific biomedical applications in the field of contact lenses for over forty years

(Alvarez-Lorenzo, Yanez et al. 2006), thus this hydrogel support is also widely

available and cost-effective. Furthermore, as this is a completely synthetic wound

dressing, it minimises transport and storage issues associated with biological

compounds, which are also responsible for increased consumer prices.

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It is clear that the studies outlined in this thesis have led to a range of novel findings in

the fields of chronic wound healing and wound dressing development. However, it has

also raised many issues that need further clarification. Most importantly, there is a

need to further investigate the chronic wound environment in its entirety, i.e. in both

the chronic wound fluid and the wound bed. This is needed as while many studies

focus on CWF as an indicator of the wound’s clinical status (Ladwig, Robson et al.

2002), little is known about the actual wound bed environment. The main reason for

this is that ulcer biopsies are extremely invasive, as they require the creation of a new

wound in an already non-healing ulcer. Therefore, care needs to be taken to optimise

the information generated from these smaller patient studies. Furthermore, it has to be

noted that both of these samples, i.e. CWF and wound bed biopsies, are likely to be

extremely heterogeneous diagnostic samples. Clearly, multiple patient samples need

to be analysed to gain statistically significant results. In addition, improved models

need to be developed – to allow initial studies to be performed in in vitro or ex vivo

environments, while still providing relevant information that can be correlated to

human in vivo situations, e.g. a three-dimensional ex vivo skin model with impaired

wound healing properties. In terms of proteases specifically, further investigation is

required into the other MMPs present in CWF samples. In Chapter 3, MMP-9 has

been identified as the predominant protease in CWF and has also been shown to

provide a good indicator of the wound’s clinical status. However, other MMPs are still

present in these depleted samples. Therefore, complete MMP-9 depletion of the

samples through a series of immunoprecipitation steps could reveal less abundant

MMPs, and these may well provide improved biomarkers of ulcer prognosis.

Once the complete protease profile of chronic wounds has been investigated, further

development of the wound dressing itself is required. Hence, future studies will

investigate the reaction between methacryloyl chloride and alendronate in more detail,

along with examining other potential chemical linkages between a synthetic hydrogel

and the chosen bisphosphonate. In terms of future in vitro work, these functionalised

alendronate hydrogels will be analysed with a particular focus on potential extracts, in

a similar manner to the initial primary keratinocyte toxicity testing. For the purpose of

this study, they were only examined in a three-dimensional ex vivo skin model as this

a more representative of their intended future human biomedical application.

Furthermore, longer-term stability testing of this chemical bond needs to be performed

127.

in a simulated wound environment to ensure that no alendronate will be released into

the wound bed. This point is critical as the only wound bed-based patient samples that

have been analysed thus far revealed that there were decreased levels of MMPs in the

wound bed compared with the CWF (Cook, Stephens et al. 2000). Therefore, the

release of MMP inhibitors in the wound bed could potentially impede the wound

healing process even further. In addition, recent research conducted by others in our

Program has been directed at linking the alendronate to a synthetic hydrogel through

custom-made PEG molecules. This approach may provide better flexibility and an

enhanced method for controlling the location of the tethered alendronate within the

dressing, thereby improving its potential clinical applications.

The results outlined in this thesis, which examined the relationship between the

clinical status of an ulcer and its resultant wound fluid, along with improved wound

dressing developments, have provided further insight into the chronic wound healing

environment and also provided the stimulus for further research within the Tissue

Repair and Regeneration Program at QUT. Of most significance, an Australian

Provisional Patent (2007903101) was recently filed by Tissue Therapies Limited on

behalf of QUT to protect the invention of a tethered bisphosphonate wound dressing

to promote chronic ulcer healing. Moreover, along with the IHBI ECR grant

mentioned previously, work has also been undertaken in a joint Australian Research

Council (ARC) Linkage project with the University of Queensland and Tissue

Therapies Limited to further investigate the concept of a bioactive wound dressing

that can deliver growth factors and, at the same time, inhibit protease activity in CWF.

Furthermore, Prof Graeme George is also co-supervising a more recent PhD student,

Jason Yeh, who is investigating the use of different molecular weight PEG molecules

to act as porogens in PHEMA hydrogels synthesised through gamma irradiation. The

main differences in this project is the deliberate addition of EDGMA to act as a

crosslinker, which then allows for further investigation into the crosslinking itself.

It is interesting to note that there are indeed potential other applications for this

technology. For example, there are many situations where excessive protease activity

may be detrimental to medical implants and biomaterials, a common case being

aseptic prosthesis loosening in total hip replacements (Kido, Pap et al. 2007). For this

particular application, a protease inhibitor that is tethered to the implant or biomaterial

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itself may provide improved performance of the material and minimise implant

failures by attenuating local proteolytic activity. Alternatively, an MMP-inhibitor

tethered to non-absorbable surgical sutures could also provide patients with an

improved healing outcome. In this situation, by inhibiting MMPs around the sutures

the overall wound breaking strength may potentially be improved, thereby reducing

the time for the wound to heal completely. In fact, Witte et al. (1998) previously

performed a similar MMP-inhibitor study on Sprague-Dawley rats and showed that

mechanical strength of the surgical incision was improved with inhibition of MMPs,

while not causing an increase in collagen deposition (Witte, Thornton et al. 1998).

From this information, it is clear that there are many other potential biomedical

applications for this technology.

In summary, the in vitro results reported in this thesis suggest that this novel

functionalised hydrogel holds promise as a dressing treatment for chronic wounds. As

demonstrated herein, MMPs are the major class of proteases responsible for the

degradation of collagen in the wound bed. Furthermore, this activity can be largely

attributed to the high levels of MMP-9 present in CWF. In addition, levels of MMP-9

correlate with a wound’s clinical status. Importantly, MMP-9 can be inhibited with the

bisphosphonate alendronate, in the form of a sodium salt, a methacrylated analogue,

and in a tethered state. This last point is critical in terms of development of an optimal

wound dressing treatment that can inhibit MMPs in the wound fluid, but will still

allow MMPs to perform biological functions important to healing in the wound bed

itself, such as activation of growth promoting factors. In conclusion, the emphasis on

minimising potential adverse side-effects by tethering the MMP inhibitor, rather than

releasing, or topically applying, the inhibitor to the wound bed, allows for an

improved, more device-orientated wound dressing treatment for these chronic ulcers.

CHAPTER 6

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