Investigation of the biointerfaces of nanostructured surfaces · 2017. 3. 1. · surfaces...

Investigation of the biointerfaces

of nanostructured surfaces

Submitted in total fulfilment of the requirements for the degree of

Doctor of Philosophy

by

Thi Hong Vy Pham

Department of Chemistry and Biotechnology

Faculty of Science Engineering and Technology

Swinburne University of Technology

2016

ii

Abstract

Recent developments in nanotechnology have opened a new era for

nanostructured materials due to their unique physical chemical and biological

properties The surface of certain nanostructured materials can be manipulated to

impose certain metabolic activities onto cells coming in contact with these

substrates Implantable materials with a particular surface micro- andor

nanostructure often promote human cell attachment and tissue integration however

these structures can also stimulate the attachment of pathogenic bacteria which may

come in contact with the substrate prior to or during surgical processes If

biomaterial surfaces become infected with pathogenic bacteria it is likely that the

implantation of such surface will result in an infection requiring the removal of the

device and treatment of the infection With the increase in the use of medical

implants an in-depth investigation into the events taking place at the interface when

nanostructured materials come into contact with biological systems is of

considerable importance

This project investigated the surface properties of different nanostructured

surfaces derived from titanium graphene and black silicon and their effects to

different types of cells The nano-smooth titanium surfaces were fabricated by using

an equal channel angular pressing technique Two bacterial strains namely

Staphylococcus aureus and Pseudomonas aeruginosa exhibited different attachment

affinities towards these substrates It was found that Gram-positive S aureus

attachment was not restricted on surfaces that possessed an average roughness less

than 05 nm In contrast P aeruginosa cells were found to be unable to colonise

surfaces possessing an average roughness below 1 nm unless sharp nanoprotrusions

of approximately 20 nm in diameter were present It is postulated that the attachment

of P aeruginosa cells onto surfaces possessing these nanoprotrusions was facilitated

by the ability of the flexible cell membrane to stretch over the tips of the

nanoprotrusions

Two types of graphene films containing variable edge lengths and different

angles of orientation between the graphene sheets were fabricated It was found that

these graphene surfaces exhibited substantial bactericidal activity towards S aureus

and P aeruginosa bacteria The density of the edges was found to be one of the most

iii

important parameters contributing to the antibacterial behaviour of the graphene

nanosheet films Both experimental and computational simulation results have

proved that the graphene nanosheets triggered the formation of pores in the bacterial

cell walls resulting in a subsequent imbalance in the osmotic pressure causing cell

death

The surface of nanostructured black silicon was pre-infected with live

pathogenic bacteria allowed to equilibrate then inoculated with eukaryotic cells to

determine whether the bacterial cells would adversely affect the growth of the

eukaryotic cells It was found that the fibroblasts were able to successfully compete

with the bacteria for growth over the surface with no signs of infection being

evident after seven days The eukaryotic cells were able to grow over the

pathogenic bacteria which were mechanically ruptured by the action of the surface

nanopillars present on the black silicon causing cell death It was also

demonstrated that the black silicon surface promoted the attachment and

proliferation of human fibroblast epithelial and osteoblast cells In addition an in-

vivo analysis performed in mouse trials demonstrated that the topology of the black

silicon did not trigger severe inflammatory responses When applied to

erythrocytes however these surfaces proved to be highly active causing the

autogenous lysis of the cells coming into contact with the surface The

biocompatibility and a lack of an inflammatory response of the black silicon

together its ability to eliminate bacterial contamination without the need for

antimicrobial agents suggests that this surface topography would make an

excellent model for the design of biomaterial surfaces particularly those used for

the fabrication of medical implants

iv

Acknowledgement

I would like to express my sincere gratitude to my principal supervisor

Professor Elena P Ivanova for her inspiration in scientific research since I started

my Bachelor degree followed by her continuous guidance support and

encouragement throughout this project I am grateful to have been part of her

research team and to have been trained by wonderful and talented people that

motivated me to become a better researcher Similarly I would like to give my

deepest thanks to Professor Russell J Crawford for his insightful and educational

suggestions on the academic style of writing Dr Shannon Notley and Professor

Pauline P Doran for co-supervising this project and for all of their inspirational ideas

that contributed to the structure of my project I have also gained useful experience

in designing experiments and writing scientific papers from Professor David

Mainwaring Dr Vi Khanh Truong Dr Mohammad Al Kobaisi and Dr Wendy

Zeng Without their expertise this project would not have been able to be completed

and Irsquom very thankful for their participation

To my family both in Vietnam and in Australia there are not enough words

for me to say how much you all mean to me To my parents I have not yet been a

good daughter despite your endless sacrifice emotional support and encouragement

throughout all my ups and downs in Australia and in my PhD To my brother thanks

for always being there for me during my darkest time and for putting up with the

lsquosufferingrsquo I caused all those days To my grandma who always thinks about me and

prays for me thanks for always reminding me to become a good person and a good

family member no matter who I am out there To my boyfriend thanks for helping

me to overcome a most difficult time in both my emotional and professional life for

keeping me on track so that I could make it to this achievement To the rest of my

relatives thanks for countless wonderful memories of Tết for giving me so much

advice in coping with this foreign world for sending me so many beautiful gifts and

home foods which significantly lessened my homesickness when I was studying in

Australia

v

To all my friends here in Swinburne and Australia especially chi Nga Do

Matthew Quinn Simon Grossemy anh Hiep Pham (chi) Dr Song Ha Nguyen Dr

Hayden Webb Chris Bhadra Dr Jafar Hasan Jaimys Arnott Vanya and all others

you have made my research and my daily life more enjoyable with many laughs fun

quotes and stories BBQs parties secrets gossip advice and scientific suggestions

(some of which have now been published in scientific journals) My thanks go

especially to Matt and Simon My PhD experience has been greater with you guys

around

I would like to thank Dr Vladimir Baulin Dr Saulius Juodkazis and

Professor Yuri Estrin for their collaboration in computational modelling black

silicon fabrication and titanium preparation respectively A special thank you to

Chris Bhadra for her contribution in preparing the black silicon samples as well as

Matthew Quinn who prepared the graphene films used in this study Thank you to

Dr James Wang for his assistance in performing SEM experiments Thank you to

Dr Alex Fulcher for his expertise in imaging live cells using the confocal

microscope at Monash Microimaging (MMI) facilities Monash University

For technical assistance I would like to give special thanks to chu Ngan

Chris Key Chris Anthony Soula Rebecca Katharine Adcroft Savithri and Angela

for helping me with multiple tasks during the course of my research I have learned

valuable technical strategies from them that can seldom be found in textbooks or

manuals

Lastly I would like to give my sincere gratitude to Professor David

Mainwaring for the opportunity to become a part time research assistant for a project

in CRC Polymers since completing my PhD laboratory work This position has not

only provided my financial support but also extended my original expertise in cell

biology to organic chemistry and given me a chance to work with industry

representative within the academic environment I also would like to thank Dr

Pandiyan Murugaraj who is a senior Postdoctoral Fellow for CRC Polymers for his

assistance he has guided me through this challenging work with patience and care

vi

Declaration

I Vy TH Pham declare that this thesis is original work and contains no material

that has been accepted for the award of Doctor of Philosophy or any other degree or

diploma except where due reference is made

I declare that to the best of my knowledge this thesis contains no material previously

published or written by any other person except where due reference is made I

warrant that I have obtained where necessary permission from the copyright owners

to use any third party copyright material reproduced in the thesis or to use any of my

own published work in which the copyright is held by another party

Signature

________________________________________________________________

vii

List of Publications

Publication arising from this thesis

Book chapters

1 Vy T H Pham Chris M Bhadra Vi Khanh Truong Russell J Crawford

Elena P Ivanova (2015) Design antibacterial surfaces for biomedical implant in

Antibacterial Surfaces Springer ISBN 9783319185934 pp 89-111

2 Hayden K Webb Chris M Bhadra Vy T H Pham Russell J Crawford Elena

P Ivanova (2014) The design of superhydrophobic surfaces in

Superhydrophobic surfaces Elsevier ISBN 9780128013311 pp 27-44

Peer-reviewed articles

1 Vy T H Pham Vi Khanh Truong Ronald Unger Shahram Ghanaati Mike

Barbeck Patrick Booms Alex Fulcher Chris M Bhadra Vladimir Baulin C

James Kirkpatrick David E Mainwaring Saulius Juodkazis Russell J

Crawford Elena P Ivanova (2016) ldquoRace for the surfacerdquo eukaryotic cells can

win ACS Applied Materials amp Interfaces vol 8 no 34 pp 22025-22031

2 Vy T H Pham Vi Khanh Truong Matthew DJ Quinn Shannon M Notley

Yachong Guo Vladimir Baulin Mohammed A Kobaisi Russell J

Crawford Elena P Ivanova (2015) Graphene induces formation of pores that

kill spherical and rod-shaped bacteria ACS Nano vol 9 no 8 pp 8458-8467

3 Vi Khanh Truong Vy T H Pham Alexander Medvedev Rimma Lapovok

Yuri Estrin Terry C Lowe Vladimir Baulin Veselin Boshkovikj Christopher J

Fluke Russell J Crawford Elena P Ivanova (2015) Self-organised

nanoarchitecture of titanium surfaces influences the attachment of

Staphylococcus aureus and Pseudomonas aeruginosa bacteria Applied of

Microbiology and Biotechnology vol 99 no 16 pp 6831-6840

4 Vy T H Pham Vi Khanh Truong David Mainwaring Yachong Guo Vladimir

A Baulin Mohammed A Kobaisi Gediminas Gervinskas Saulius Juodkazis

Wendy R Zeng Pauline P Doran Russell J Crawford Elena P Ivanova (2014)

viii

Nanotopography as a trigger for the microscale autogenous and passive lysis of

erythrocytes Journal of Materials Chemistry B vol 2 no 19 pp 2819-2826

Conference and poster presentation with published abstract

1 Vy T H Pham Vi Khanh Truong Alex Fulcher Chris M Bhadra David E

Mainwaring Saulius Juodkazis Russell J Crawford Elena P Ivanova (2015)

ldquoIn-vitro interactions of eukaryotic cells with the complex nanopillar geometry

of antibacterial surfacesrdquo 5th International Symposium of Surface and Interface

of Biomaterials amp 24th Annual Conference of the Australasian Society for

Biomaterials and Tissue Engineering 2015

2 Vi Khanh Truong Vy TH Pham Alexander Medvedev Hoi Pang Ng Rimma

Lapovok Yuri Estrin Veselin Boshkovikj Christopher J Fluke Russell J

Crawford Elena P Ivanova (2014) ldquoSelf-organization of nanoscale architecture

of titanium surfaces influencing Staphylococcus aureus and Pseudomonas

aeruginosardquo Australian Society of Microbiology 2014

Other publications

1 Duy H K Nguyen Vy T H Pham Mohammad Al Kobaisi Chris M Bhadra

Anna Orlowska Shahram Ghanaati Berardo Manzi Vladimir Baulin Saulius

Juodkazis Peter Kingshott Russell J Crawford Elena P Ivanova (2016)

Adsorption of human plasma proteins onto nanostructured black Silicon

surfaces Langmuir vol 32 no 41 pp 10744ndash10751

2 The Hong Phong Nguyen Vy T H Pham Song Ha Nguyen Vladimir Baulin

Rodney J Croft Brian Phillips Russell J Crawford Elena P Ivanova (2016)

The bioeffects resulting from prokaryotic cells and yeast being exposed to an 18

GHz electromagnetic field PLoS ONE vol 11 no 7

3 Chris M Bhadra Vi Khanh Truong Vy T H Pham Mohammad Al Kobaisi

Gerdiminas Seniutinas James Y Wang Saulius S Juodkazis Russell J

Crawford Elena P Ivanova (2015) Antibacterial titanium nano-patterned arrays

inspired by dragonfly wings Scientific Reports vol 5 p 16817

ix

4 Veselin Boshkovikj Hayden K Webb Vy T H Pham Christopher J Fluke

Russell J Crawford Elena P Ivanova (2014) Three dimensional reconstruction

of surface nanoarchitecture from two-dimensional datasets AMB Express vol

4 no 1 p 3

5 Kun Mediaswanti Cuie Wen Elena P Ivanova Francois Malherbe Christopher

C Berndt Vy T H Pham James Wang (2014) Biomimetic creation of surfaces

on porous titanium for biomedical applications Advanced Materials Research

vol 896 pp 259-262

6 Kun Mediaswanti Cuie Wen Elena P Ivanova Christopher C Berndt Vy T H

Pham Francois Malherbe James Wang (2014) Investigation of bacterial

attachment on hydroxyapatite ndashcoated titanium and tantalum International

Journal of Surface Science and Engineering vol 8 no 2-3 pp 255-263

7 Kun Mediaswanti Cuie Wen Elena P Ivanova Christopher C Berndt Francois

Malherbe Vy T H Pham James Wang (2013) A review on bioactive porous

metallic biomaterials Biomimetics Biomaterials and Tissue Engineering vol

18 no 1

x

Table of Contents Abstract ii

Acknowledgement iv

Declaration vi

List of Publications vii

List of Abbreviations xv

List of Figures xvii

List of Tables xxvii

1 Chapter 1

Introduction 1

11 Overview 2

12 Aims and objectives 3

6 Chapter 2

Literature review 6

21 Overview 7

22 Nanostructured surfaces ndash the new future 8

221 Nanostructured surfaces and biological applications 8

222 Concerns regarding nano-cytotoxicity 17

223 Selected nanostructured surfaces for this studied 22

2231 Ultrafine grain titanium 22

2232 Graphene film 25

2233 Black silicon 28

23 Bacterial interactions with nanostructured surfaces 30

231 Bacterial colonisation 31

2311 Mechanisms responsible for bacterial colonisation 31

2312 Impacts of bacterial infection 34

232 Current approaches in preventing bacterial infections 36

xi

2321 Antifouling surfaces 37

2322 Chemically bactericidal surfaces 39

2323 New approach mechanically bactericidal surfaces 41

24 Mammalian cell interactions with nanostructured surfaces 44

241 Cell attachment spreading and migration 45

242 Cell proliferation 49

243 Cell differentiation 50

25 Competitive colonisation of bacteria and mammalian cells for the ldquorace for

the surfacerdquo 52

251 Race for the surface 52

252 Current investigations 53

56 Chapter 3

Materials and methods 56

31 Overview 57

32 Fabrication of nanostructured surfaces 57

321 ECAP modified titanium 57

322 Graphene films 58

323 Black Silicon preparation 59

33 Characterization of nanostructured surfaces 59

331 Surface crystallinity 59

332 Surface elemental composition 60

3321 X-ray photoelectron spectroscopy 60

3322 Raman spectroscopy 61

3323 Energy dispersive x-ray spectroscopy 61

333 Surface hydrophobicitywettability 61

334 Surface morphology 62

335 Surface topography 62

xii

3351 Optical profilometry 62

3352 Atomic force microscopy 63

34 Preparation of biological samples 65

341 Culturing of bacterial cells 65

342 Preparation of red blood cells 66

343 Culturing of eukaryotic cells 66

344 Im- and explantation in CD-1 mice 67

345 Culturing of COS-7 cells on pre-infected surface 68

35 Biological assays 68

351 Scanning electron microscopy 68

352 Confocal laser scanning microscopy 69

353 Quantification of bacterial biofilm 71

354 BCA assay 71

355 MTT assay 71

356 Histological analyses 71

357 Qualitative and quantitative histomorphometrical analyses 72

74 Chapter 4

Investigation of bacterial interactions on nano and micro-structured titanium surfaces

74

41 Overview 75

42 Surface characterisation of ECAP modified titanium 75

43 Interactions of bacteria on ultrafine grain titanium surfaces 84

44 The effects of topographical parameters on bacterial attachment 88

45 Conclusion 91

92 Chapter 5

The bactericidal effects of graphene nanosheets 92

51 Overview 93

xiii

52 Characterisation of graphene film 93

53 Bactericidal effects of graphene nanosheet films 100

54 Mechanism of antibacterial effects of graphene nanoflakes 104

55 Conclusion 108

110 Chapter 6

The response of eukaryotic cells on black silicon 110

61 Overview 111

62 The response of fibroblast cells to black silicon surfaces 112

63 The response of epithelial osteoblast fibroblast and endothelial cells to the

bSi surface 119

64 Co-culture of endothelial and fibroblast cells 122

65 Inflammatory responses of black silicon surface 123

66 Conclusion 126

128 Chapter 7

The response of erythrocytes on black silicon surfaces 128

71 Overview 129

72 Time-dependent interactions of erythrocytes with nanopillar surfaces 129

73 Modelling of RBC membrane ndash nanopillar interactions 138

74 Conclusion 146

147 Chapter 8

Competitive colonisation of bacteria and eukaryotic cells onto the surface of

bactericidal black silicon 147

81 Overview 148

82 Real time antibacterial activity of bSi 149

83 Competitive colonisation of pathogenic bacteria and COS-7 on bSi 151

84 Conclusion 156

157 Chapter 9

General discussion 157

xiv

91 Overview 158

92 Proposed mechanisms of bacterial attachment on nanoscopically smooth

and rough surfaces with distinct surface architecture 159

93 The responses of different mammalian cell types to the nanopillar

structured black silicon surface 164

94 Competitive colonisation of bacteria and mammalian cells onto the surface

of black silicon 165

168 Chapter 10

Conclusions and future directions 168

101 Summary and conclusions 169

102 Future directions 170

103 Final remarks 171

Bibliography 173

Appendix 227

xv

List of Abbreviations

ABC Avidin-Biotin Complex

AFM Atomic force microscopy

AR As-received

ATCC American Tissue Cell Culture

BCA Bicinchoninic acid

BSA Bovine serum albumin

bSi Black silicon

CLSM Confocal laser scanning microscopy

CP Commercially pure

CTAB Hexadecyltrimethylammonium bromide

CT Connective tissue

DAB 33-diaminobenzadine

DAPI 4acute6acute-diamidino-2-phenylindole

DiI 11-dioctadecyl-3333-tetramethylindocarbocyanine perchlorate17 18

DMEM Dulbeccos Modified Eagles medium

ECAP Equal channel angular ppressing

E coli Escherichia coli

EDS Energy dispersive X-ray spectroscopy

EDTA Ethylenediaminetetraacetic acid

EPS Extracellular polymeric substances

FBS Fetal bovine serum

FDA Food and Drug Administration

GT Graphite

GN-R Graphene ndash rough side

GN-S Graphene ndash smooth side

HE Hematoxylin and eosin

HUVEC Human umbilical vein endothelial cells

MSCRAMM Microbial surface components recognizing adhesive matrix component

xvi

MTT 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide

PBS Phosphate buffer saline

PDMS Polydimethylsiloxane

(p)HF (primary) human fibroblast

P aeruginosa Pseudomonas aeruginosa

RBC Red blood cell

RIE Reactive ion etching

(r)GO (reduced) Graphene oxide

ROS Reactive oxygen species

S aureus Staphylococcus aureus

S epidermidis Staphylococcus epidermidis

SBC Swinburne Biosafety Committee

SCMF Single chain main field

Si Silicon

SEM Scanning electron microscopy

XPS X-ray photoelectron microscopy

XRD X-ray diffractometry

TEM Transmission electron microscopy

WCA Water contact angle

xvii

List of Figures

Figure 21 (A) A range of typical nanostructured materials that has been studied and

manufactured for biological applications (B) A 3 times 3 array vertical nanowire

electrode platform was used to record and stimulate intracellular neuronal activities

of cortical cell (HEK293) (C) Nanowire structured titanium was shown to enhance

human fibroblast attachment by providing more anchoring points also acting as

contact guidance for cell orientation (D) Distinct formation of active synapses

(green) in primary rat hippocampal neurons on fibrous peptide scaffolds (E)

Extended configuration of filopodia in primary bovine aortic endothelial cells which

were shown to probe the titania nanotube surface and protrude into the nanotube

holes enhancing cellular propagation (F) Internalization of few-layer graphene into

mouse macrophages and (G) the proposed molecular dynamic simulations of a

spontaneous penetration process initiating at a sharp corner monolayer graphene

sheet through a lipid bilayer (H) Size-dependent uptake of Herceptin-gold

nanoparticles (GNPs) which selectively bind to and control the expression of a

cancer receptor (ErbB2)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip15

Figure 22 (A) The generation of reactive oxygen species Incomplete oxidative

phosphorylation and other oxidative reactions result in the production of superoxide

radicals (O2macrbull) and hydrogen peroxide (H2O2) Reaction between superoxide and

nitric oxide (NO) produces proxynitrite (ONOOmacr) Hydrogen peroxide is converted

to hydroxyl radical (bullOH) by cytosolic transition metal cations in the Fenton

reaction (B) Sources (black arrows) and targets (red arrows) of ROS ROS are

produced during oxidative phosphorylation in mitochondria by oxidative enzymes

including cytochrome P450 in the endoplasmic reticulum and by xanthine oxidase

(XO) and reduced metal ions in the cytosol Cellular targets attacked by ROS include

DNA proteins membrane lipids and mitochondriahelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip19

Figure 23 Schematic diagram of a typical ECAP facility the X Y and Z planes

denote the transverse plane the flow plane and the longitudinal plane

respectivelyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip helliphelliphelliphellip24

Figure 24 Schematic diagram of the synthesis of graphene film using the method of

sonication-assisted liquid exfoliation (A) Sonication of graphite powder (1) in

CTAB at the concentration of 06 mM (B) After 6 hours graphite was exfoliated

xviii

into two-dimension single or few layers graphene sheet (C) Graphene dispersion

was dialysed against water to remove excess CTAB and aggregated graphite (D)

Graphene solution was vacuum filtered with alumina membrane to generate

graphene thin film helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip26

Figure 25 A schematic depiction of the reactive ion etching process A system is

built from two electrodes (1 and 4) that create an electric field (3) used to accelerate

ions of gas mixtures (2) toward the surface of the samples (5)helliphelliphelliphelliphelliphelliphelliphellip30

Figure 26 Schematic diagram and scanning electron images of the main stages in

the progress of bacterial biofilm formation (a) In the initial stage of attachment one

or a few planktonic bacteria sense and approach a surface with favourable

conditions This stage is regarded to be a reversible process (b) Bacteria produce

extracellular polymeric substances and irreversibly adhere to the substratum forming

a biofilm (c) Proliferation of bacteria occurs leading to (d) maturation of the

biofilm (e) In the last stage of biofilm formation bacteria are released from the

biofilm and are distributed to the surrounding environmenthelliphelliphelliphellip33

Figure 27 Total arthroplasty operations performed and total prosthetic infections

resulting from surgery as a function of year of operationhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35

Figure 28 Schematic representation of the different strategies currently being used

in the design of antibacterial surfaceshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38

Figure 29 Nanomaterials with surface structures that have shown reduced bacterial

growth or antifouling property (A) Cross patterned poly(dimethyl siloxane)

elastomer (PDMS) fabricated by photolithography (B) Nanowire titanium oxide

formed by hydrothermal treatment in high alkaline concentration (C) The Sharklet

AFTM design of PDMS consisting of 2 microm wide rectangular ribs at different lengths

varied from 4 microm to 16 microm (D) Lamella-like structures of polystyrene surfaces with

2 microm spatial period and a line-like structure at 6-8 microm period (E) Anodized

nanotubular titanium with inner diameters of 80 nm was fabricated by acid etching

(F) High aspect ratio nanopillar structure generated on silicon surface known as

black silicon with the pillar of 500-600 nm height42

Figure 210 A basic illustration of the ldquorace for the surfacerdquo concept In the

competition for surface colonization bacteria are expected to be inhibited from

surface attachment preventing the formation of biofilm (left) At the same time host

xix

cells should be able to eliminate any pathogenic microorganisms that may be present

to allow appropriate levels of tissue integration ensuring the success of an

implantation process (right) These effects can be supported by modifying the

implant surface using antimicrobial coatings or through the generation of a

bactericidal surface pattern which should be biocompatible to the relevant host

tissue cellshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53

Figure 31 (A) The atomic force microscope can reveal the topography of a sample

surface by raster-scanning a small tip back and forth over the surface The tip is on

the end of a cantilever which deflects when the tip come across the surface features

This deflection is sensed by a laser beam which can reflect the end of the cantilever

onto a segmented photodiode which magnifies and record the cantilever deflections

(B) Illustration of AFM contact mode versus tapping modehelliphelliphelliphelliphelliphelliphelliphelliphellip64

Figure 41 X-ray diffractogram of as-received and ECAP modified Tihelliphelliphelliphellip77

Figure 42 TEM images of the ultrafine grains of ECAP grade 2 (A amp B) and grade

4 (C amp D) Scale bar 100 nmhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78

Figure 43 Surface topography of as-received and ECAP modified titanium grade 2

and 4 analysed by optical profiling (top) and AFM (middle) with corresponding

surface line profile Typical AFM scanning areas are shown in 1 microm times 1 micromhelliphellip80

Figure 44 Surface architecture of grade 2 and 4 ECAP modified titanium surfaces

demonstrated by AFM height images (top) compared with phase tapping images

(bottom) which revealed the size shape and organisation of titanium ultrafine

nanograins (orange) grain boundary (blue) and sub-nanograin structure (green)

Transition of titanium surface architecture from as-received materials to ECAP

processed surfaces can be found in the following link

(httpyoutubeHlwcTV4DXmk)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip82

Figure 45 The protrusion of grade 2 and 4 ECAP modified titanium surfaces ((a)

and (b) respectively) with statistical distribution performed by ImageJ (e) Greyscale

AFM scans of both surfaces were transformed into (c) and (d) to facilitate the

distribution analysishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip83

Figure 46 The responses of Staphylococcus aureus on the as-received and ECAP

modified titanium surfaces after 18 h incubation SEM images (top) represent the

typical cell attachment and morphology Three-dimensional CLSM images (middle)

xx

represent cell viability and EPS production (live cells were stained green dead cells

were stained red EPS were stained blue) The CLSM images were used for further

analysis of biofilm performed by COMSTAT softwarehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip85

Figure 47 The responses of Pseudomonas aeruginosa on the as-received and

ECAP modified titanium surfaces after 18 h incubation SEM images (top) represent

the typical cell attachment and morphology Three-dimensional CLSM images

(middle) represent cell viability and EPS production (live cells were stained green

dead cells were stained red EPS were stained blue) The CLSM images were used

for further analysis of biofilm performed by COMSTAT softwarehelliphelliphelliphelliphelliphellip86

Figure 48 Statistical quantification of bacterial viability on titanium surfaceshellip87

Figure 49 S aureus (right) and P aeruginosa (left) biovolume and average biofilm

thickness on surfaces of as-received and ECAP titanium quantified using

COMSTAT (Heydorn et al 2000) helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip88

Figure 410 Statistical analysis showing the relationship between the average

roughness and kurtosis of titanium surfaces and the amount of attached bacteria

cells There was no clear correlation between the attachments of both S aureus and

P aeruginosa to the Sa values within the sub-nanometric range while the Skur

appeared to be proportional with the number of the adherent cellshelliphelliphelliphelliphelliphelliphellip89

Figure 51 The UV-Visible absorption spectra of aqueous graphene suspension

during the 6 hour sonication-assisted exfoliating processhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip94

Figure 52 Chemical analysis of the exfoliated graphene films using (a) Raman

spectroscopy showing the doublet G peak which corresponds to the multilayer

graphene sheets and (b) EDS confirming the elemental composition of graphene

films and the absence of bromine from the CTAB used in the manufacture

processhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip95

Figure 53 X-ray diffractogram of a sample of peeled graphite block (green)

compared with graphene GN-R and GN-S films (blue middle and bottom lines

respectively)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip96

Figure 54 The surface morphologies of graphene nanoflakes visualised using SEM

(1 μm times 1 μm area) The contrast of the images was enhanced to reveal the sheet

xxi

edges allowing the size distribution of edge lengths of both the rough (GN-R) and

smooth (GN-S) graphene surfaces to be determined helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip97

Figure 55 Surface topographies of GT GN-R and GN-S films visualized by SEM

AFM and Raman spectroscopy illustrating the typical geometry size and thickness

of graphite layers and graphene flakes on both the upper and lower sides of the film

This reflects the different dimensions in the arrangement of the flakes AFM images

were taken over scanning areas of 5 microm times 5 microm with the corresponding surface line

profile representing the thickness of graphite layers and graphene flakeshelliphelliphelliphellip99

Figure 56 Scanning electron micrographs showing the typical attachment of S

aureus and P aeruginosa cells onto GT GN-R and GN-S films The damaged

bacteria have been highlighted with colour to enable a direct comparison with the

intact cells observed on the surface of the GThelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip101

Figure 57 Typical (A) CLSM images and (B) quantification of viable vs non-

viable cells and (C) total number of attached cells present on the surfaces of GT

GN-R and GN-S Live cells were stained green dead cells were stained red (scale

bars are 10 μm) helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip102

Figure 58 Schematic diagram illustrating the interaction between the graphene

micro- (GN-R) and nano-structures (GN-S) with the P aeruginosa (A amp B) and S

aureus (C amp D) cells These possible configurations have been determined according

to the AFM topographical cross sectional profiles and the respective bacterial

morphologieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip103

Figure 59 Free energy difference ΔF between phospholipid bilayer and inserted

graphene sheets with varying hydrophobicity (interaction parameter (εobj) of (a) -5

(b) -6 and (c) -75 kT) as a function of the distance from the bilayer centre to the

edge of the surface Distance 40 corresponds to the unperturbed bilayer before it has

made contact with the surface (zero energy reference state) the blue stripe

corresponds to the solution of insertion of the surface into the bilayer with no change

in the bilayer configuration the orange stripe corresponds to the solution with a pore

in the bilayer (positive energy) Selected density profiles correspond to different

positions of graphene surface the colours of the bilayer represent the volume

fraction of tails and heads from 0 to 1helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip107

xxii

Figure 61 SEM images of primary human fibroblast (pHF) cells cultured on the

bSi Si and plastic control surfaces compared to the growth of fibroblast-like cell

lines over incubation periods of 1 3 and 7 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip114

Figure 62 CLSM images of pHF cells on bSi and control Si surfaces Actin

filaments were stained with Alexa fluor 488 Phalloidin (green) Vinculin ndash a

component of the focal adhesion point were stained with an anti-vinculin primary

antibody and with Alexa Fluor 546 conjugated anti-mouse IgG (red) The cell nuclei

were stained with DAPI (blue) helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip115

Figure 63 (A) Single cell interactions of COS-7 cells on bSi surface visualised

using time-lapse sequential CLSM over 3 hours (C) SEM images of freeze fractured

COS-7 cells attached onto the bSi surfaces (B) Visualisation of the interface

between a single cell and bSi surface The arrows show the local contact point of the

cells with the surface nanopillars Snapshots are taken from real-time interactions

between the COS-7 cells with the bSi surface Cells were stained with CellTracker

CMFDA (green) helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip117

Figure 64 Intracellular protein production by COS-7 cells on the bSi and control

surfaces quantified by the BCA assay over a 7 day growth period (Significantly

different if P = 1 (t gt 005) insignificantly different if P = 0 (t lt 005))helliphelliphellip118

Figure 65 The number of attached COS-7 cells on both the bSi and control

surfaces as quantified by MTT assays over a 7 day growth period (Significantly


Figure 66 Monocultures of human epithelial (A549) osteoblast cells (MG63)

fibroblast and endothelial cells growing on the surfaces of plastic and bSi after 24 h

and 96 h of incubation Cells were stained with Calcein-AM After a 24 hr growth

period on the bSi surfaces the epithelial and osteoblast cells exhibited a slightly

reduced attachment and spreading whereas the fibroblast and endothelial cells were

present on the surface in much fewer numbers and exhibited a mostly rounded-up

phenotype After 96 h the epithelial and osteoblast cells on both the plastic and bSi

surfaces had formed a nearly confluent monolayer Only very few of the initially

added endothelial cells remained viable after 96 hhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip121

Figure 67 Formation of interconnected microcapillary-like structures (red arrows)

of co-cultures between primary human fibroblasts and endothelial cells growing on

xxiii

plastic and black silicon surfaces Cells were fixed and stained with endothelial cell-

specific PECAM-1 and the nuclei were stained with DAPI (blue) helliphelliphelliphelliphelliphellip123

Figure 68 Representative microphotographs of the tissue reactions to the surfaces

of bSi (A and B) and the Si control (C and D) implanted samples within the

subcutaneous connective tissue (CT) of the CD-1 mouse at day 15 after implantation

(A) On the surfaces of bSi a thin layer of mononuclear cells (arrows) and

extracellular matrix was clearly seen Within the surrounding CT increased numbers

of mononuclear cells (red arrows) were detected (B) The immunohistochemical

detection showed that only small numbers of the cells adherent to the bSi surfaces

were macrophages (black arrows) Most of the cells within the surrounding CT were

also identified as macrophages (green arrows) (C) At the surfaces of the Si implants

a thicker layer (arrows) composed of mononuclear cells was detected In the peri-

implant CT more mononuclear cells (red arrows) were detected (D) Most of the

cells adherent to the Si surfaces were identified as macrophages (black arrows)

Numerous macrophages (green arrows) were detected within the peri-implant CT All

scale bar are 10 micromhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip124

Figure 69 The number of macrophages associated with the biomaterials Bar chart

shows the results of the analysis for the histomorphometrical measurements of

material-adherent macrophages per mm Silicon showed a significantly increased

number of material-adherent macrophages as compared to black silicon ( P lt 001)

helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip125

Figure 71 SEM images showing an overview of the time-dependent erythrocyte

interactions with bSi nanopillar-arrayed surfaces Images were taken at different time

intervals for up to three hours of contact Scale bars are 20 micromhelliphelliphelliphelliphelliphelliphellip130

Figure 72 Typical SEM images of the dynamic interaction of erythrocytes with

three control surfaces glass gelatin-covered glass and silicon wafer over 3 hours of

incubation Images were selected as being representative from 10 different areas of 3

independent experiments Scale bars are 20 micromhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip131

Figure 73 Comparative quantification of the dynamic attachment of RBC on bSi

and on the control surfaces (a) Data were plotted as an average of the total number

of attached cells from 10 different areas in 3 independent experiments (b) The

xxiv

separated quantitative plotting of intact biconcave RBCs versus deformed and

ruptured RBCs which appeared like lsquocell printsrsquo on the bSi surfaceshelliphelliphelliphelliphellip133

Figure 74 SEM micrographs (top and side view) showing the step-by-step

morphological changes from a healthy biconcave shape to a completely damaged

cell as a result of the action of the nanopillarshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip134

Figure 75 Snapshots of the real time (video) interactions of erythrocyte attachment

to bSi Optical images showed cells appearing in the frames when in contact with the

bSi surface disappearing after rupture when they moved out of camera focus The

real-time movie can be found at

httpwwwrscorgsuppdatatbc4c4tb00239cc4tb00239c2mpghelliphelliphelliphelliphelliphellip134

Figure 76 CLSM analysis confirmed (a) the rupturing of RBC in contact with bSi

and (b) the intact healthy RBC attached to the control surfaces Cells were stained

with 11-dioctadecyl-3333-tetramethylindocarbocyanine perchlorate Segments of

ruptured cell membrane can be seen which may be regarded as the lsquocell

footprintrsquohelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip135

Figure 77 Raman analysis of attached and ruptured erythocytes on the bSi surfaces

(i) Two-dimensional mapping of RBCs interacting with bSi surfaces using Raman

spectroscopy lsquoArsquo Raman spectrum of the area where RBCs are not present lsquoBrsquo the

spectrum of RBC prior to disruption and lsquoCrsquo the ruptured RBC (ii) Corresponding

three-dimensional image of the Raman spectroscopic map Erythrocytes were

incubated with bSi for 30 minutes in all experiments (iii) Spectra in the area of RBC

Raman activity from 1100 cm-1 to 3500 cm-1 which provides discrimination from

the bSi nanopillar resonance peak at 480 cm-1helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip136

Figure 78 Characterisation of the bSi nanopillar arrayed surfaces (a) Top view

SEM image of bSi (scale bar 500 nm) (b) Area distribution of the pillars

quantified at widest cross-section showing a maximum at 49 nm in area at the

widest pillar width aggregation represented by the shoulder and tailing in the

distribution extending to ~100 nm (c) Fast Fourier 2D Transform of SEM image (a)

yields an intense ring extended to four broad orthogonal lobes from this secondary

structure (d) Radial grey scale intensity (0-255) profile showing the intense sharp

ing in the centre peaks at a frequency distance of 185 nm characteristic of the

average distance between pillars with extended shoulders representing secondary

xxv

pillar ordering (e) Side view of bSi nanopillars and (f) schematic representation

showing dimensions calculated from average plusmn variance of 50 measurements of five

SEM imageshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip140

Figure 79 Interfacial topology between the bSi and erythrocyte membrane

architecture (a) Reconstruction of the RBC cytoplasmic membrane surface as

determined by reconstructing the AFM image of immobilized RBC (obtained from

(Parshina et al 2013)) through image analysis consisting of adjustment of bSi SEM

(20 nm from nanopillar tip) and the AFM image of RBC to comparable contrasts

colour thresholding boundary delimitation by variance transformation

backgrounding and summation of area distributions The freestanding RBC lipid

bilayer (black) represented approximately 50 of the geometrical area defined by

typical junctional nodes shown by the yellow points (b) Size distribution of the bSi

nanopillars and the corresponding freestanding lipid bilayer areas between where it

was anchored to the spectrin network of the RBCshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip141

Figure 710 Single Chain Mean Field density profile of a lipid bilayer in contact

with regularly distributed nanopillars (A) General view of the lipid bilayer and the

tips of the pillars and the simulation box representing the mesh of the 3D periodic

structure The box size represents the spacing between nanopillar tips (B) A

sequence of solutions corresponding to relative positions of the bilayer with respect

to the nanopillar The distances are given in Angstrom while the colours of the

bilayer represent the volume fraction of tails and heads from 0 to 1 (below)helliphellip143

Figure 711 Free energies driving nanopillar insertion Free energy difference ΔF

between unperturbed bilayer and the bilayer with inserted attractive cone as a

function of the distance from the centre of the bilayer to the tip of the cone The red

stripe corresponds to the solution of an unperturbed bilayer and a cone before contact

(reference state zero energy) the grey stripe corresponds to a cone touching the

bilayer without piercing the bilayer the green stripe corresponds to a cone having

induced the formation of a pore in the bilayerhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip145

Figure 81 SEM images of the damaged bacterial cells on the nanopillar structured

surface of bSi (a amp b) and intact bacterial cells on non-structured silicon wafer

control surfaces (c amp d) scale bars are 2 microm Sequential time lapse confocal

xxvi

microscopic images showing the dynamic bactericidal activities of bSi interacting

with P aeruginosa (e) and S aureus (f) over 6 hours scale bars are 5 micromhelliphellip150

Figure 82 SEM images of COS-7 cell growth onto the infected bSi surface and Si

wafer control surfaces after 1 3 and 7 days of incubation Both surfaces were

infected with P aeruginosa and S aureus cells for 6 hours at their respective

infective doses prior to the surfaces being exposed to the COS-7 cellshelliphelliphelliphellip153

Figure 83 Visualization of the co-cultured COS-7 and bacterial cells on the bSi and

silicon wafer control surfaces Live COS-7 cells were stained with calcein AM

(green) dead COS-7 cells were stained with ethidium homodimer-1 (red) bacteria

were stained SYTOreg 9 (blue) helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip154

Figure 84 Quantification of the number of COS-7 cells present on the infected bSi

and silicon wafer control surfaceshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip155

Figure 91 Comparison between the uniform evenly distributed ultrafine grains of

the grade 2 titanium structure (A) and the presence of spatially distributed

nanoprotrusions on the grade 4 titanium surface (B) formed by the equal channel

angular pressing (ECAP) modification processhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip161

Figure 92 Interaction behaviours between the bacterial cell membrane and the

graphene surface (a) The increase in size of the non-viable S aureus (viable cells

are green non-viable cells are red) indicates an osmotic pressure imbalance within

the damaged cells after the insertion of graphene sheets (scale bar 10 μm) (b)

Sequence of simulated interaction between the graphene sheet and phospholipid

membrane resulting in pore formationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip163

Figure 93 Prevalence of the five most frequent pathogens as a function of the

origin of the orthopaedic infection in a collection of 272 clinical isolates obtained

from 242 patients in the period between 2007 and 2011 K amp H knee and hip

arthroprotheses respectively EF amp IF External and internal fixation MD medical

devicehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip166

xxvii

List of Tables

Table 21 Typical examples of nanostructured materials and their applications 10

Table 42 Titanium surfaces elemental composition inferred from XPS analysis 76

Table 43 Contact angle and surface free energy of the as-received and ECAP

modified titanium surfaces 76

Table 44 AFM surface roughness analysis of the as-received and ECAP modified

titanium surfaces on two nanoscale scanning areas 81

Table 55 Topographical analysis of graphite (GT) together with rough (GN-R) and

smooth (GN-S) graphene surfaces 97

1

Chapter 1

Introduction

2

11 Overview

The effect of substrate surface structure on the attachment of different

biological systems has long been a focus of research for biological and biomedical

applications It has been established that the extent of most biological interactions

with substrates is heavily controlled by the initial cell-surface interactions that take

place at the nano-length scale An understanding of the cellular events that occur

when biological organisms come into contact with a substrate would offer the ability

to control a number of complex cellular behaviours Materials can now be

engineered precisely to the nano-level to target the nano-components of cells thus

allowing an unprecedented level of control of cell functions These initial

interactions play a critical role in determining subsequent cellular communications

functionality and tissue regeneration with the surface These factors in combination

determine the ultimate success of a biomaterial This concept has led to a new era of

nanostructured surfaces and nanomaterials which can be engineered to target and

control many complex cell behaviours for various applications (Kayser et al 2005

Valiev et al 2008 Zhang amp Webster 2009)

One research direction over the past few years has been focusing on the

modification of surface nanostructures to control the extent of colonisation of

pathogenic bacteria onto substrate surfaces with the intention of identifying new

methods for controlling bacterial infection Biomaterial-associated infection has been

recognised as one of the most devastating issues in medical science (Donlan 2001

Schierholz amp Beuth 2001 Clohisy et al 2004 Zimmerli 2006 Del Pozo amp Patel

2009 Montanaro et al 2011) Complications that may arise from the colonisation of

medical implants by pathogenic bacteria include increased antibiotic-resistance

caused by biofilm formation induced hyper immune responses leading to the

necessity of implant removal and in some cases mortality In addition infection of

biomedical devices results in significant health care costs (Costerton et al 1999

Donlan 2001 Donlan amp Costerton 2002 Clohisy et al 2004 Zimmerli 2006 Del

Pozo amp Patel 2009 Moriarty et al 2011) Much of the research being conducted is

to improve the antibacterial properties of biomedical surfaces using a variety of

antimicrobial coatings and surface functionalization in addition to modern sterilising

techniques (Seymour amp Whitworth 2002 Darouiche 2004 Langlais et al 2006

Zhao et al 2009) Improvements have been made to these processes however

3

instances of increased levels of bacterial resistance are also often reported (Davies

2003 Campoccia et al 2006 Hetrick amp Schoenfisch 2006) Recently advances in

nanotechnology have allowed nanostructured surfaces to be engineered such that

they exhibit antibacterial properties where the primary preventative mechanism is

based on the physical interactions taking place between the nanostructured surface

and the bacterial cells without the need for additional chemical treatments (Akhavan

amp Ghaderi 2010 Hasan et al 2013a Ivanova et al 2013 Li et al 2014 Hasan et al

2015) Such surfaces have the potential to be alternatives for chemical-additive based

antimicrobial surfaces

Another characteristic of an implant material is the necessity for the material

to be compatible with the host system where the host tissue cells can fully integrate

with the surface (Williams 2008 Norowski Jr amp Bumgardner 2009 Anselme 2011

Busscher et al 2012 Niinomi et al 2012) Different types of surface nanostructures

have been shown to influence many cellular processes such as cell adhesion

migration proliferation differentiation and other specific cellular activities

depending on cell types (Sniadecki et al 2006 Zhang amp Webster 2009 Bacakova et

al 2011 Murty et al 2013 Bonde et al 2014) The mechanisms of these effects are

however not yet fully understood Recent investigations have reported a competitive

situation in which host cells are placed in a situation where they are required to

compete with pathogenic bacteria for the effective colonisation of a surface

(Subbiahdoss et al 2010b Busscher et al 2012) This phenomenon has been termed

the ldquorace for the surfacerdquo (Gristina 1987) Although the concept of this event was

introduced long ago to date limited information has been made available regarding

the mechanisms responsible for driving these competitive activities One of the main

reason for this is that it is difficult to design the appropriate experimental conditions

in which bacterial attachment in the presence of in-vitro and in-vivo host integration

can be studied (Subbiahdoss et al 2009 Busscher et al 2012 Neoh et al 2012)

12 Aims and objectives

The ultimate aim of this study was to understand the effects of varying

surface parameters at the nanoscale on the colonisation of bacteria and mammalian

cells Three substrate materials were selected according to their physical and

chemical properties and their ability to be used as prospective biomedical

4

applications The materials were fabricated and modified to generate specific micro-

and nanostructures The attachment behaviours of different cell types on the surface

of these substrates were investigated to achieve three following objectives

The first objective was to investigate the influence of surface nanostructure

on bacterial attachment colonisation and biofilm formation The attachment

response of pathogenic bacteria was measured on two distinct surface structures

nanoscopically smooth titanium and microscopically rough graphene film The

surface structures were characterised using a wide range of techniques including

scanning electron microscopy X-ray photoelectron spectroscopy energy dispersive

spectroscopy X-ray diffractometry Raman spectroscopy optical profilometry and

atomic force microscopy The attachment response of various bacterial cells onto

these surfaces was assessed by analysing their attachment behaviours cell viability

and biofilm formation

The second objective was to investigate the responses of mammalian cells to

black silicon a surface that has been demonstrated to exhibit highly efficient broad

spectrum antibacterial properties The bactericidal activities of the nanopillars on the

black silicon surface were shown to be mechano-responsive which makes this model

a prospective alternative to chemical-based antibacterial surfaces A range of

different cell types were employed to assess the biocompatibility of black silicon in

vitro including primary human fibroblast fibroblast cell line (COS-7) osteoblast

cells (MG-63) epithelial cells (A549) and primary human endothelial cells Single

cell interactions with the bSi nanopillars was investigated by imaging the dynamic

attachment process and the filopodia development of COS-7 fibroblast-like cells

using real-time sequential confocal microscopy The in vivo response of the black

silicon surface was also investigated using CD-1 mice

The third objective was to investigate whether or not the antibacterial

properties of black silicon could support the growth of mammalian cells while live

bacteria were present on the surface A novel experiment was introduced to assess

the competition between bacteria and mammalian cells in order to demonstrate the

effects of the black silicon surface structure in preventing bacterial infection and

preserving biocompatibility The ldquorace for the surfacerdquo was studied by pre-infecting

the black silicon surface with live pathogenic bacteria after which time COS-7 cells

were introduced to compete with the bacteria The behaviours of both cell types

5

regarding cell morphology viability and proliferation were analysed to determine if

the surface structure of the black silicon would be suitable for implant applications

In the following chapters the current knowledge regarding the interactions of

bacterial and mammalian cells with different types of nanostructured surfaces will be

discussed Following this discussion the methodology that was employed to conduct

the experiments will be detailed followed by the results and the discussion of the

investigations that was mentioned as above

6

Chapter 2

Literature review

7

21 Overview

The study of the activity of biological organisms at the surface of a material

the lsquobiointerfacersquo has long been a major research topic in the field of life sciences

The outcomes of these studies have provided fundamental knowledge for a wide

range of biochemical medical and pharmaceutical applications which have brought

significant financial benefits for the related industries To date it has been established

that most cell-surface interactions begin at the nanoscale level which involves the

structure of the underlying substrata and biological components such as proteins

cells ligands DNA and macrophages (Valiev et al 2007 Mahapatro 2012 Zhu et

al 2013)

This chapter will review the current knowledge of the interactions taking

place between bacterial and mammalian cells with different types of nanostructured

surfaces The first section of this chapter will introduce some of the most common

nanostructured materials that have been extensively studied for biological

applications followed by consideration of the possible cytotoxicity of these

materials to human health The second section will focus on newly engineered

nanostructured surfaces that can exhibit antibacterial properties The advantages of

the characteristics of such materials will be compared with those of other

conventional methods that have been used in an attempt to prevent biomaterial-

associated infections The influence of surface nanostructure on the behaviour of

mammalian cells will also be discussed mainly in reference to cell adhesion

proliferation and differentiation Based on this literature review a selection of three

nanostructured surfaces will be introduced in order to investigate these newly

engineered nanostructured surfaces particularly in light of the mechanisms by which

these parameters affect the responses of cells A competitive situation in which the

bacteria and mammalian cells are placed in a circumstance in which they need to

compete for their effective colonisation to a surface will also be discussed Section

232 of this chapter was published in a book chapter which was listed in the List of

Publications

8

22 Nanostructured surfaces ndash the new future

221 Nanostructured surfaces and biological applications

In the last decade nanostructured materials have been extensively researched

and commercially produced for a wide range of novel and improved applications in

optics physics electronics agriculture cosmetics textiles food and medicine

(Zhang amp Webster 2009 Murty et al 2013 Zhu et al 2013) These materials are

generally defined as materials that have at least one dimension smaller than 100 nm

(Nel et al 2006 Sniadecki et al 2006 Von Der Mark et al 2010 Tang et al 2012)

The extremely small size of nanostructured materials results in a physically large

surface area per unit of volume leading to significant differences in physical

chemical electrical and biological properties compared to the bulk form (Federico

2004 Sniadecki et al 2006 Gonsalves et al 2007 Murty et al 2013 Bonde et al

2014) These unique characteristics if intelligently designed could provide a

plethora of new solutions and benefits to human life and the global ecology

Different forms of nanostructured materials that have been developed include

nanoparticles nanofibers nanotubes nanowire nanorods nanoplatelets

nanopatterned surfaces and thin solid films with nanoscale thickness (Sniadecki et al

2006 Wang amp Lin 2007 Teli et al 2010 Murty et al 2013) Some of the most

recent studies of nanostructured materials that have been researched and applied in

life sciences are presented in Table 21 and Fig 21 The synthesis of nanostructures

is often classified into two groups depending on the method by which they were

produced these are known as bottom-up and top-down approaches Bottom-up

approaches start with molecules atoms or simple chemical components that are

subjected to other physical or chemical processes to allow them to combine their

basic units into nanostructures (Huang et al 2007 Sainiemi et al 2007 Coelho et al

2009 Thakkar et al 2010) Techniques belonging to this category include molecular

self-assembly atomic layer deposition vapour condensation electrodeposition and

chemical functionalisation An example is the formation of nanoparticles from either

self-assembly ultrasonic colloidal dispersion or sol-gel methods (Jiang et al 2008

Faraji amp Wipf 2009 El-Rafie et al 2012 Cronholm et al 2013) Top-down

approaches on the other hand use physical or chemical techniques to modify a

macroscopic material into a nanostructured material These techniques include

9

different types of lithography such as photolithography X-ray lithography electron

beam and ion beam lithography molecular beam epitaxy chemical and plasma

etching (Sjoumlstroumlm et al 2009 Zhang amp Webster 2009 Von Der Mark et al 2010

Tay et al 2011 Kim et al 2013) An example is a range of different nanopatterns

that can be precisely printed onto a solid substrate such as a silicon wafer in a

precise size and shape These patterns include nanocones nanostars nanocylinders

and nanopillars (Brammer et al 2008 Brammer et al 2011 Ercan et al 2011

Ezzati Nazhad Dolatabadi et al 2011 Chung et al 2013 Vasudevan et al 2014

Bhadra et al 2015) Top-down methods are generally more expensive and time

consuming and are frequently used in laboratory research methods rather than in

large scale production due to the requirement of sophisticated equipment

Fabrication using bottom-up methods in contrast is fast and more economically

efficient and thus is more often used in commercial situations (Federico 2004 Liu et

al 2011b) Depending on the base materials and the structures required each

technique can offer specific advantages to control the surface morphology size

shape orientation and geometry including the addition of other functional groups if

these are required to meet the demands of different applications (Huang et al 2007

Coelho et al 2009 Webb et al 2011a) It has been estimated that the use of

nanomaterials contributes to approximately $1 trillion to the global economy (Nel et

al 2006 Tang et al 2012)

A majority of nanostructured materials has been engineered for biochemical

and medical applications The interactions between biomedical devices such as

synthetic tissue engineering scaffolds and implant materials are often investigated at

different length scales including macro micro and nano-scales (Niinomi 2008

Williams 2008 Anselme 2011) On macro and micro scales it has been

demonstrated that effective organ and tissue integration are a function of the implant

chemical physical characteristics and surface microtopography (Chen et al 1997

Cukierman et al 2001 Tay et al 2011) The effects of material surface on the

activities of other molecular components such as protein adsorption blood clotting

focal adhesion development and gene expression however require an assessment of

the biointerfaces at nanoscale level (Nag et al 2005 Gonsalves et al 2007 Williams

2008 Anselme et al 2010 Von Der Mark et al 2010 Anselme 2011 Bolisetty amp

Mezzenga 2016 Chang amp Olsen 2016 Ngandu Mpoyi et al 2016 Reshma et al

10

2016 Xiao et al 2016) Since the importance of the nanoscale interface has gained

the recognition of researchers the research in this field has increased leading to

promising applications of nanostructured materials in guiding cells (Bucaro et al

2012) probing biomolecules (Shalek et al 2010 Na et al 2013) gene transfection

(Na et al 2013) cellular force measurements (Krivitsky et al 2012) biosensors

(Engel et al 2010 Krivitsky et al 2012) antibacterial surfaces (Ivanova et al 2013)

and drug delivery (Kayser et al 2005 Dasgupta et al 2014)

Table Error Use the Home tab to apply 0 to the text that you want to appear here1 Typical examples of nanostructured materials and their applications

Types of

nanostructures

Base

materials

Research and

biomedical applications

References

Nanoparticles

Gold Cancer diagnostics and

therapeutic treatments

(Huang et al 2006 Jain

et al 2006 Chen et al

2007 Boisselier amp

Astruc 2009 Kang et al

2016 Wu et al 2016b

Zhang et al 2016)

Platinum Catalysts (Narayanan amp El-Sayed

2003 Mei et al 2005

Narayanan amp El-Sayed

2005 Wang et al 2008)

Titanium Cosmetics and personal

care products

orthopaedic coatings

(Tsuang et al 2008

Simchi et al 2011

Zhao et al 2011)

Zinc UV shielding in wool and

cotton fabrics

antimicrobial agents

food additives

(Fan amp Lu 2005

Becheri et al 2007 Xie

et al 2010 Espitia et al

2012)

Silver Antimicrobial agents

antibacterial cotton

fabrics

(Sondi amp Salopek-Sondi

2004 El-Rafie et al

2012)

11

Types of

nanostructures

Base

materials

Research and


References

Quantum dots InAs amp

GaAs

Diode lasers booster

amplifiers biological

imaging labelling and

sensors

(Lodahl et al 2004

Dieter 2005 Medintz et

al 2005)

Nanotubes Carbon Electronic conductors

field emission electron

guns and cathodes

radioactive labelling

drug delivering tools

(Huang et al 2003

Minoux et al 2005

Barhate amp Ramakrishna

2007 Liu et al 2007

Ezzati Nazhad

Dolatabadi et al 2011

Yu et al 2014b)

Titania Antibacterial surfaces for

bone implant

(Ercan et al 2011

Minagar et al 2013

Damodaran et al 2015

Nair amp Elizabeth 2015)

Nanofibers Alumina Waste water treatment

air filters

(Huang et al 2003


2007)

Polyaniline Chemical vapor sensors (Huang et al 2002 Li

et al 2008a)

Nanopores Hydroxyapatite

composites

Orthopaedic implants

bonecartilage tissue

engineering bone

disease treatments

(Wang et al 2007

Venugopal et al 2010)

Nanoplatelets

nanoflakes

Graphite and

graphene

composites

Enhancing mechanical

characteristics in polymer

production

(Potts et al 2011

Sengupta et al 2011)

Graphene

oxide and its

composite

Antimicrobial materials

in the form of solution or

thin films

(Peltonen et al 2004

Prinz et al 2008

Akhavan amp Ghaderi

12

Types of

nanostructures

Base

materials

Research and


References

2010 Tian et al 2014

Luan et al 2015)

Nanoclay Polymer

composites

Improved plastic

production for lighter

weight and better scratch

resistance

(Markarian 2005 Zhao

et al 2008)

Nanopillars

Nanowires

Silicon Field effect transistor

photovoltaic system and

solar cells

(Hu amp Chen 2007

Garnett amp Yang 2010

Gervinskas et al 2013

Malinauskas et al 2013

Buividas et al 2015)

Biocompatible synthetic

platforms for cell

guiding signalling

promoting cell growth

and biomolecule

delivering tools

(Stevens amp George

2005 Pimenta et al

2007 Qi et al 2007

Bucaro et al 2012 So

Yeon amp Eun Gyeong

2013 Pan et al 2014

Prinz 2015)

Antibacterial surfaces (Fellahi et al 2013

Ivanova et al 2013 Li

et al 2014)

Gallium nitride Strong emission nano-

optoelectronic and

sensing devices

(Kouklin amp Liang 2006

Lo et al 2011)

Gallium

phosphide

Culturing substrata of

neurons for enhancing

neurite growth and

neurotransmission

(Persson et al 2013)

13

It has been established that the physical and chemical properties of

nanostructured surfaces play a significant role in dictating cellular responses and

other related host cell activities thus determining the success of an implant and other

clinical treatments These properties include surface topography chemistry

crystallinity wettability and surface energy induced by the size shape orientation

geometry and density of the nanostructure of the surface (Rack amp Qazi 2006 Witkin

amp Lavernia 2006 Valiev et al 2008 Zhang amp Webster 2009 Bhushan amp Jung

2010) The small size of surface nanostructures is known to increase their ability to

cross various biological barriers without causing substantial damage to biological

organisms due to the comparable size between the nanopatterns and biological

components (Wang amp Lin 2007) Host components such as plasma proteins

macrophages blood cells membrane ligands receptors and antigens which

represent the first point of contact with implanted biomaterials have been shown to

exhibit positive responses to many nanostructured surfaces (Holmes et al 2000

Webster et al 2001 Faghihi et al 2006 Jung amp Donahue 2007 Tay et al 2011

Minagar et al 2013) If these initial interactions occur at the interface in an

appropriate manner they will further regulate the processes of cell attachment

orientation migration proliferation and differentiation ensuring appropriate cell

functionalities and tissue regeneration (Tran amp Webster 2009 Teli et al 2010 Bai amp

Liu 2012 Binsalamah et al 2012 Egli amp Luginbuehl 2012 Wang et al 2012a)

These interactions are not always reported in a consistent manner due to a large

number of parameters involved some of which are known however many remain

unknown but are involved in the complex activities taking place at the biointerface

Many studies have demonstrated that even a small variation in one or few parameters

of the surface structure at nanoscale may lead to a significant change in the

behaviour of cells (Degasne et al 1999 Webster et al 2000 Webster et al 2001)

A few examples of current biomaterials that have been used to control and

manipulate cell activities are presented in Fig 22 Most of recent studies have

demonstrated the favourable responses by mammalian cells to the structures of

nanoparticles nanotubes nanorods and nanopillars made by metal metal oxide and

semiconductor materials The effects of nanostructured surfaces to cells vary from

exhibiting similar to moderately or significantly enhanced cell responses depending

on the size shape and density of the nanostructures Meanwhile the response of cells

to other newly discovered two-dimensional materials such as graphene graphene

14

derivatives and molybdenum disulphide (MoS2) nanosheets remains highly

controversial Robinson et al constructed a silicon nanowire array integrated to an

electronic circuit to culture and record the activities of rat cortical neurons (Qi et al

2009) These nanowire arrays can act as a scalable intracellular electrode platform to

measure and stimulate the action potentials between hundreds of neurons They can

also map multiple synaptic connections (Fig 21B) The authors suggested that the

small dimension combined with the efficiency and the flexibility of the system

would allow this system to be further integrated with on-chip digitization and signal

multiplexing providing a possibility for the nanowire electrode to be used as an

implantable microelectrode for neuronal prosthetics (Qi et al 2009) A similar

enhancement of active synapses and extensive growth of neurites was reported with

rat PC12 cells and primary rat hippocampal neurons on a self-assembling peptide

scaffold (Fig 21D) (Holmes et al 2000) In another study that was searching for

improved coronary stent materials TiO2 nanotube substrata were found to

significantly increase the migration of primary bovine aortic endothelial cells

(BAECs) by extended cell filopodia and extracellular matrix induced by the

nanotube structure (Fig 26E) (Brammer et al 2008) A similar enhancement of cell

focal adhesion was also observed with primary human fibroblasts attached to a

nanowire structured titanium surface (Fig 26C) (Bhadra et al 2015) The authors

suggested that the increased contact area of the nanowire structure provided more

anchoring points for cell adhesion thus leading to the extension of the cytoskeleton

network and subsequent stimulation of growth

15














16


cancer receptor (ErbB2) Licence agreement

httpcreativecommonsorglicensesby30 (Wang amp Lin 2007) Macmillan

Publishers Ltd [Nature Nanotechnology] (Qi et al 2009)

httpcreativecommonsorglicensesby40 (Bhadra et al 2015) Copyright 2000

National Academy of Sciences (Holmes et al 2000) Copyright 2008 American

Chemical Society (Brammer et al 2008) Copyright 2008 American Chemical

Society (Akhavan et al 2011) and (Jiang et al 2008) respectively

One of the most common nanostructured materials that has been largely

applied in diverse application fields are nanoparticles (examples of which are

presented in Table 21 and Fig 21H) Nanoparticles have also been used as

experimental tools to track real time dynamic biological processes in organs tissues

and single cells at the molecular level such as fluorescent nanoparticles (Lewin et al

2000 Beaurepaire et al 2004 Slowing et al 2006 Hsiao et al 2008 Idris et al

2009) quantum dots (Gao et al 2004 Howarth et al 2005 Medintz et al 2005

Michalet et al 2005 Tada et al 2007) or radioactive labelled nanoparticles (Liu et

al 2007 Lin et al 2014 Ormsby et al 2014)

It should be noted that ldquonano-biordquo interfaces include the continuous dynamic

physicochemical interactions kinetics and thermodynamic exchanges between the

surface of nanostructured materials and the surfaces of biological components such

as cell membrane permeability conformational flexibility of three dimensional

proteins circulation and respiration activities of blood cells cell adhesion process or

the signal transmission between neuronal cells (Holmes et al 2000 Hong et al

2001 Jung amp Donahue 2007 Mahapatro 2012 Klymov et al 2013 Zhu et al

2013) Thus the study of a material biointerface requires multi-discipline research

efforts in order to gain a complete understanding in this challenging field Firstly the

materials under investigations need to be carefully designed and fabricated to

achieve the desired nanostructure The surface chemical and physical properties

should be comprehensively analysed to confirm the improved characteristics of

nanostructured materials compared to that of their bulk form Thanks to the

continuous development in nanotechnology a number of analytical tools have made

surface characterisation become simpler and faster from macro to atomic scales

17

providing significant improvements in visualising surface structures and analysing

biointerfacial events Throughout this study a range of advanced microscopic and

spectroscopic was extensively performed to characterise the selected nanostructured

surfaces and to analyse the cellular responses to these surfaces (see chapter 3) High

performance computational simulation a merging field between experimental and

computer science was also performed Theoretical simulation has become an

important tool in providing an understanding of the behaviours of a system to

explain the mechanisms of interacts based on mathematical and physical modelling

(Kitano 2002 Southern et al 2008)

222 Concerns regarding nano-cytotoxicity

Along with the abovementioned plethora of benefits that nanostructured

materials are providing to human life there is growing concern regarding the safety

of these materials for human exposure The advantageous properties of many

nanostructured materials have encouraged a large amount of research and the

commercial use of these materials without a significant amount of consideration of

their potential cytotoxicity (Fu et al 2014 Theodorou et al 2014) Up until now an

increasing number of studies have noted the short term toxicity of several types of

nanostructured materials and it is unclear if this toxicity could be tolerated for long

term exposure (Stadtman amp Berlett 1997 Nel et al 2006 Song et al 2010 Khanna

et al 2015) The concern has arisen from the fact that unlike in laboratory

conditions humans may be insecurely exposed to nanostructured materials in their

normal life through a number of different ways including daily inhalation ingestion

or skin and eye contact (Oberdoumlrster et al 2005 Theodorou et al 2014) The

benefits associated with the nanosize of the surface components of these materials

that have been mentioned in previous sections in facilitating their diffusion into cell

membranes allowing them to penetrate into the larger biological system disrupting

regular activities however may also have problematic consequences (Nel et al

2006 Fu et al 2014) For example Zinc oxide (ZnO) is one of the most commonly

used metal oxides in both industrial and commercial applications including skin and

hair care products sunscreens pigments coatings ceramic products and paints (Fan

amp Lu 2005 Blinova et al 2010 Ivask et al 2014) ZnO nanoparticles have

however also been reported to induce the production of reactive oxygen species

(ROS) trigger inflammation inhibit cellular growth and even lead to cell death

18

(Reddy et al 2007 Xia et al 2008) Another example is titanium dioxide (TiO2)

nanorods which can be widely found in photocatalytic applications waste water and

air treatments textiles pharmaceuticals and biomedical fields (Chen amp Mao 2007

Markowska-Szczupak et al 2011 Liu et al 2015b) however TiO2 based products

have also been shown to cause enhanced systemic inflammation and oxidative stress

increased heart rate and systolic blood pressure promoting long term thrombotic

potential and hepatotoxicity in pulmonary exposure conditions (Nemmar et al 2011

Roberts et al 2011) Therefore the importance of the safety of nanostructured

materials should not be underestimated

A key mechanism causing a majority of the toxic effects of nanostructured

materials to cellular functions has been linked to the overproduction of reactive

oxygen species (ROS) (Stadtman amp Berlett 1997 Poli et al 2004 Valko et al

2006) In the regular activities of cellular mitochondria molecular oxygen is reduced

through various oxidative phosphorylation and other oxidative reactions to produce

ATP and water providing energy for multiple activities of cells During this process

some ldquoleakagerdquo of electrons from the mitochondrial respiratory chain may lead to the

incomplete reduction of a small amount of oxygen molecules resulting in the

formation of hydrogen peroxide (H2O2) superoxide anion radicals (O2macrbull) and other

reactive oxygen species (ROS) (Fig 22A) (Yin et al 2012 Madl et al 2014

Khanna et al 2015) It is clear that ROS are the by-products of cellular oxidative

metabolism from which 1-3 of molecular oxygen can possibly turn to superoxide

(Halliwell amp Gutteridge 1986) While superoxide is generally not highly active itself

it will react quickly with the nitric oxide radical (NObull) produced by nitric oxide

synthase to form the potent oxidant peroxynitrite (ONOOmacr) (Stadtman amp Berlett

1997 Fu et al 2014 Khanna et al 2015) Hydrogen peroxide is also a weak

oxidising agent and is therefore poorly reactive but slowly decomposes to form the

highly reactive hydroxyl radical (bullOH) (Barber et al 2006) This can be accelerated

in the presence of reduced metal ions such as ferrous ion Fe 2+ (Fenton reaction)

(Pryor amp Squadrito 1995 Beckman amp Koppenol 1996) Both peroxynitrile and

hydroxyl radicals are highly reactive and can cause oxidative damage to proteins

lipids and DNA (Fig 22B)

19





to hydroxyl radical (bullOH) by cytosolic transition metal cations in the Fenton reaction

(B) Sources (black arrows) and targets (red arrows) of ROS ROS are produced

during oxidative phosphorylation in mitochondria by oxidative enzymes including

cytochrome P450 in the endoplasmic reticulum and by xanthine oxidase (XO) and

reduced metal ions in the cytosol ROS can target and damage cellular components

such as DNA proteins membrane lipids and mitochondria Adapted with permission

from Elsevier (Barber et al 2006)

Cells can tolerate a certain amount of ROS by a self-defence mechanism

including the production of antioxidant enzymes such as superoxide dismutase

catalase and peroxidase (Fridovich 1995 Barber et al 2006 Ivask et al 2014)

Overproduction of ROS triggering by other environmental factors can lead to serious

consequences due to the unregulated physiological redox reactions The destructive

20

effects of ROS to biological system include oxidative modification of proteins to

generate protein radicals (Stadtman amp Berlett 1997) initiation of lipid peroxidation

(Stadtman amp Berlett 1997 Butterfield amp Kanski 2001 Poli et al 2004) DNA-strand

breaks modification to nucleic acids (Bhabra et al 2009 Singh et al 2009

Yamashita et al 2010) modulation of gene expression through activation of redox-

sensitive transcription factors (Shi et al 2004) and modulation of inflammatory

responses through signal transduction leading to temporary or permanent toxic

effects and eventually cell death (Xia et al 2006) DNA is one of the most critical

cellular target of ROS Oxidative DNA damage involves base and sugar lesions

DNA-protein crosslink single and double-strand breakage and the formation of

abasic sites (Valko et al 2006) Highly reactive radicals such as hydroxyl radicals

can damage DNA quickly in the vicinity whereas the less-reactive ROS may interact

with DNA at a distance (Fu et al 2014) This DNA damage can lead to unregulated

cell signalling changes in cell motility cytotoxicity apoptosis and cancer initiation

and promotion (Nel et al 2006 Fu et al 2014 Madl et al 2014 Khanna et al 2015

Soenen et al 2015) It has been demonstrated that ROS and oxidative stress are

associated with many age-related degenerative diseases (Stadtman amp Berlett 1997

Butterfield amp Kanski 2001 Droumlge 2002 Sohal et al 2002 Valko et al 2006)

including amyotrophic lateral sclerosis arthritis cardiovascular disease

inflammation Alzheimerrsquos disease Parkinsonrsquos disease diabetes and cancer

(Kawanishi et al 2002 Valko et al 2007 Yin et al 2009)

Nanostructured materials possess high surface area leading to high

bioactivities upon contact with cellular systems making cells more sensitive to

cytotoxicity induced by ROS An example is the oxidative stress of silica

nanoparticles demonstrated by Akhtar et al in a dose dependant manner mediated

by the induction of ROS and lipid peroxidation in the cell membrane (Akhtar et al

2010) In a later work they also found that nano-CuO induces cytotoxicity in mouse

embryonic fibroblasts releasing lactate dehydrogenase (LDH) and causing similar

oxidative stress (Akhtar et al 2012) Other nanostructured materials made by metal

oxide such as silver (Cronholm et al 2013) iron (Wang et al 2009) and cobalt

(Wang et al 2011b) have also been reported with ROS induced cytotoxicity in

different levels depending on the materialrsquos concentrations time of exposure as well

as their physical and chemical properties

21

Another recognised mechanism is the physical damage of nanostructure

materials which leads to the physically destruction of cell membranes and other

cellular components An example is the penetration of two-dimensional graphene

materials into cell that have attracted a lot attention recently One of the proposed

mechanisms stating that the sharp edges of graphene micro- or nano-sheets can act as

ldquoknivesrdquo to spontaneously pierce through the phospholipid bilayer of cell membrane

causing the leakage of intercellular substances and eventually cell death (Peltonen et

al 2004 Akhavan et al 2011 Dallavalle et al 2015 Mangadlao et al 2015 Yi amp

Gao 2015) A demonstration of this destructive mechanism was shown by the

experimental and simulation work of Li et al 2013 presented in Fig 21FampG

(Section 221) Song et al reported a low toxicity of Fe nanowire however at high

concentrations (10000 nanowires per cell) the nanowires can pierce through the cell

membrane causing disruption to the interior cytosolic matrix (Song et al 2010) An

interesting study of Muumlller et al however claims that the toxicity of ZnO nanorods to

human monocyte macrophages is independent to high aspect ratio nature of the

material The dissolution of ZnO is rather triggered only at a particular lysosomal pH

of 52 leading to fast uptake of the nanorods into cell interior causing Zn2+ toxicity

and eventually cell death (H Muumlller et al 2010) The author suggests that within a

safe delivery range of zinc (8 ndash 11 mg per day for adults) the dissolution rate of ZnO

can be modulated to apply for drug targeting Similar toxic effect of other metal

oxide such as gold nanorods were reported to be potentially beneficial in cancer

diagnostic and therapies (Huang et al 2006 Hauck et al 2008 Patra et al 2009

Raja et al 2010)

Although the risk of cytotoxicity and genotoxicity do exist by studying the

precise mechanism and the parameters inducing the toxic effects efforts have been

made to raise the awareness and to control the mass exposure to potentially toxic

materials Moreover researchers nowadays can control the design of nanostructured

materials to impose either positive or negative effects to different types of cells The

cell-material system can be tailored to suit the different demands of application for

instance it could be fabricated and modified to cause destructive effects to bacterial

cells at the same time to promote favourable effects to human cells and tissue such

as the materials used in implant applications In order to do so the biointerface of

22

these materials needs to be well understood including the effects of versatile surface

parameters to different biological components that would come into play

223 Selected nanostructured surfaces for this studied

In the attempt to contribute to the current knowledge of the biointerfaces of

nanostructured materials three different materials have been selected for this study

including ultrafine grain titanium modified by equal channel angular pressing

graphene thin film constructed by exfoliated graphene nanosheets and nanopillar

arrayed silicon surface generated by reactive ion etching The materials were

selected based on their reported excellent physical and chemical properties that make

them ideal for many prospective applications The modification techniques chosen

for each material have been shown to be able to create specific surface

nanostructures and geometry that can lead to a specific desirable cell response The

bioactivities of these modified surfaces and the respective mechanisms will be

investigated in the following chapters

2231 Ultrafine grain titanium

Titanium has been used in biomedical and implant industry since post-

World War II due to its excellent combination of high mechanical strength low

density high resistance to corrosion complete inertness to body environment low

modulus and enhanced biocompatibility with human bone and other tissues (Boyan

et al 1999 Guillemot 2005 Niinomi 2008 Stynes et al 2008 Truong et al 2010

Von Der Mark et al 2010 Biesiekierski et al 2012) In terms of hard tissue

replacements titanium and titanium alloys are widely used in artificial elbow hip

knee joints and dental implants (Albrektsson et al 1994 Keegan et al 2007 Lee amp

Goodman 2008 Coelho et al 2009 Nasab et al 2010 Siddiqi et al 2011 Wang et

al 2011a Cousen amp Gawkrodger 2012) Among the commonly used titanium based

materials such as commercially pure (cp) titanium (Ti) Ti-6Al-4V Ti-6Al-7Nb Ti-

13Nb-13Zr Ti-12Mo-6Zr-2Fe etc the use of cp Ti is more preferable due to the

long term toxicological effects of most Ti alloys caused by the release of vanadium

and aluminum Both Al and V ions released from the Ti-6Al-4V alloy were found to

be associated with long-term health problems such as Alzheimerrsquos disease

neuropathy and osteomalacia (Eisenbarth et al 2004 Nag et al 2005) In addition

23

vanadium is toxic both in the elemental state and oxides V2O5 which are present at

the implant surface (Maehara et al 2002)

In the last few years researchers have applied a modification technique called

equal channel angular pressing (ECAP) with commercially pure titanium to enhance

the mechanical strength of bulk metallic materials (Ravisankar amp Park 2008

Semenova et al 2008 Valiev et al 2008 Filho et al 2012) Commercially pure

titanium that has undergone ECAP processing has been demonstrated to exhibit

improved tensile (Kim et al 2007a Filho et al 2012 Sordi et al 2012) and fatigue

strength to even greater than that generally achieved by combining alloys with the

metal (Kim et al 2006 Chon et al 2007 Zhang et al 2011 Semenova et al 2012)

The process of ECAP also known as equal channel angular extrusion

(ECAE) was first introduced by Segal and his co-worker in the 1970s and 1980s at

an institute in Minks in the former of Soviet Union (Segal 1974 Segal et al 1981)

In the 1990s reports and overviews began to appear documenting the potential for

using ECAP to produce ultrafine-grained metals with new and unique properties

(Valiev et al 1993 Furukawa et al 2001) The principle of ECAP is shown in Fig

23 (Berbon et al 1999 Nakashima et al 2000) For the die shown in Fig 23 the

internal angle is bent through an abrupt angle Φ equal to 90deg and an additional

angle Ψ equal to 0deg represents the outer arc of curvature where the 2 channels

intersect The sample in the form of a rod or bar is machined to fit within the

channel and the die is placed in the form of press so that the sample can be pressed

through the die using the plunger The nature of the imposed deformation is simple

shear which occurs as the sample passes through the die (Chon et al 2007

Ravisankar amp Park 2008 Zhang et al 2011 Filho et al 2012) As can be seen from

Fig 23a the theoretical shear plane is shown between two adjacent elements within

the sample numbered 1 and 2 these elements are transposed by shear as depicted in

the lower part of the diagram

24


denote the transverse plane the flow plane and the longitudinal plane respectively

Adapted with permission from Elsevier (Nakashima et al 2000)

Despite the interference of a very intense strain as the sample passes

through the shear plane the sample is processed through the die without

experiencing any change in the cross-sectional dimensions Three separate

orthogonal plans are also defined in Fig 23b where these planes are the X or

transverse plane perpendicular to the flow direction the Y or flow plane parallel to

the side face at the point of exit from the die and the Z or longitudinal plane parallel

to the top surface at the point of exit from the die respectively (Berbon et al 1999

Nakashima et al 2000)

Since the cross-sectional area remains unchanged the same sample may be

pressed repetitively to attain exceptionally high strains (Nakashima et al 2000

Furukawa et al 2001 Chon et al 2007 Filho et al 2012) For example the use of

repetitive pressings provides an opportunity to invoke different slip systems on each

consecutive pass by simply rotating the samples in different ways between the

various passes (Segal 1995) Changes in the grain size and mechanical strength of

titanium due to formation of micro- and nanoscale grain structure by ECAP

processing were evaluated in previous work (Chen et al 2010 Truong et al 2010

Dheda amp Mohamed 2011 Zheng et al 2011 Hoseini et al 2012a Hoseini et al

2012b) Valiev et al demonstrated that a reduction of the average grain size from 25

microm to 150 microm can be achieved with commercially pure grade 4 titanium by ECAP

25

followed by a forging and drawing treatment (Valiev et al 2008) As a consequence

of grain refinement the tensile strength of titanium increased from 700 to 1240 MPa

exceeding that for annealed Ti-6Al-4V (940 MPa) (Valiev et al 2008) A superior

fatigue life was also achieved (Valiev et al 2008) Estrin and co-worker

demonstrated a reduction of grain size of commercially pure titanium grade 2 from

45 microm to approximately 200 nm after 4 passes of ECAP followed by polishing with

1 microm diamond paste and colloidal silica (Estrin et al 2009 Estrin et al 2011)

The ECAP-processed material offers two important benefits (Valiev et al

2007) Firstly it makes it possible to avoid the use of expensive and cytotoxic

alloying elements as the required strength can be obtained by grain refinement

rather than by solid solution strengthening and precipitate hardening Secondly the

enhanced strength permits downsizing implant thus making surgery less invasive

This is particularly important in dental implants and orthopaedic products such as

screws and plates (Vinogradov et al 2001 Faghihi et al 2006 Kim et al 2007a)

2232 Graphene film

Graphene is defined as an atomic thick planar sheet of sp2-hybridized carbon

atoms that pack into a two-dimensional (2D) honeycomb lattice made out of

hexagons (Park amp Ruoff 2009 Novoselov et al 2012 Mao et al 2013 Roy-

Mayhew amp Aksay 2014 Perrozzi et al 2015) Due to its excellent physical and

chemical properties including aqueous processability amphiphilicity surface

functionalizability surface enhanced Raman scattering property and fluorescence

quenching ability graphene oxide and graphene have been studied for a wide range

of applications such as field-effect-transistor based biosensors (Ohno et al 2010)

gene delivery system (Chen et al 2011 Kim et al 2011) drug delivery system (Liu

et al 2008) antibacterial substrate (Liu et al 2011a Tu et al 2013) scaffold for

tissue regeneration (Fan et al 2014) and neuron regeneration (Junker et al 2013) A

number of methods have been proposed to synthesise graphene based materials such

as chemical vapour deposition micromechanical exfoliation of graphite also known

as the ldquoScotch taperdquo or peel-off method epitaxial growth on electrically insulating

surfaces and the colloidal suspension method (Lotya et al 2009 Park amp Ruoff 2009

Sengupta et al 2011 Lu et al 2012 Sham amp Notley 2013 Punith Kumar et al

2015) Among these methods colloidal suspension provides a scalable time-

26

efficient affordable and the possibility of mass production for graphene and

chemically functionalized graphene products (Park amp Ruoff 2009 Notley 2012

Sham amp Notley 2013) The exfoliation of graphite powder using cationic and anionic

surfactants has shown to increase the concentrations of resulting graphene

(Haumlllstroumlm et al 2007 Notley 2012 Sham amp Notley 2013) In Chapter 5 an

adaptation of this method will be used to generate graphene thin film as illustrated in

Fig 24 The resulting surfaces possess nanosheet structures which exhibit variable

antibacterial properties







graphene thin film

The interest for the nanostructure of graphene surfaces has risen from recent

studies reporting the antibacterial properties of graphene materials (Peltonen et al

2004 Akhavan amp Ghaderi 2010 Liu et al 2011a Gurunathan et al 2012

27

Krishnamoorthy et al 2012 Tang et al 2013 Tu et al 2013 Hui et al 2014 Yu et

al 2014a) Most of these studies have investigated the antibacterial effects of

graphene oxide (GO) and reduced graphene oxide (rGO) (Liu et al 2011a

Gurunathan et al 2012 Liu et al 2012) combined with silver derivatives (Ma et al

2011 Shen et al 2012 Tang et al 2013 de Faria et al 2014 Yu et al 2014a) or

polymer composites (Park et al 2010 Cai et al 2011 Santos et al 2011 Tian et al

2014 Wang et al 2014) The mechanism responsible for the antimicrobial action of

graphene products continues to be a subject of debate The discussion mainly focuses

on two points the first emphasizes the role of sharp edges of graphene micro or

nanosheets which act as ldquobladesrdquo to cut through the cell membrane causing the

leakage of intercellular substances and eventually cell death (Akhavan amp Ghaderi

2010 Hu et al 2010a Akhavan et al 2011 Liu et al 2011a Li et al 2013b Tu et

al 2013 Wu et al 2013 Tian et al 2014) This mechanism is sometimes referred to

as the lsquoinsertion modersquo or lsquomembrane stress effectrsquo which was described in several

theoretical simulations and experimental studies An example is the work of

Akhavan et al who reported the direct contact between the bacterial cell wall and

sharp edges of GO and rGO is the cause of their bactericidal activities against Gram-

negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria

(Akhavan amp Ghaderi 2010) This group also reported bacterial inactivation by

aggregated GO nanosheets through a trapping mechanism preventing them from

exchanging materials with outer environment and cell division (Akhavan et al

2011) A detailed mechanism of this insertion mode was described by several

computational simulations however inconsistencies in the data have been reported

The first reported work of Li et al suggested a spontaneous localized piercing of the

graphene microsheets at the sharp edges and corner sites followed by full penetration

into the lipid bilayer membrane (Li et al 2013b) Their simulations showed that the

nearly orthogonal orientation of graphene sharp edges with respect to phospholipid

bilayer had the lowest interactive free energy and was therefore the most preferred

penetrating configuration These findings were supported by Yi et al who further

developed that the graphene sheets in micro-size preferred to adopt a near-

perpendicular configuration whereas the nanosized sheets required a parallel

position of the entire sheet along the lipid bilayer to be embedded into the cell

membrane due to the hydrophobic attraction between the lipid tails and the graphene

surface (Yi amp Gao 2015) These results are however in contrast with Dallavallersquos

28

model which demonstrated that within the nanometer range the smaller the

graphene sheets the more freely they could diffuse into the lipid membrane and

preferentially adopt a perpendicular orientation while the larger nanosheets

preferred to arrange themselves across the membrane embedding themselves into

the hydrophobic part of the membrane (Dallavalle et al 2015) It should be noted

that these theories have been proposed based on computational modellings and have

not yet been supported by experimental data

The second theory however states that the destructive effect of graphene

layers arises from their chemical properties The recent work of Mangadlao et al

argued that the antimicrobial efficiency of graphene is independent to the sharp

edges but relies on the contact between the GO basal planes and microorganisms

(Mangadlao et al 2015) This work reported an 89 killing efficiency of GO film

against E coli while eliminating the exposure of GO sharp edges to bacteria by using

the Langmuir-Blodgett depositing method The similar work of Hui and co-workers

also demonstrated that masking of the GO basal plane would decrease the

antimicrobial efficiency of the GO nanosheets by decreasing the direct contact

between E coli and the GO basal plane (Hui et al 2014) A few mechanisms have

been suggested for this mode of action including reactive oxygen species

(Gurunathan et al 2012) oxidative stress (Liu et al 2011a Hui et al 2014) or direct

extraction of the phospholipid membranes (Li et al 2013b Tu et al 2013) Another

recent model of Luan et al demonstrated that the hydrophobic nature of graphene

could disrupt the hydrophobic protein-protein bonding leading to the destabilization

of the protein complex consequently causing functional failure (Luan et al 2015) A

killing mechanism based on bacterial metabolic activity which could reduce GO to

bactericidal graphene through their glycolysis process was also reported (Akhavan

amp Ghaderi 2012 Nanda et al 2016) The mechanisms suggested in most of these

works similar to those supporting the mechanism of ldquoinsertion moderdquo are mainly

based on theoretical data and hence further work is required in this area

2233 Black silicon

Natural surfaces that possess high aspect ratio features frequently display

unique properties For example the Psaltodaclaripennis cicada wing surfaces have

been shown to exhibit both superhydrophobicity and bactericidal activity against

29

Gram-negative bacteria where significant physical deformation accompanied

inactivation (Ivanova et al 2012 Hasan et al 2013b) Diplacodesbipunctata

dragonfly wings exhibited a broad spectrum bactericidal activity against Gram-

negative and Gram-positive bacteria even their spores Inspired by nature a

synthetic analogue of dragonfly wings known as black silicon was fabricated

using deep reactive ion etching (DRIE) (Ivanova et al 2013)

DRIE is common technique used to fabricate high-aspect-ratio features in

substrate surfaces (Laermer amp Urban 2003 Xie et al 2011 Krivitsky et al 2012

Xie et al 2012) The DRIE-process was firstly invented at Bosch and later on further

developed together with Surface Technology Systems Plc (UK) and Alcatel

Vacuum Technology (France) with the implementation of inductively coupled

plasma (ICP) tools (Laermer amp Urban 2003 Huang et al 2007) This process was

found to result in a novel method to etch surfaces an alternative to classical wet

etching This method uses chemically reactive plasma to remove material deposited

on wafers The plasma is generated under low pressure (vacuum) by an

electromagnetic field High-energy ions from the plasma attack the wafer surface

causing a surface reaction In the standard approach all gas species are introduced at

the same time and the etching results depend on the glow discharge having one

radical species present to achieve the surface etching and another present to protect

the side walls during the process (Laermer amp Urban 2003 Gervinskas et al 2013)

In this study a gas mixture of SF6 and O2 was used to fabricate the high-aspect-ratio

features of the black silicon (Sainiemi et al 2007 Wu et al 2010) By adjusting the

O2 and SF6 flow rates in the plasma etching process different surface morphologies

of the high-aspect-ratio structure can be obtained (Fig 25)

30



ions of gas mixtures (2) toward the surface of the samples (5)

The black silicon surface is comprised of a nanopillar array structure with a

specific geometry that leads to its excellent antibacterial properties The integrity of

bacterial cells is disrupted by the action of the nanopillar arrays indicating a physical

deformation leading to membrane stress and eventually cell death This effect was

proven to be independent of surface chemistry and hydrophobicity and apparently

only mechanical in nature (Hasan et al 2013b Ivanova et al 2013) The

antibacterial effects achieved without the need for antibiotics or other chemical

additives have made the topography of black silicon become a prospective candidate

for the design of biomedical device surfaces The effects of black silicon on

mammalian cells however remain unknown Reports of cell responses to other high

aspect ratio surfaces that are available in the literature also remained controversial

as discussed in Section 24 thus these issues warranted further investigation In the

next sections the current knowledge regarding the interactions that take place

between bacteria and mammalian cells with these nanostructured surfaces will be

discussed in details

23 Bacterial interactions with nanostructured surfaces

Bacterial colonisation onto surfaces has long been a focus of extensive

research due to its impact upon various aspects of life Successful bacterial

- - - - - - - -

- -

31

colonisation often leads to the formation of a biofilm which subsequently causes

contamination in plumbing systems oil refineries paper mills housing systems

clinical devices and other infrastructure (Costerton et al 1999 Donlan 2001

Subramani et al 2009) Marine fouling is precipitated by the formation of bacterial

biofilm on the hulls of ships followed by the attachment of progressively larger

marine organisms This fouling increases the cost of fuel by increasing the drag of

seafaring vessels by up to 40 (Alexander et al 2013) In clinical practices biofilms

are the main cause of persistent infections triggering vigorous immune responses

releasing of harmful toxins into human systems leading to device failure and even

death (Donlan amp Costerton 2002 Costerton et al 2005 Ploux et al 2010) Due to

these serious consequences many years of research have been dedicated to find

more efficient methods to prevent bacterial contamination and infection These

preventive methods would not only benefit various industries but more importantly

to improve the quality of life for humans To date the vast majority of strategies

used to prevent bacterial infection and biofilm formation are generally classified into

two main categories including bactericidal materials of which the surfaces can be

designed to release antimicrobial compounds or antifouling materials which are

capable of inhibiting bacterial adhesion This section will summarise the current

accepted mechanisms responsible for biofilm formation and their subsequent clinical

impacts followed by a discussion of the current approaches being used for the

treatment of bacterial infections

231 Bacterial colonisation

2311 Mechanisms responsible for bacterial colonisation

The initial adhesion of bacteria to the surface of a substrate material is

believed to be the critical event in the pathogenesis of foreign body infections

(Gristina 1987 Costerton et al 1999 Davies 2003 Harris et al 2004 Hetrick amp

Schoenfisch 2006 Moriarty et al 2011 Singh et al 2012b) It appears that only a

low dose of inoculum is required to result in the infection of an implant In an animal

model study it was found that 100 colony forming units (cfu) of S aureus were

sufficient to infect 5 of the subcutaneous implants (Zimmerli et al 1982 Zimmerli

2006) Most of the microorganisms causing implant infections are present in the host

flora of which the most frequent are Staphylococci Streptococci Pseudomonas

32

species and coliform bacteria (Rupp amp Archer 1994 Boulangeacute-Petermann et al

1997 Davies 2003 Costerton et al 2005 Harris amp Richards 2006 Mitik-Dineva et

al 2009 Gasik et al 2012)

In the process of biofilm formation bacterial cells undergo five different

phases of surface adhesion co-aggregation and colonization as described in Fig 26

A bacterial biofilm is a self-organised community encapsulated in an extracellular

polymeric substance (EPS) layer composed of polysaccharides proteins and other

metabolic products Bacteria within a biofilm maintain their own communication

channels metabolic flows and a highly flexible genetic exchange between colonized

bacteria in response to any unfavorable changes in environmental conditions

(Costerton et al 1999 Donlan 2001 Davies 2003 Costerton et al 2005) These

mechanisms were demonstrated in early reports that showed the significant

differences in phenotypic and genotypic characteristics of bacteria when they are in

the sessile and planktonic stages (Donlan amp Costerton 2002 Davies 2003) These

phenotypic and metabolic adaptations enable bacterial communities to become much

more resistant to immune systems antimicrobial stresses as well as

chemotherapeutic treatments (Costerton et al 1999 Donlan 2001 Campoccia et al

2006 Subramani et al 2009 Zhao et al 2009 Singh et al 2012b)

33








biofilm and are distributed to the surrounding environment Adapted with permission

from Annual Reviews and Elsevier (Stoodley et al 2002 Rosche et al 2009)

There are many powerful antibiotics and antimicrobial agents that have been

developed to treat infections since the first discovery of penicillin in 1928 Despite of

the remarkable commercial success of these treatments in terms of their efficiency

and patient recovery serious consequences arising from bacterial infection are still

frequently reported due to the fact that once bacteria have developed a biofilm they

are several orders of magnitude more difficult to eliminate from the colonised

34

surfaces compared to when they are present in a planktonic form Thus it has been

suggested that the most critical step in preventing biomaterial-associated infections is

to prevent the initial attachment of bacteria thus prevent the formation of a biofilm

(Costerton et al 1999 Clohisy et al 2004 Esposito amp Leone 2008 Levent et al

2010 Ploux et al 2010 Moriarty et al 2011)

2312 Impacts of bacterial infection

An ever-increasing demand for implants makes it imperative that

development efforts in the area of biomaterials have been accelerating The need for

implants in dental spinal hip and knee replacements arises as a result of the damage

or degradation of the mechanical properties of bones due to excessive loading or a

deficiency in the normal biological self-healing process being present (Niinomi

2008 Geetha et al 2009 Biesiekierski et al 2012 Vanderleyden et al 2012) With

an aging global population and the desire for an active lifestyle the demand for such

implants is expected to increase It was estimated that 800000 total hip and total

knee arthroplasties were performed in the United States in 2006 alone (Zimmerli

2006 Del Pozo amp Patel 2009) This increase in implants was associated with a

corresponding increase in bacterial infections one of the most serious challenge in

clinical practice especially in the implantation of biomedical devices (Donlan 2001

Mela et al 2001 Clohisy et al 2004 Costerton et al 2005 Lucke et al 2005 Del

Pozo amp Patel 2009 Levent et al 2010) In a report of Del Pozo and Patel (shown in

Fig 27) in total hip arthroplasty operations the number of which is increasing up

to 13 of recipients were treated for infections while in total knee arthroplasty

operations reported primary infections were up to 2 of total operations (Del Pozo

amp Patel 2009) In another report about 10 of the arthroplasties performed required

revision at a later date due to implant failures (Kurtz et al 2008) with 8ndash15 of

these revision operations being a direct result of an infection (Kurtz et al 2008

Puckett et al 2010) Implant-related infections were linked with a mortality rate of

7ndash63 for total hip arthroplasty operations and 25 of total knee arthroplasty

operations Similarly an average infection rate of 2ndash5 was reported for joint

prosthesis operations and fracture-fixation devices (Darouiche 2004) In another

report which involved an eight-year analysis of dental implants an implant failure

rate of 2ndash3 in America arose as a result of bacterial contamination (Costerton et al

2005)

35


resulting from surgery as a function of year of operation (Reproduced with

permission from (Del Pozo amp Patel 2009) Copyright Massachusetts Medical

Society)

The complications associated with of implant-associated infections are due

to the resistance of pathogenic bacteria to the host defence system and the antibiotics

being used to treat the infection This resistance often leads to the failure of the

treatments without surgical intervention (Zimmerli et al 1982 Mela et al 2001

Schierholz amp Beuth 2001 Zimmerli 2006 Norowski Jr amp Bumgardner 2009

Subramani et al 2009 Zhao et al 2009 Neoh et al 2012) It has been estimated that

orthopaedic surgical site infections prolonged total hospital stays by a median of 2

weeks per patient approximately doubled the rehospitalisation rates and increased

healthcare costs by more than 300 (Whitehouse et al 2002) Patients with

orthopaedic surgical site infections were found to experience significant reduction in

their quality of life limitation in their physical functions with some cases requiring

the removal of the implant or even death (Whitehouse et al 2002 Campoccia et al

2006 Hetrick amp Schoenfisch 2006 Qiu et al 2007 Del Pozo amp Patel 2009) Long-

term tragic consequences of bacterial infection has urged the search for more

effective methods in treating and more importantly in preventing biomaterial

infections

36

232 Current approaches in preventing bacterial infections

A variety of approaches have been developed for the construction of

biomaterials that can exhibit improved antibacterial properties and at the same time

support the integration of the host tissue The resulting biomaterials have improved

the success rates of implants which is not only advantageous for the patients but

also alleviates the economic burden of implant-related infections on society

(Costerton et al 1999 Davies 2003 Darouiche 2004 Costerton et al 2005

Norowski Jr amp Bumgardner 2009 Neoh et al 2012) Administration of peri-

operative antibiotic prophylaxis has become a routine procedure in orthopaedic

surgery to reduce infection rates (Seymour amp Whitworth 2002 Lucke et al 2005

Schmidmaier et al 2006 Esposito amp Leone 2008 Vester et al 2010) Systemic

delivery of antibiotics may however raise concerns about later renal and liver

complications (Darouiche 2004 Costerton et al 2005) To achieve a long term

release of antibiotics without exceeding the limit that can result in local toxicity

delivery methods such as antibiotic coatings and antibiotic loaded cements have been

used (Langlais et al 2006 Schmidmaier et al 2006) A major problem associated

with antibiotic prophylaxis is the possibility that these compounds will contribute to

the development and spread of antibiotic resistant organisms such as methicillin-

resistant Staphylococcus aureus (MRSA) (Costerton et al 1999 Poelstra et al 2002

Seymour amp Whitworth 2002 Davies 2003 Darouiche 2004 Costerton et al 2005

Campoccia et al 2006)

In view of this concern much effort in recent years has focused on the

development of anti-infective implant surfaces that do not rely on antibiotics but

instead rely on the modification of the physicochemical properties of the implant

material such that the surface topography interferes with the microbial colonization

process (Jung amp Donahue 2007 Coelho et al 2009 Anselme et al 2010 Bacakova

et al 2011 Wu et al 2011 Almaguer-Flores et al 2012 Singh et al 2012b May et

al 2016) The current designs for antibacterial material surfaces can be classified

into two major groups according to their mode of action The first is antifouling

surfaces which have the ability to repel or prevent bacteria from adhering to their

underlying substrata The second is bactericidal surfaces which have the ability to

damage or kill any pathogenic bacteria coming into contact with the surface

(Campoccia et al 2013b a)

37

2321 Antifouling surfaces

As previously mentioned it is clear that an infection arising from the

presence of pathogenic bacteria on an implant would not have occurred if the

bacteria involved were unable to initially colonise the medical device The complex

mechanisms associated with bacterial attachment have long been studied in order to

gain an understanding into the methods by which antibacterial surfaces can be

designed such that this event can be prevented A wide range of chemico-physical

properties and methods for attaching functional groups onto both the substrate and

pathogens have been modified in order to modulate the attachment of these bacteria

(Fusetani 2004 Bazaka et al 2011 Fusetani 2011 Webb et al 2011a Bazaka et al

2012 Hasan et al 2013a) More recently surface architectures that contain specific

surface porosity roughness and geometry have been used to produce biomaterial

surfaces that are resistant to microbial colonisation (Anselme et al 2010 Webb et al

2011a Bazaka et al 2012 Crawford et al 2012 Meng et al 2014)

Biomaterial devices are often exposed to body fluids and a rich protein

environment at the site of surgical implantation (Arciola et al 2003 Campoccia et

al 2013a b) It is known that a variety of host proteins can promote bacterial

attachment and the subsequent formation of biofilms These microbial surface

components have the ability to recognize adhesive matrix molecules or

MSCRAMMs and include collagen fibrinogen fibronectin laminin vitronectin

clumping factor A and B bone sialoprotein elastin IgG and other possible

components (Patti et al 1994 Foster amp Houmloumlk 1998 Hauck et al 2006 Lambris et

al 2008 Montanaro et al 2011 Arciola et al 2012 Lv et al 2013 Foster et al

2014) Biomaterial surfaces are therefore required to support the adsorption of host

adhesins onto their surface to ensure the successful subsequent integration of tissue

whilst at the same time being able to repel the bacteria

Chemical methods can be used to construct microbe-repellent surfaces by

attaching antifouling molecules to the surfaces of implant materials Common

chemical modification approaches include rendering the surfaces superhydrophobic

superhydrophilic or coating them with highly hydrated or non-charged chemicals

each of these being unfavourable for bacterial adhesion under certain circumstances

(Fig 28)

38


in the design of antibacterial surfaces (Adapted with permission from Elsevier

(Campoccia et al 2013a)

One of the most common coatings to render a surface hydrophilic is

poly(ethylene glycol) (PEG) The inhibition mechanism of such PEG-containing

surfaces is based on the dynamic motion and steric repulsion of hydrated polymer

chains which prevents bacterial attachment (Harris et al 2004 Maddikeri et al

2008) In addition polycationic polymers exhibiting antifouling effects have been

used by directly coating or grafting them onto biomedical devices (Chua et al 2008

Shi et al 2008 Hu et al 2010b Subbiahdoss et al 2010c Siedenbiedel amp Tiller

2012) Heparin coatings have also been shown to exhibit a high antiadhesive effect

for bacteria by increasing the hydrophilicity of the surfaces The heparin forms a

highly hydrated layer between the pathogens and the substrate (Ruggieri et al 1987

Arciola et al 1993) In this work it was reported that the heparin could inhibit the

extent of S epidermidis binding to fibronectin thus preventing the subsequent

colonisation of the surface

Another approach where quorum-sensing inhibitors (eg furanones and their

derivatives) are incorporated onto biomedical device surfaces was used to disrupt

the processes responsible for the formation of a biofilm (Fig 28) (Fusetani 2004

39

2011) This approach however has significant drawbacks in terms of the long-term

stability of the coating and the possible cytotoxicity of these additives in biomedical

applications Current approaches use surface topography as the factor by which the

degree of bacterial adhesion and subsequent biofilm formation can be controlled or

prevented Techniques such as this represent a more robust method for creating

surfaces that repel or control the extent of microbial attachment (Webb et al 2011a

Bai amp Liu 2012 Bazaka et al 2012 Crawford et al 2012 Hasan et al 2015) For

example superhydrophobic surfaces have been shown to exhibit antifouling

characteristics and can be obtained by physically modifying the micro- and

nanostructures of biomaterial surfaces by mimicking natural surface structures such

as that of the lotus leaf (Crick et al 2011 Fadeeva et al 2011 Truong et al 2012)

By tailoring the precise and specific surface topographical parameters these surfaces

have shown promising results in their ability to limit the initial adhesion of

pathogenic bacteria

2322 Chemically bactericidal surfaces

Another common approach in the prevention of biofilms on biomedical

devices is the utilization of bioactive antibacterial agents that act by killing the

bacteria upon contact These techniques involve coating the substrate with various

immobilized antimicrobial substances such as antibacterial peptides (Brouwer et al

2011 McCloskey et al 2014 Salwiczek et al 2014) (Mei et al 2012 Schaer et al

2012) nitric oxide (Nablo et al 2005 Fox et al 2010) or antibacterial metals such

as silver zinc cobalt aluminium and copper (McLean et al 1993 Kawashita et al

2000 Heidenau et al 2005 Wan et al 2007 Prantl et al 2010 Lemire et al 2013

Stafford et al 2013) These substances are not released from the substrate thus they

can directly interact with any pathogenic bacteria coming in contact with the surface

(Williams amp Worley 2000) Bioactive antibacterial coatings have been used

extensively in applications that require the surface to be self-sterilizing over

extended periods (Williams amp Worley 2000 Campoccia et al 2013a)

Silver and its derivatives are some of the earliest bactericidal agents that have

been largely applied in a wide range of applications (Richards 1981 Dueland et al

1982 McLean et al 1993 Nomiya et al 1997 Kawashita et al 2000 Zhao et al

2009 Bayston et al 2010) Other metals that have also been reported to exhibit

40

bactericidal effects mostly in their composite form including zinc cobalt

aluminium and copper (Wan et al 2007 Wang et al 2007 Prantl et al 2010

Samanovic et al 2012 Stafford et al 2013) The use of antimicrobial metals is

however often associated with a certain degree of cytotoxicity This can have an

impact on the host cell response leading to the loss of cell viability and the failure of

tissue integration This occurs mainly as a result of corrosion of the metal in the

physiological environment which causes the release of metal ions at relatively high

concentrations leading to local toxicity and occasionally metal accumulation in the

target organs (Vasilev et al 2009 Campoccia et al 2013b Lemire et al 2013) The

mechanisms responsible for the antibacterial activity of metals and metal ions are not

fully understood Gordon et al suggested that silver interacts with thiol groups

causing the inactivation of critical enzymes in the respiratory chain and the induction

of hydroxyl radicals (Gordon et al 2010)

Another emerging strategy for the manufacture of antimicrobial surfaces is

the incorporation of biocide-releasing surfaces such as those containing

nanoparticles The extent of the bactericidal effect of these surfaces depends on the

size shape concentration and chemical composition of the nanoparticles (Cui et al

2012 Hajipour et al 2012 Zhang et al 2013) While the exact mechanisms of the

antimicrobial activity are also not fully understood most nanoparticles are seen to

generate reactive oxygen species and damage the cell membranes (Cui et al 2012

Hajipour et al 2012 Zhang et al 2013) For example gold nanoparticles exhibit

bactericidal effects against E coli by inhibiting ATP synthase activity followed by

the inhibition of the ribosome subunit in tRNA binding (Cui et al 2012) There is

still a lack of knowledge on the toxicology of nanoparticles with most of the

available data being inconsistent and largely non-reproducible (Yildirimer et al

2011 Campoccia et al 2013a) The negative impact of nanoparticles in biomedical

applications includes the induction of apoptosis introduction of toxic effects to the

genome and the possible translocation of nanoparticles to distant tissues and organs

with an associated risk of systemic effects (Yildirimer et al 2011 Campoccia et al

2013a) The major problem however is that biofilms display an increased tolerance

towards antimicrobial agents which substantially restricts the ability to treat biofilm

ndash related infections in clinical settings While the increased resilience of biofilms

towards antibiotics is multifactorial this resistance can be attributed to the presence

41

of persistent bacteria those that can enter into a specific phenotype state that allows

them to survive in the presence of 1000 times the minimum inhibitory concentration

of bactericidal antibiotics (Olson et al 2002 Davies 2003) Persistent cells have

recently been the subject of increased investigation with a view to limiting their

biofilm-associated antibiotic tolerance The more preferable strategy for preventing

the formation of biofilms is to develop ways by which the initial bacterial adhesion

step can be inhibited which will subsequently limit the growth of the biofilm

(Fusetani 2004 Hasan et al 2013a)

2323 New approach mechanically bactericidal surfaces

For the reasons previously described surfaces which could be designed to

exhibit antimicrobial properties without relying on its physico-chemical

characteristics would represent a significant step forward in developing antibacterial

implants (Webb et al 2011a Crawford et al 2012 Hasan et al 2013a Ivanova et

al 2013 Denisov et al 2016 Sjoumlstroumlm et al 2016 Sugnaux amp Fischer 2016 Wu et

al 2016a) This idea has led to an era of researching new material surfaces that can

physically disrupt or prevent bacterial colonisation by tailoring the surface

topography and architectures Numerous promising results have been reported in the

last decades with respect to surfaces that possess micro and nano-structures

generated onto different materials such as polymers semiconductors and metals to

serve various applications A few examples are presented in Fig 29

42










black silicon with the pillar of 500-600 nm height Licence agreement can be found

from Elsevier (Vasudevan et al 2014) (Bhadra et al 2015)

httpcreativecommonsorglicensesby40) (Reproduced with permission from

(Chung et al 2007) Copyright 2007 American Vacuum Society) (Valle et al 2015)

(Ercan et al 2011) (Ivanova et al 2013)

httpcreativecommonsorglicensesby40) Scale bar are 5 microm insert is 2 microm

Vasudevan et al demonstrated a reduced number of adherent bacteria on a

range of micropatterned polydimethylsiloxane (PDMS) surfaces produced by

photolithography (Vasudevan et al 2014) They observed that Enterobacter cloacae

a bacterium responsible for catheter associated urinary tract infections have less

surface coverage on a variety of micropatterned surfaces including cross pillars

hexagonal pits hexagonal pillars and SharkletTM pillars compared to smooth PDMS

surface The most efficient pattern was recorded to be the cross micropillar structure

43

(Fig 29A) by a significant 89 reduction of bacterial coverage with respect to the

flat control surface The authors suggested that a possible mechanism is due to

spontaneous attachment of bacteria to the recessed regions only when approaching a

patterned surface which could possibly reduce the overall percentage surface areas

of bacterial fouling (Vasudevan et al 2014) Similarly Ercan et al showed a lower

bacterial viability on titanium surfaces with nanotube structure ranging from 20 nm

to 80 nm fabricated by anodization method (Fig 29E) They found that the 80 nm

Ti nanotube surface combined with heat treatment exhibited highest antimicrobial

capacity against S aureus and S epidermidis compared to the surfaces with either

larger surface features without heat treatment or non-modified surfaces (Ercan et al

2011) Another work of Bhadra et al performed also with titanium substrata

showed a selective bactericidal effect of nanowire structured titanium with an

average nanowire bundle size of 402 nm (Fig 29B) The surface exhibited 50

killing efficiency against P aeruginosa while this value is 20 against S aureus

while exhibiting positive responses to fibroblast cell attachment and proliferation

(Bhadra et al 2015) Chung et al presented a Sharklet AFTM design (Fig 29C) a

biomimetic microstructure of shark skin on PDMS elastomer substrata which can

delay the biofilm maturation process (Chung et al 2007) They showed that S

aureus required triple the amount of time to connect the isolated multilayered

colonies between the recessed and protruding features and fully cover the Sharklet

AFMTM structured surfaces with biofilm compared to the time required for attaching

to smooth surface The authors suggested that this delay would be beneficial for the

host immune system to have a higher chance in eliminating the bacteria at the early

adhesion stage The host cell can primarily integrate with the surface before

appreciable bacterial biofilm appears however the mechanism of this effect is

unknown (Chung et al 2007) A reduction in S aureus adhesion on a lamella

microstructure of polystyrene film (Fig 29D) under both static and fluid flow

conditions was also reported but the mechanism responsible was also unclear (Valle

et al 2015)

Among most of these surface structures the antibacterial effects were proven

either being low to moderately effective or being selective depending on some

critical factors such as bacteria species contact time or the requirement of additional

treatments One of the more promising surfaces which has been proved to efficiently

44

killed a broad range of bacterial species including Gram-negative Gram-positive and

their spores in a purely mechano-responsive manner is the nanopillar structure of

black silicon surface introduced by Ivanova et al (Fig 29F) (Ivanova et al 2013)

The surface was inspired by the natural self-cleaning bactericidal surface structure

of insect wings such as those of cicada (Psaltoda claripennis) and dragonfly

(Diplacodes bipunctata) wings (Ivanova et al 2012 Pogodin et al 2013) and was

shown to possess comparable antibacterial activities In the current work the effects

of black silicon nanopillar nanostructures on mammalian cell behaviour were

investigated to provide an insight into the potential use of the black silicon surface

nanostructure in biomedical applications Current knowledge of the effects of similar

nanopillarnanowire structured surfaces on mammalian cell activities will be

discussed in the next section

24 Mammalian cell interactions with nanostructured surfaces

The mammalian cell is a unique self-regulating self-replicating micro-

system wherein various proteins are synthesized and spontaneously or actively

assembled to construct the cellrsquos structure and regulate its functionality (Geiger et al

2001 Sniadecki et al 2006 Bryant amp Mostov 2008) Nanotechnology has emerged

to be as useful tool in the pursuit of an understanding of the fundamental

relationships between cells and their underlying substrates (Sniadecki et al 2006)

The appropriate understandings of the cellular systems combined with modern cell

manipulation techniques provide researchers the ability to control alter or reverse

various biological activities thus offer solutions to problems such as those relate to

disease cancer or infection issues (Boyan et al 1999 Valiev et al 2007 Anselme

2011 Tay et al 2011)

It has been established that cells can sense and respond to nanotopographic

cues in an explicit and selective manner Engineered nanostructured surfaces often

act as external chemical and physical stimuli to the bacteria triggering the

development of the extracellular matrix (ECM) inducing the cell-cell

communications and trigger signalling cascades that lead to a specific cellular

response (Sniadecki et al 2006 Wang amp Lin 2007 Zhu et al 2013) High aspect

ratio materials are among the most common nanostructured materials that possess

unique characteristics (Qi et al 2009 Robinson et al 2012 Gervinskas et al 2013

Bonde et al 2014 Dasgupta et al 2014 Elnathan et al 2014) Physical and

45

chemical parameters of the nanostructured surfaces can be precisely controlled to

manipulate complex cellular functions including cell adhesion migration

proliferation and differentiation (Bettinger et al 2009 Brammer et al 2011 Kim et

al 2012b Mendes 2013 Na et al 2013 Piret et al 2014 Prinz 2015) An increasing

number of recent studies have investigated the interactions of high aspect ratio

surfaces with various cell types however the specific responses of each cellular

system were reported with high levels of inconsistency mainly due to complex

parameters involved from both the nanomaterials and the biological system under

investigation (Stevens amp George 2005 Kim et al 2007b Qi et al 2009 Shalek et

al 2010 Roberts et al 2012 Robinson et al 2012 Kim amp Yang 2013 Bonde et al

2014 Elnathan et al 2014 Lee et al 2014 Prinz 2015) In the following sections

the current understandings on the interactions between high aspect ratio surfaces and

mammalian cells will be summarised focusing on the effects of this surface

nanostructure to the process of cell adhesion proliferation and differentiation

241 Cell attachment spreading and migration

Cell adhesion is mediated by large protein scaffolds known as focal adhesion

points These adhesion points are tightly associated with an actin cytoskeleton and

together they control a range of cellular responses such as morphology migration

and adhesion which cells use both for sensing and responding to their environment

(Burridge amp Chrzanowska-Wodnicka 1996 Cukierman et al 2001 Geiger et al

2001 Bonde et al 2014) When foreign materials are inserted into the body such as

implant or medical devices a complex series of biological events occur at the

material surface Water molecules bind to the surface and incorporate hydrated ions

such as Cl- Na+ and Ca2+ followed by the adsorption of a protein layer produced by

the blood plasma (Stevens amp George 2005 Sniadecki et al 2006 Anselme 2011

Neoh et al 2012) The exact mixture of adsorbed proteins and their conformational

states are largely controlled by the material surface and the proteins mediating the

subsequent cellular adhesion Blood cells at the surface of the implant are activated

and release cytokines and other soluble growth and differentiation factors which

will later regulate a host of biological events including cell proliferation and

differentiation (Amano et al 1997 Sniadecki et al 2006 Humphries et al 2007

Anselme 2011 Bacakova et al 2011 Neoh et al 2012)

46

Regarding to the study of cell behaviours on nanopillar structured surfaces it

has been reported that cell adhesion greatly depends on the dimension of nanopillars

present on the surfaces For example a study of Kim et al showed that nanowires of 6

microm in length and 09 microm in diameter are able to promote the growth of mouse

embryonic stem cells and human embryonic kidney cells (HEK 293T) for up to 7 days

despite their spontaneous penetration into the cells (Kim et al 2007b) In contrast Kim

and Yang demonstrated that similar nanowires (58 microm) were less favourable for the

attachment and spreading of human cervical cancer (Hela) cells than those observed on

medium (360 microm) and short (130 microm) nanowires of a similar diameter (~ 1 microm)

determined by the lower number of attached cells accompanied with the decreased

expression of focal adhesion complex (Kim amp Yang 2013) Another contradicting

behaviour is presented in a report from Li and co-workers who quantified the traction

forces of Hela and L929 cell lines versus primary mechanocytes concluding that the

cancer cells exhibited up to 50 larger traction forces than primary mammalian cells on

silicon nanowires (3 microm in length 140 microm or 280 microm in diameter) which is likely lead

to enhanced cell migration (Li et al 2009) Similar silicon nanowires were however

shown to favour the adhesion of human hepatic cells but restricted cell spreading due to

the relative large interval space between the nanowire clusters making it difficult for

cells to reach out from the first local contact nanowire clusters (Qi et al 2009)

The discrepancy exists not only in the case of silicon materials Piret et al

reported that gallium phosphide nanowires (4 microm in length and 80 nm in diameter) at

different densities did not exhibit significant effects on the growth of glial cells (Piret

et al 2013) Meanwhile a report of neuron cell interactions with gallium phosphide

nanowires however demonstrated an extended axonal outgrowth of various cell types

including peripheral sensory neurons Schwann cells fibroblasts and satellite cells

(Haumlllstroumlm et al 2007) From these inconsistencies it is clear that not only the aspect

ratio but other parameters such as density spatial distribution clustering capacity

and specific geometry of the nanowires or nanopillars would exhibit their own

effects on the cellular responses of different cell types which would require further

investigation (Kim et al 2007b Shalek et al 2012 Kim amp Yang 2013 Piret et al

2013)

Some of the later reports have emphasized the important effects of the surface

nanopillar density to the adhesion of cells While medium and low density nanopillars

have been largely shown to support (and in some cases promote) cell adhesion (Abdul

47

Kafi et al 2012 Bezuidenhout et al 2014 Chang et al 2014) high density nanopillar

surfaces were in some cases able to support cell adhesion but were generally observed to

inhibit cell adhesion (Choi et al 2007 Qi et al 2007 Lee et al 2009 Qi et al 2009

Sjoumlstroumlm et al 2009 Zhao et al 2010) Kim et al demonstrated that 90 of seeded

cells were able to be retained on a nanopillar substratum while the flat control surfaces

captured less than 25 of the cells (Kim et al 2012b) The reversible detachment of

cells from nanopillar surfaces has been investigated under dynamic flow or increasing

centrifugal speed conditions which demonstrated that the nanopillar surfaces

significantly reduced the extent of cell detachment (Qi et al 2009 Chang et al 2014

Elnathan et al 2014) It has been suggested that the difference in surface areas caused by

the different dimensions of the nanowire substrates is the key factor explaining the

variable adhesion behaviours (Bonde et al 2014) When contacting a high density of

nanopillars or nanowires cells are forced to adhere directly to the nanopillars themselves

and are not able to reach the underlying flat surfaces thus experiencing a reduced

available contact area (Qi et al 2009) This leads to a reduced extent of cell-surface

adhesion It was shown that the focal adhesion points were preferably formed on the

surface between the nanopillars (Chang et al 2014) If this surface area was too small to

ensure the formation of an adequate number of focal adhesion points the cells were not

be able to adhere to the surface

It was reported that stem cells cultured on a high density nanowire array with

an interspacing distance of approximately 1 microm formed a radial spreading and

flattened morphology suggesting that focal adhesion contacts were established in all

directions within this range of interwire spacing (Bucaro et al 2012) Cell

morphology was reported to be highly polarized with long and narrow axon-like

extensions Within the range of interwire spacing of 4 microm cells expressed a stellate

morphology and multiple cell extensions (Bucaro et al 2012) More recently Jahed

et al reported that the cell ˗ nanopillar interactions were also dependent on cell

location on the nanopillar substrata and nanopillar geometries in addition to their

size and spacing (Jahed et al 2014) They showed that when 3T3 fibroblasts adhere

to a nickel substratum with 600 nm-diameter nanopillar surface signs of membrane

rupture were observed at the edges of the cells with membrane protrusions

appearing on the nanopillar arrays while all the pillars were buried under the cells

with no signs of membrane rupture (Jahed et al 2014) They also demonstrated that

220 nm mushroom-shaped nanopillars which were at a distance of 5 microm from the

48

cell edges could be detected and pulled toward the cell body by a single filopodium

Mushrooms-shaped nanopillars in direct contact with the cell body were also tilted

towards the nucleus of the cell most likely due to the traction forces (Jahed et al

2014) It was suggested by the authors that this specific geometry could be applied in

determination of the direction of spatially localized filopodia forces at various stages

of sensing attachment and spreading while most of other metallic nanopillars were

considered unsuitable for cell traction force measurements due to their rigidity and

plasticity (Tan et al 2003 Wang amp Lin 2007 Jahed et al 2014)

The adhesion of most cell types onto substrate surfaces is mediated by

membrane receptors known as integrins The process involves mechanical as well as

biochemical interactions with the actin cytoskeleton Different cell types undergo

different adhesion processes depending on their cell functions surrounding tissues

and other stimuli in the environments (Burridge amp Chrzanowska-Wodnicka 1996

Geiger et al 2001 Humphries et al 2007) In the inactive state the integrins

distribute within the cell membrane until a binding site becomes available Physical

clustering of multiple integrins will occur with more proteins being recruited at the

adhesion site to expand the cell surface area and increase the adhesion strength

These large structures of adhesive proteins and integrins are known as lsquofocal

adhesionsrsquo (Geiger et al 2001 Sniadecki et al 2006) Focal adhesions are flat often

elongated and mediate adhesion to the substrate or other tissue by anchoring bundles

of actin filaments through a plaque that consist of ligand binding proteins such as

vinculin tubulin paxillin fibronectin vitronectin and laminin (Burridge amp

Chrzanowska-Wodnicka 1996 Geiger et al 2001 Sniadecki et al 2006) Forces that

trigger the growth of focal adhesions can be internally generated by intracellular

contractile machinery or can be induced by external stimulants (Bershadsky et al

1996 Chrzanowska-Wodnicka amp Burridge 1996) It is believed that these focal

adhesions are responsible for mechanical and biochemical sensing activities in the

ECM also regulating the biochemical processes taking place in the cytoskeleton

(Burridge amp Chrzanowska-Wodnicka 1996 Geiger et al 2001) Focal adhesions can

be considered both as sensors of force and as sites from which cytoskeletal forces

originate through the anchored actin-microfilament (Engler et al 2006 Buxboim et

al 2010)

49

242 Cell proliferation

The ability of cells to proliferate is an important measure of cell health and

also provides an indication as to the suitability of the substrate for further

applications Cell proliferation is commonly defined as a combination of the number

of cell divisions and the increase in number of cells because a low number of cells

observed over time does not necessarily indicate a low cell division rate as the

number of detached and dead cells would not necessarily be considered (Bonde et al

2014)

It is known that nanotopography can regulate cell proliferation in a cell-

material specific manner the direct correlation between the dimensions of a

nanostructure and the proliferation of cells however remains unclear Early research

has demonstrated that cell proliferation in human cell lines is sensitive to the surface

nanoarchitecture when culturing cells on substrates consisting of randomized

nanoscale bumps or nano-islands of various heights less than 100 nm (Lim et al

2005 Schindler et al 2005) Similarly Shinobu and co-workers showed a normal

proliferation rate of Hela cells on a nanopillar-containing polystyrene film with the

nanopillars being 500 nm in diameter and 1 microm in height (Shinobu et al 2005)

Their analysis also showed that the ratio of apoptotic cells on nanopillar surface over

time is 28 which is lower than that of Hela cells cultured on a commercial

culturing dish (33) and that observed on flat polystyrene surfaces (35) More

recently Bond et al found a higher proportion of cells proliferated on InAs

nanowire arrays compared to those cultured on a flat control surface (Chang et al

2014) This study is in agreement with a number of other studies which

demonstrated the capability of nanostructured surfaces to promote cell proliferation

(Christopherson et al 2009 Bacakova et al 2011 Abdul Kafi et al 2012 Im et al

2012 Minagar et al 2013) In contrast other studies such as those of Persson et al

illustrated a decreased rate of fibroblast cell proliferation would occur on substrates

containing long nanowires (38 microm and 67 microm in heights average density of 1

nanowire per microm2) A possible explanation suggested by the authors is that cells are

forced to maintain their membrane integrity over the high surface area of the surface

containing long nanowires which lead to cell stress elevation of cell respiration

rates and in the high production of ROS (Persson et al 2013 Persson et al 2015)

Theses discrepancies indicated that the effect of the surface nanotopography on the

50

extent of cellular proliferation is very complex involving not only the surface

chemistry of the substrate but also on other parameters such as the density

nanopattern dimensions and geometry of the nanotopography which warrants further

investigation

243 Cell differentiation

Previous studies also showed that surface nanotopography plays an important

role in cell differentiation A number of reports have recorded the effects of

nanostructured materials on the biochemistry of cells indicated by the expression of

certain housekeeping genes and other specific markers which are often related to the

differentiation of cells (Sniadecki et al 2006 Dalby et al 2007 Oh et al 2009

Sjoumlstroumlm et al 2009 Brammer et al 2011 Lavenus et al 2011 Migliorini et al

2011 Im et al 2012) In a few studies the nanopillar substrata were reported to

exhibit a negative response to cell genetic functions (Persson et al 2013 Piret et al

2014 Pan et al 2015) For example Piret et al found that although mouse retinal

cells exhibited good adhesion and long term survival on silicon nanowire substrata

for up to 18 days in-vitro the cells underwent remarkable phenotypic changes

including the absence of neurites and the under-expression of the retinal cell markers

β-tubulin-III TRPV4 Brn3a Chx10 PKC recoverin and arrestin The authors

suggested that this neurotoxicity could be attributed to residual contaminants trapped

in the nanowire array of the substrata (Piret et al 2014)

In contrast a majority of available studies have demonstrated the positive

effects that nanopillar-containing surfaces have on cell biochemistry and

differentiation (Sjoumlstroumlm et al 2009 Loya et al 2010 Shalek et al 2010 Lu et al

2012 Rasmussen et al 2016) Shalek et al showed that the initial penetration of

cells by silicon nanowires did not cause significant differences in the expression of

housekeeping genes in Hela cells and fibroblast cells The mRNA expression of

ACTB B2M GAPDH GUSB and HPRT1 genes were found to be very similar to

those expressed on the flat control surfaces (Shalek et al 2010) Another gene

analysis of cortical neural stem cells attached onto 4 microm long GaP nanowires showed

that an approximately two-fold upregulation of Cd9 Rnd2 KiFap3 and Apoc 1

genes occurred which was associated with increased levels of cell adhesion actin

cytoskeleton formation microtubules processes and cell metabolism respectively

51

(SanMartin et al 2014) An upregulation of the stress marker (Hspa8) and a redox

activity regulator (Cybasc3) was also observed (SanMartin et al 2014) The work

performed by Migliorini et al emphasized that the height of nanopillars appeared to

be a critical physical factor that affected the differentiation of embryonic stem cells

into neurons (Migliorini et al 2011) 615 of cells expressing the early

differentiation of the β-tubulin class III and nestin markers were those grown on

substrates containing square nanopillars of 360 nm in height 250 nm in width with a

period of 500 nm compared to the those cultured on flat or shorter nanopillars These

authors also reported that neurites grew mostly on the top of the higher pillars (lt 360

nm) without reaching the bottom surface while those grown on the shorter

nanopillars (50 80 and 120 nm) appeared to have a random coverage along the pillar

body (Migliorini et al 2011) Another case of enhanced osteogenic differentiation

mesenchymal stem cells (MSC) was reported by Brammer and co-workers when

MSCs were cultured on a hydrophobic nanopillar substratum (25 microm in height 20

nm in width) (Brammer et al 2011) The physical nanostructure appeared to have

the potential to promote osteo-differentiation bone mineralization and protein

deposition of MSC without the need for inducing reagents such as growth factor

The authors also suggested that the increased number of adherent and cell-cell

contacts occurring on the nanopillar surfaces lead to the formation of an aggregated

ldquobone nodulerdquo per se which was not observed on flat or microstructured surfaces

resulting in differentiating stimulation (Brammer et al 2011) Hence these studies

suggest that nanotopographic cues of precise dimensions could be used to bias

precursor pluripotent and adult stem cells toward particular fates These results

would be highly useful in processes designed to modulate the surface

nanotopography for use in implant devices Several hypotheses have been proposed

to explain the molecular mechanisms driving these processes however there is still a

lack of extensive experimental proof of this phenomenon which necessitates further

investigation (Kim et al 2012a)

52

25 Competitive colonisation of bacteria and mammalian cells for the ldquorace

for the surfacerdquo

251 Race for the surface

In 1987 Anthony Gristina first introduced the concept of the ldquorace for the

surfacesrdquo describing the competition taking place between bacterial cells and host

cells as they seek to colonize the surface of a biomedical or implant surface (Gristina

1987) If pathogenic bacteria are present on an implant surface when inserted into the

host body they would be competing together for the colonization of the surface In

an ideal scenario the host cell would be expected to win the race over the bacterial

cells defending the substratum surface from the invading pathogens and vigorous

immune responses ensuring an appropriate tissue integration (Fig 210) (Gristina

1987 Gristina et al 1990 Busscher et al 2012) If bacteria become primary

colonizers of the surface biofilm formation will occur leading to infection Host

tissue cells would then be unable to compete for nutrition surface adhesion and

tissue integration with the implanted material The successful formation of bacterial

biofilm will protect the communities of bacteria from environmental stresses such as

host defense responses antibiotics and other antimicrobial treatments by inducing a

phenotypic resistance state making them extremely difficult to eliminate (Gristina

1987 Neoh et al 2012) Thus the initial contact of both cell types to the surface is

often regarded as the most critical step in the prevention of bacterial infection at the

same time stimulating tissue integration before appreciable bacterial colonization

(Davies 2003 Costerton et al 2005 Moriarty et al 2011 Arciola et al 2012

Busscher et al 2012 Neoh et al 2012) The first six hours of contact has been

identified as the ldquodecisive periodrdquo when the implant is particularly susceptible to

surface colonization (Poelstra et al 2002 Davies 2003 Hetrick amp Schoenfisch

2006) Preventing bacterial invasion during this period is critical to the long term

success of an implant

53









tissue cells Adapted from (Chang et al 2014) with permission of The Royal Society

of Chemistry

252 Current investigations

Although the concept of the race for the surface is widely known limited

studies have been reported with respect to material surfaces that can simultaneously

stimulate the host response and prevent bacterial infection The mechanism driving

these competing events also remains unknown (Gristina 1987 Busscher et al 2012

Neoh et al 2012)

A majority of studies have measured the interactions of bacteria and

mammalian cells with certain biomaterial surfaces separately which does not allow

an insight into the behaviors of both cell types in a competitive situation (Qiu et al

2007 Engelsman et al 2009 Neoh et al 2012 Campoccia et al 2013a Chang et al

2014) Several experimental methods have been proposed in an attempts to

demonstrate the race for the surface under in-vitro and in-vivo conditions

(Subbiahdoss et al 2009 Subbiahdoss et al 2010a Subbiahdoss et al 2010b

54

Subbiahdoss et al 2010c Saldarriaga Fernaacutendez et al 2011 Yue et al 2014) For

example the research group of Busscher and co-workers have demonstrated

different in vitro experimental designs in co-culturing bacteria and mammalian cells


Subbiahdoss et al 2010c Yue et al 2014) In 2009 a model was proposed in which

S epidermidis growth could be partially inhibited whilst simultaneously allowing a

limited growth of U2OS osteosarcoma cells under dynamic flow conditions

(Subbiahdoss et al 2009) Further work reported that neither the alteration in surface

wettability nor the addition of polymer coatings could effectively prevent the

overgrowth of pathogenic bacteria on biomaterial surfaces (Subbiahdoss et al

2010a Subbiahdoss et al 2010c) A post-contamination model was then introduced

illustrating the successful attachment of U2OS osteosarcoma cells to a substrate in

the presence of S epidermidis cells only if the mammalian cells were present at a

high initial cell density and were allowed to adhere to the surface 24 hours prior to

the exposure of the bacteria to the system (Subbiahdoss et al 2010b) It was however

unclear whether the U2OS cells could maintain their long term viability and cellular

functionality after the bacteria were added to the system An in vivo model was also

presented for the study of contaminated biomaterials by using a genetically modified

bioluminescent bacterial strain The bioluminescence was shown to be non-invasive

for visualizing the infected sites over time (Engelsman et al 2009)

Trentin et al reported the selective reduction of the S epidermidis biofilm

together with the simultaneous growth of Vero cells when both cells were being co-

cultured on a surface coated with an antifouling agent (Trentin et al 2015) This

coating chemical however exhibited low sensitivity against other bacterial strains

such as P aeruginosa S aureus and K pneumonia and may in fact promote

bacterial resistance over time due to its chemical-based mode of action The model

proposed by Chow et al used a co-culture of heat-inactivated E coli and lung cancer

cells (H59) to determine the receptors responsible for mediating postoperative

pneumonia associated with cancer treatments These authors found that the presence

of the E coli enhanced the adhesion and migration of the eukaryotic cells in vitro

and significantly increased the formation of in vivo hepatic metastases (Chow et al

2015) These experimental models can predict only the behaviors of bacteria and

ma0mmalian cells in the race for the surface within certain strict experimental

55

conditions that might not be similar to actual conditions being experienced during

medical implantation processes Also through the body of literature reported in this

topic there is a lack of data demonstrating whether a biomaterial surface could be

developed that can simultaneously prevent bacterial infection whilst actively

promoting host cell integration

56

Chapter 3

Materials and methods

57

31 Overview

In this study the experiments were designed to systematically investigate the

interactions of bacteria and mammalian cells on the surfaces Two typical bacteria

that are recognised as two of the main causes of biomaterial-associated infection

were chosen for this study including Staphylococcus aureus CIP 658T and

Pseudomonas aeruginosa ATCC 9027 (Rupp amp Archer 1994 Schierholz amp Beuth

2001 Harris amp Richards 2006 Del Pozo amp Patel 2009 Mitik-Dineva et al 2009

Moriarty et al 2011) Different cell types including erythrocytes primary human

fibroblast fibroblast cell line osteoblasts epithelial and endothelial cells will be

assessed for their adhesion spreading proliferation and metabolic activities onto the

selected nanostructured surfaces In order to understand the effects of different

parameters of surface nanostructures to the cell behaviours the surfaces of the

selected materials were comprehensively characterized using a wide range of

techniques followed by the analysis of cellular responses using complementary

microscopic and spectroscopic techniques

32 Fabrication of nanostructured surfaces

321 ECAP modified titanium

Commercially pure (CP) ASTM grade 2 and grade 4 titanium materials (Ti)

with an average grain size of 20 and 30 microm respectively were used to generate

surface nanostructure Billets from these materials 10 mm in diameter and 35 mm in

length were processed by equal channel angular pressing (ECAP) to produce an

ultrafine grain structure as described previously (Estrin et al 2009 Truong et al

2009 Truong et al 2010 Estrin et al 2011) The ECAP process selected together

with the application of back-pressure under the temperature regime selected ensured

samples were produced that contained a uniform distribution of predominantly

equiaxed grains

Small disc-shaped specimens were prepared from ECAP-processed material

by sectioning a cylindrical billet (10 mm in diameter) into 1 mm thick slices using

wire cutting by electric discharge in order to prevent changes in microstructure

These specimens were progressively ground on silicon carbide grinding papers to a

grit size of P2000 (84 microm) This process was used to ensure the production of a

58

planar surface with only shallow scratches and free of deformation pits thus

achieving an excellent surface finish In contrast with traditional metallography the

diamond polishing stage was omitted and the samples were polished directly with

colloidal silica (OP-S) mixed with hydrogen peroxide (30) at a ratio of 20 parts to

1 The resulting specimens were subsequently rinsed and ultrasonically cleaned first

in MilliQ H2O (with resistivity of 182 MΩ cm-1) to remove the silica suspension

used for polishing and then in ethanol In this study ECAP-modified grade 2 and

grade 4 Ti specimens mirror-polished according to the above schedule were

denoted Ti EG2 and Ti EG4 respectively

322 Graphene films

Graphite powder and hexadecyl trimethyl ammonium bromide (CTAB) were

purchased from Sigma Aldrich Graphene sheet films were fabricated using liquid

phase exfoliation followed by subsequent film formation as previously described

(Notley 2012 Sham amp Notley 2013) A suspension of graphene was exfoliated in an

aqueous solution of CTAB The surfactant assisted in the exfoliation by reducing the

surface tension of the liquid phase to match the cohesive energy of graphite The

surfactant also inhibited re-aggregation through adsorption onto the planar surface of

the graphene A stock solution of 06 mM CTAB was prepared in MilliQ water by

heating at 40 degC with continuous stirring for 30 minutes The solution was preheated

for 10 minutes prior to each experiment

Each sample was prepared by dispersing 10 graphite (wv) in 06 mM

CTAB The exfoliation was performed via ultrsonication using a Cell Disruptor

model W-220F sonicator from Heat Systems-Ultrasonics Inc at 60 W for 6 hours

UV-Visible absorption (Varian Cary 6000i UV-Visible spectrophotometer) and zeta

potential (the value of zeta potential was determined from the electrophoretic

mobility using the Smoluchowski equation) (ZetaPALS Brookhaven Instruments

Corp) Measurements of the suspension were taken every hour during the

exfoliation process The UV-visible spectra of the graphene suspension confirmed

the presence of a highly conjugated arrangement of carbon atoms in graphene sheets

with a peak in the absorption band at 270 nm (see chapter 5 Fig 51) which is in

agreement with previously published work (Notley 2012 Sham amp Notley 2013)

After 6 hours of sonication the solution was left to stand for 24 h to allow for the

formation of any unstable aggregates and then centrifuged for 20 minutes at 1500

59

rpm The supernatant was dialyzed against MilliQ water for 2 days to remove excess

CTAB using 002 microm cellulose dialysis tubing During dialysis the pH was strictly

controlled at 9 to maintain the small negative charge on the edges of exfoliated

graphene sheets

The dialyzed 200 mL solution was vacuum filtered through an alumina

membrane (002 microm Anapore Whatman) with excess MilliQ water used to remove

any remaining traces of CTAB When the resulting graphene film was completely

dried it was gently removed from the membrane The section of the film that was

furthest from the membrane was referred to as ldquoGN-Rrdquo (graphene ndash rough side) and

the inner side closest to the membrane was referred to as ldquoGN-Srdquo (graphene ndash

smooth side) Highly oriented pyrolytic graphite (GT) was used as the control in all

experiments The surface was prepared by single peeling of the top layers of

commercial graphite using Kaptonreg tape (DuPontTM) The peeled graphite film was

attached to a glass surface for handling during in all experiments

323 Black Silicon preparation

The bSi was prepared using a p-type boron doped 100 mm diameter silicon

(Si) wafer with specific resistivity of 10 ndash 20 Ω cm-1 a (100) oriented surface and a

thickness of 525 microm plusmn 25 microm (Atecom Ltd Taiwan) The samples were subjected to

reactive ion etching (RIE) using SF6 and O2 over a 5 minute period to produce the

bSi using an Oxford PlasmaLab 100 ICP380 instrument (Oxford Instruments

Concord MA USA) RIE processing was performed in mixed mode with etching

and passivation occurring simultaneously under the following conditions SF6 gas

flow rate of 65 standard cm3 min-1 (sccm) O2 gas flow rate of 44 sccm a pressure of

35 mTorr 100 W RIE power electrode temperature of 20 degC and a 10 Torr helium

backside cooling pressure The surface reflection over changed almost linearly from

10 to 20 over the visible spectral wavelength range 400 nm ndash 800 nm

33 Characterization of nanostructured surfaces

331 Surface crystallinity

X-ray diffractometry (XRD) is a versatile non-destructive technique that

reveals the crystallographic structure of natural and manufactured materials

(Whitaker 1986 Hurst et al 1997 Crosa et al 1999 Shah et al 2006 Beckers et al

60

2007 Elzubair et al 2007 Graetzel et al 2012) A crystal lattice is a regular 3-

dimensional distribution such as monoclinic triclinic cubic tetragonal hexagonal

etc of atoms in space (Slingsby et al 1997 Paris et al 2011 Tomita et al 2012)

These crystals are adjacent to each other and form parallel planes separated from one

another by a distance d with specific orientation both parameters are characteristic

for a particular material When a monochromatic X-ray beam with wavelength λ is in

contact with a crystalline material at an angle Ɵ (theta) diffraction occurs only when

the distance travelled by the rays reflected from successive planes differs by a

complete number n of wavelengths (Slingsby et al 1997 Paris et al 2011 Tomita et

al 2012) By varying the angle Ɵ the Braggrsquos law conditions are satisfied by

different d-spacing in polycrystal materials A diffractogram is constructed by

plotting the characteristic angular positions with the intensities of the diffracted

peaks If the materials compose of different phases the diffractogram is generated by

the combination of each crystallinersquos pattern (Slingsby et al 1997 Paris et al 2011

Tomita et al 2012)

332 Surface elemental composition

Surface chemical composition can be assessed by X-ray photoelectron

spectroscopy (XPS) Raman spectroscopy and energy dispersive x-ray spectroscopy

(EDX) For each material two or more techniques were used to confirm the

chemical composition of the material surfaces

3321 X-ray photoelectron spectroscopy

XPS was performed using an Axis Ultra spectrometer (Kratos Analytical

Ltd UK) equipped with a monochromatic X-ray source (Al Kα hν = 14866 eV)

operating at 150 W The relative atomic concentration of the elements detected by

XPS was quantified on the basis of the peak area in the recorded spectra with the

account of sensitivity factors for the Kratos instrument used Peaks in the high-

resolution regions of the spectra were fitted with synthetic Gaussian-Lorentzian

components after removal of a linear background (using the Kratos Vision II

software)

61

3322 Raman spectroscopy

Raman micro-spectrometer (WiTEC) with a 532 nm laser wavelength (hυ =

233 eV) was used to determine the chemical components of the material surfaces A

100times magnification objective (numerical aperture = 10) was used to acquire a grid

of 100 spectra times 100 spectra for a scanning area of 10 microm times 10 microm The integration

time for a single spectrum was 015 s For each type of surfaces scanning was

repeated twice on 5 independent samples

A water immersion lens with 60times objective magnification (numerical

aperture = 09) was used to map the attachment of erythrocytes present on the

surface of bSi Optical microscope was used to record the real time attachment of

RBCs on the nanopillar surface

3323 Energy dispersive x-ray spectroscopy

The absence of surfactant on the graphene surface after the dialysis and

filtration processes was also confirmed using energy dispersive x-ray spectroscopy

(EDX) The absence of both nitrogen and bromine peaks in the surface scans confirm

the complete removal of CTAB through the rinsing process

333 Surface hydrophobicitywettability

The surface hydrophobicity is determined by measuring the contact angle of

a liquid on a water droplet resting on a substrate (Smolders amp Duyvis 1961 Van Oss

et al 1988b) The hydrophobicity of surfaces can be evaluated by surface free

energies To calculate surface free energies of the substrate surfaces the Lifshitz-van

der WaalsAcid-base (LW-AB) approach was employed (Busscher et al 1984 Van

Oss et al 1988a Van Oss 1993) The method is involved in the measurement of the

contact angles of two different polar solvents and one contact angle of a non-polar

solvent on the substrate In this study three diagnostic liquids including MilliQ

water formamide (Sigma) and diidomethane (Sigma) were applied in the sessile

drop method (Smolders amp Duyvis 1961 Van Oss et al 1988b) with a FTA 1000C

device equipped with a nanodispenser (First Ten Angstroms Inc) Every contact

angle measurement was recorded within 10 seconds in 50 images with a Prosilica

Model Navitar 444037 camera and the contact angle was determined using the

processing software FTA Windows Mode 32

62

334 Surface morphology

The surface morphology was visualised using high resolution scanning

electron microscopy (SEM) This technique uses a focus beam of high-energy

electrons to generate a variety of signals at the surface of solid specimens (Schatten

2011) The signals that derived from the interactions between electrons and sample

reveal the external morphology that makes up the sample Data can be collected over

a selected area of the surface and a twondashdimensional (2D) image is generated that

displays spatial variations in these properties (Cizmar et al 2008) SEM can offer a

magnification of up to 200000times

The specimens were imaged from top or cross-section at appropriate

magnifications to reveal the surface micro or nanostructures The captured

micrographs were used for the analysis of the surface patterns including the size

shape orientation distribution and density of the surface features using ImageJ

software (Abragravemoff et al 2004 Henriques et al 2010)

335 Surface topography

The surface topography can be analysed using optical profilometer and

atomic force microscopy (AFM) Optical profilometry is a non-contact method based

on the superimposition of waves or interferometry which provides surface

topographical information from millimetre to micro sizes (Deck amp de Groot 1994)

AFM is a more versatile technique which can directly provide a lateral resolution

down to nano- and molecular ranges (Gross et al 2009) In this study optical

profilometry was used to image and evaluate the overall homogeneity of the

surfaces while AFM was used to analyse the micro and nano-topographical

characteristics of the material surfaces

3351 Optical profilometry

A Wyko NT1100 optical profiling system (Contour GT Bruker Corp USA)

were used in the white light vertical scanning interferometry (VSI) In this mode the

superimposition of fringes were generated by multiple of waves as the fringes move

different areas being measured come into focus allowing a reconstruction of the

surface topography (Arecchi et al 1979 Pettigrew amp Hancock 1979) A 50times

objective lens was used combined with 2times digital multiplier which results in a

63

scanning area of approximately 1043 microm times 782 microm to scan multiple regions of the

investigated surfaces The obtained images were processed using the Vision

software

3352 Atomic force microscopy

Atomic force microscopy (AFM) is a surface analytical technique which

allows detection and measurement of the topographical features of a sample (Binnig

et al 1986 Merrett et al 2002 Li et al 2004 Butt et al 2005 Whitehead et al

2006) AFM allowed the imaging of the topography of conducting insulating and

biological surfaces in either solid or liquid conditions with nano- and atomic

resolution (Binnig et al 1986 Lal amp John 1994 Li et al 2004 Butt et al 2005

Dorobantu et al 2012) An AFM consists of a sharp tip on a flexible cantilever on

the back of which a laser is reflected to a position-sensitive detector (Binnig et al

1986 Butt et al 2005 Webb et al 2011b) Either the tip or the sample is mounted

on a piezoelectric scanner and as the tip is raster scanned across the sample surface

the force between the tip and the sample is measured by monitoring the deflection of

the cantilever A topographic image of the sample is obtained by plotting the

deflection of the cantilever versus its position on the sample (Binnig et al 1986 Butt

et al 2005)

64






(B) Illustration of AFM contact mode versus tapping mode (Hansma)

There are two standard modes of AFM scanning including contact mode and

tapping mode Contact mode is when the AFM tip is in contact with the surface and

the piezoelectric scanner maintains constant force between the tip and the surface

Tapping mode refers to the oscillations of the tip caused by an applied voltage the

amplitude and phase difference between the driving voltage and tip oscillation reflect

the topography of the sample surface (Fotiadis et al 2002 Garciacutea amp Peacuterez 2002

Giessibl 2003 Dufrecircne 2004 Webb et al 2011b) In some cases imaging in contact

mode can damage or distort some delicate components of a sample surface while

tapping mode can minimize this problem by having the tip oscillate over the sample

making only brief intermittent contacts (Fotiadis et al 2002 Bar amp Meyers 2004 Li

et al 2004) The tapping mode also provides additional information about the

property of the surface in the phase image that can be generated along with the

height image

In this study AFM scans were conducted using an Innovareg scanning probe

microscope (Veeco Bruker USA) Scans were performed in the tapping mode at

65

ambient temperature and pressure using silicon cantilevers (MPP-31120-10 Veeco

Bruker USA) with a spring constant of 09 Nm-1 and a resonance frequency of

approximately 20 kHz Scanning was performed perpendicular to the axis of the

cantilever at a scan speed of 1 Hz Different scanning areas were chosen depending

on the different types of surfaces and the dimensions of surface features to generate

the best scan of the surfaces Data processing softwares including NanoScope

Analysis 140r1 and Gwydion (available from httpgwyddionnet) were used to

analyse the AFM data (Nečas amp Klapetek 2012) For the titanium surfaces the AFM

phase tapping mode was also employed to visualise the organisation of the ultrafine

Ti grains The measured phase differences corresponded to variations in the surface

properties such as surface composition stiffness and viscoelasticity (Bar amp Meyers

2004 Aicheler et al 2011 Webb et al 2011b Crawford et al 2012 Webb et al

2012)

The surface topographical data were analysed using different parameters

including the average roughness (Sa) root-mean-squared roughness (Sq) and

maximum roughness (Smax) Two spatial parameters skewness (Ssk) and kurtosis

(Skur) were also used to provide an insight into the distribution of surface features

Skewness is the measure of the symmetry of the height distribution ie a surface

with equal amount of peaks and valleys would have zero skewness (Gadelmawla et

al 2002 Tayebi amp Polycarpou 2004 Webb et al 2012) Kurtosis is a parameter

reflecting the shape of peak distribution Surface with normal peak distribution has a

kurtosis of 3 while a surface possessing the Skur value larger than 3 appears to have

relatively narrow sharp peaks and valleys (and the inverse applies) (Gadelmawla et

al 2002 Webb et al 2012)

34 Preparation of biological samples

341 Culturing of bacterial cells

P aeruginosa ATCC 9027 and S aureus CIP 658T bacterial samples were

obtained from the American Type Culture Collection (ATCC USA) and Culture

Collection of the Institute Pasteur (CIP France) respectively Bacterial stocks were

prepared in 20 glycerol nutrient broth (Oxoid) and stored at -80 ordmC Prior to each

experiment bacterial cultures were refreshed from stocks on nutrient agar (Oxoid)

and cells were collected at the logarithmic stage of growth (after 24 hours grown in

66

37degC) A fresh bacterial suspension was prepared for each of the strains by

inoculating the bacterial cells in nutrient broth with an optical density (OD) of 03

measured using a spectrophometer at the wavelength of 600 nm as previously

reported (Truong et al 2009 Ivanova et al 2010 Truong et al 2010 Ivanova et al

2011 Webb et al 2013)

The infective dose of P aeruginosa and S aureus cells was prepared

according to the guidelines of US Food and Drug Administration (FDA) (Schmid-

Hempel amp Frank 2007 FDA 2012 Ivanova et al 2013) It was specified that a

concentration of 105 cells per ml of P aeruginosa would be sufficient to cause

infection while this value is 103 cells per ml in case of S aureus The number of cells

was determined using haemocytometer

342 Preparation of red blood cells

Blood was obtained from healthy rats according to the ethical approval by the

Swinburne University of Technology Animal Ethics Committee dictated in

Biosafety Project 2014SBC01 (refer to Appendix) Fresh blood was collected in

38 (wv) sodium citrate pH 74 to prevent coagulation The anticoagulated blood

was centrifuged at 1400 rpm for 5 min to separate the blood plasma buffy coat and

the anticoagulant reagent The separated erythrocytes were washed twice in

phosphate saline buffer (PBS pH 74) and used within 6 hours

343 Culturing of eukaryotic cells

Human epithelial (A549) osteoblast cells (MG63) and fibroblast-like cells

(COS-7) were obtained from the American Type Culture Collection (LGC Standards

GmbH Wiesel Germany) The cell lines were cultured in Dulbeccos Modified

Eagles medium (DMEM Invitrogen) supplemented with 10 foetal bovine serum

(FCS Invitrogen) and 1 PenicillinStreptomycin (Invitrogen) Cells were seeded at

the density of 5000 cells per cm2 for every independent experiment The use of all

cell types were approved and stated in the Biosafety Project 2014SBC01 (refer to

Appendix)

Two sources of primary human fibroblast (pHF) were used in this study

Commercially available primary human fibroblasts were obtained from Promocell

(Germany) and cultured using ready-to-use pHF culture medium supplied by

67

Promocell supplemented with 2 FBS basic fibroblast growth factors (1 ngml)

and insulin (5 microgml) Cells were cultured to 80 confluency then were trypsinised

using the Detach kit (Promocell) Another source of pHF was used in Germany and

was isolated from human juvenile foreskin Foreskin was first digested with dispase

(25 microgml SigmandashAldrich) at 4degC for 14 h followed by trypsin (004 Seromed

Berlin Germany) in EDTA (002 Life Technologies) at 37degC for 2 h After this

endothelial cells were removed using the Dynabeads CD31 Endothelial Cell kit as

previously described (Wozniak et al 2004) The negative fraction after the removal

of the endothelial cells contained the fibroblasts The fibroblasts were cultured in the

same medium as described above

HUVEC were isolated as previously described and propagated in M199

(SigmandashAldrich Steinbach Germany) supplemented with 20 FCS (Invitrogen) 2

mM Glutamax I (Life Technologies) 1 PenicillinStreptomycin 25 microgml sodium

heparin (SigmandashAldrich) and 25 microgml endothelial growth factor supplement

(ECGS Becton Dickinson) (Wozniak et al 2004) In all cases primary cells were

used in passages between 3 ndash 6 All cells were maintained at 37˚C 5 CO2 Co-

cultures were done with HFHUVEC HF cells were added as above in the

monoculture and after 24 h medium was removed and 15 times 105 HUVEC were

added (in medium for the culturing of HUVEC described above) on top of the other

cell type

344 Im- and explantation in CD-1 mice

The in vivo pilot study was performed on 8 female 6-8 weeks old CD-1 mice

that were obtained from Military Medical Academy (Belgrade Serbia) with the

approval of the Local Ethical Committee (Faculty of Medicine University of Niš

Serbia) Animal housing under standard conditions ie regular mouse pellets and

access to water ad libitum as well as an artificial lightndashdark cycle of 12 h each was

maintained at Faculty of Medicine University of Niš Serbia

Prior to implantation the animals were randomly categorized into two study

groups with n = 4 animals per group for subcutaneous implantation of the

nanostructured bSi and non-structured Si samples for 15 days Animals of group 1

obtained implantation of the bSi samples while animals of the group 2 received the

non-structured silicon samples (ie control group)

68

The implantation of the samples was performed according to a previously

established protocol (Ghanaati et al 2010 Ghanaati et al 2012 Barbeck et al

2014a Barbeck et al 2014b Barbeck et al 2014c) Briefly an intra-peritoneal

anesthesia (10 ml of 50 mgml ketamine with 16 ml of 2 xylazine) shaving and

disinfection of the rostral region were initially conducted After that the silicon

samples were implanted in a preformed subcutaneous pocket in the subscapular

region under sterile conditions and the implantation sides were closed using 50

Prolene (Ethicon New Jersey USA) After the implantation procedure the animals

were placed individually for 15 days

Followed by this period the peri-implant tissue together with the implanted

silicon samples were collected after sacrifice of the animals via an overdose of the

above-mentioned anesthetics The implanted samples were carefully expurgated

using a surgical forceps after paraffin embedding

345 Culturing of COS-7 cells on pre-infected surface

BSi and Si control surfaces were infected with P aeruginosa and S aureus at

their infective dose (see section 341) at which it is sufficient to lead to biomaterial-

associated infection according to the Federal Food Administration (FDA USA)

COS-7 cells were grown to 70-80 confluency then were trypsinised using 025

TrypsinEDTA (Invitrogen) Cells were seeded on pre-infected bSi and Si control

substrates at the density of 5000 cells per cm2 for every independent experiment All

of the following assessments were performed after 1 3 and 7 days of seeding At

least five independent experiments were run to confirm the results

35 Biological assays

351 Scanning electron microscopy

The morphology of the bacterial and mammalian cells cultured on the

investigated surfaces were visualised using the FeSEM ndash ZEISS SUPRA 40VP

model with secondary beam energy of 3 kV to obtain high-resolution images of the

adherent cells Specimens with attached bacteria were washed twice with PBS to

remove non-adherent cells and imaged under SEM without performing the fixation

process All samples were sputter-coated with gold using a Dynavac CS300

instrument for approximately 2 minutes

69

Erythrocytes and other mammalian cells were fixed and dehydrated before

the visualisation After the incubation time the samples were washed with PBS and

fixed in 25 glutaraldehyde (Sigma-Aldrich) for 30 minutes then dehydrated in

series of ethanol (30 50 70 90 and 100) for 10 minutes of each solution

Samples can be preserved in 100 ethanol and were dried just prior to the imaging

experiment

352 Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) is a versatile optical

characterization technique which is evidenced by the ability to collect both spectral

and pictorial data (in reflection or fluorescence) over time CLSM can collect images

of individual slices using fluorescence microscopy slices in the xy xz and yz plane

During imaging the specimen is being bombarded with intense focused laser light

which can damage a sample The Fluoview FV10i (Olympus Japan) used in this

study comprises of 4 laser diodes (405 473 559 and 635 nm) which are arranged in

a compact laser combiner housed within the body of the FV10i The system can

acquire up to three fluorescence channels and a phase contrast channel

simultaneously allowing for the imaging of multiple fluorescence dyes

Visualisation of the bacteria cells was performed was performed with a 60times

water-immersing objective lens combined with 3times digital zoom (total 180times

magnification) Bacteria attached on the nanostructured surfaces were stained with a

LIVEDEADreg BacLighttrade Bacterial Viability Kit (Invitrogen) Live cells were

stained green with SYTO 9 dead cells were stained red by propidium iodide (Mitik-

Dineva et al 2009 Truong et al 2010 Ivanova et al 2012 Hasan et al 2013b)

Bacterial biofilm was stained with Alexa Fluor 633 Concanavalin A (Invitrogen)

Erythrocytes were imaged by staining the cells with 11-dioctadecyl-3333-

tetramethylindocarbocyanine perchlorate17 18 (DiI Life Technology) for 30 min

(Bonde et al 2014 Kim et al 2014) according to the protocol provided from the

manufacturer (Life Technology) The surfaces with attached erythrocytes were

washed with PBS fixed in 4 p-formaldehyde and imaged under the CLSM

Live cell imaging was conducted using the Leica SP5 Multiphoton confocal

microscope with a dipping 20times objective lens Simultaneous fluorescent imaging of

bacteria and COS-7 was achieved by labelling mammalian cells with LIVEDEADreg

70

ViabilityCytotoxicity Kit (Invitrogen) which is composed of calcein AM and

ethidium homodimer-1 for live cell and dead cell staining respectively while

bacteria were labelled with SYTOreg 17 Red Fluorescent Nucleic Acid Stain

(Invitrogen)

To perform immunocytochemistry staining cells were gently washed with

PBS fixed in 4 p-formaldehyde for 15 min permeabilized in 01 Triton X for 5

min then blocked with 1 BSA for 60 min Image-ITreg FX Signal Enhancer

(Invitrogen) were also used during fixation to enhance fluorescent stainings Fixed

cells were treated with primary anti-vinculin antibody (Sigma) overnight followed

by goat anti-mouse secondary antibody conjugated with Alexa Fluor 594

(Invitrogen) Actin filament were visualised by staining the cells with Alexa Fluor

488 conjugated Phalloidin (Invitrogen) Nucleus were labelled using DAPI

(Invitrogen) (Matschegewski et al 2010 Lavenus et al 2011 Divya Rani et al

2012) Samples with stained cells were then placed in a glass-bottomed disc for

imaging under CLSM

To visualise the formation of microcapillary-like structure of HFHUVEC co-

cultures cells on black silicon were rinsed with PBS fixed with 38

paraformaldehyde for 15 min at room temperature and then rinsed with PBS Cells

were then permeabilized with 05 Triton-X 100 for 10 min washed with PBS and

this was followed by the addition of anti-CD31 antibody (1100 PECAM-1 Santa

Cruz Biotechnology Inc Germany) Samples was allowed for incubation overnight

at 4˚C then were stained with the secondary antibody anti-mouse Alexa Fluor 488

(11000 Molecular Probes) for 1 hr at room temperature Staining of the nuclei was

performed using Hoechst 33342 fluorescent dye followed by washing with PBS A

drop of GelMount (Biomeda) was added to a glass slide and the side of bSi

containing cells was placed on the drop of GelMount Samples were examined using

Keyence fluorescent microscope

To visualize the attachment of single COS-7 cell on bSi in real time

interaction COS-7 cells pre-labeled with CellTrackerTM OrangeCMRA (Invitrogen)

were seeded and allowed to adhere onto the bSi surface 24 hours prior to a second

batch of COS-7 cells pre-labeled with CellTrackerTM GreenGMFDA Dye

(Invitrogen) being seeded onto the same bSi sample Imaging commenced from the

time the COS-7 cell labeled with CellTrackerTM Green GMFDA was seeded where

71

the z-range was determined using the COS-7 cells labeled with CellTrackerTM

Orange CMRA that had been previously adhered to the surface Images were

collected every 10 min using the Leica SP5 Multiphoton microscope with the 20times

dipping objective lens (part number 507701)

353 Quantification of bacterial biofilm

Bacterial biofilm formation was quantified using computational software

COMSTAT (Heydorn et al 2000) The software utilized three-dimensional biofilm

image stacks which were obtained from CLSM data Each image was processed to

quantitatively generate the biovolume and the thickness of biofilm based on the

amount of fluorescence detected (Heydorn et al 2000 Mitik-Dineva et al 2009

Truong et al 2010)

354 BCA assay

The total protein content as a result of cellular metabolic activities is

determined spectrophotometrically using bicinchoninic acid (BCA) protein assay

(Sigma Aldrich) The total intracellular protein synthesized by adherent cells will be

determined from a standard curve of commercial albumin run in parallel with

experimental samples (Zheng et al 2011 Divya Rani et al 2012)

355 MTT assay

Cell proliferation was assessed using a 3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide (MTT) assay (Vybrantreg MTT Cell proliferation assay

kit Invitrogen) At the prescribed time points the specimens were transferred to a

new plate and incubated with MTT reagent at 37 ordmC for 4 hours to form formazen

which was then dissolved with dimethyl sulfoxide (DMSO) The absorbance was

measured at 540 nm using a microplate reader

356 Histological analyses

The peri-implant tissue was histologically prepared for qualitative and

quantitative analyses as described elsewhere (Ghanaati et al 2010 Ghanaati et al

2012 Barbeck et al 2014a Barbeck et al 2014b Barbeck et al 2014c) These

explants were initially preserved in 4 formaldehyde solution for 24 h Afterwards

the formalin fixed tissue was cut into several segments for further embedding

72

processed in automatic tissue processor (Leica TP1020 Germany) and embedded in

paraffin blocks This procedure allowed for producing multiple 2ndash4 microm thick

sections using a rotary microtome (Leica Germany) These tissue sections were

immediately affixed on charged glass slides (VWR International SuperFrostreg Plus)

and incubated at 37degC for 12 hours

Shortly before staining a dewaxing- and rehydration process took place by

sequential immersion of the slides in xylene and graded concentration of ethanol

Initially samples were stained by hematoxylin and eosin (HE) to evaluate the section

quality After selection of the tissue blocks with the best quality Azan- and Giemsa

staining techniques were applied Furthermore murine macrophages were

immunohistochemically detected by using an anti-F480 primary antibody (rat anti-

mouse antibody clone BM8 Dianova Germany) and an autostainer (Autostainer

360 ThermoScientific Germany) Thereby endogenous peroxidase was quenched

with 3 H2O2 and epitope unmasking was done by proteinase K application while

blocking was conducted via Avidin-Biotin Complex (ABC Vector Elite Vector

Laboratories US) A mixture of Tris-buffered saline and Tween-20 was used as a

washing buffer For visualisation by light microscopy slides were additionally

stained with 33-diaminobenzadine (DAB) and for increased sensitivity of the DAB

chromogen the VECTASTAIN Elite ABC peroxidise reagent (Vector Laboratories

US) was used to control the undesirable non-specific immunolabelling Negative

controls for each slide were prepared by omitting primary antibodies

357 Qualitative and quantitative histomorphometrical analyses

Qualitative histological analysis was conducted using an established protocol

(Ghanaati et al 2010 Ghanaati et al 2012) Thereby a bright field light microscopy

(Nikon Eclipse 80i Japan) was used in order to determine interactions between the

tissue and the biomaterials Thereby the focus was on the description of the

biomaterial-induced inflammatory responses and the cells involved in this process A

DS-F1 digital camera and a digital sight control unit (Nikon Tokyo Japan) that were

connected to the above-mentioned microscope were used for making

microphotographs

Quantitative histomorphometrical analysis was performed after digitalization

of the sections was immunohistochemically stained by F480 for macrophage

73

detection A special scanning microscope system was used which composes of an

Eclipse 80i microscopy (Nikon Japan) a DS-F1 digital camera and an automatic

scanning table (EK 75 x 50 Pilot Marzhauser Germany) connected to computer

running the NIS- elements AR software (version 41003 Nikon Japan) as

previously described (Ghanaati et al 2010 Ghanaati et al 2012 Barbeck et al

2014a Barbeck et al 2014b Barbeck et al 2014c) Briefly the length of every

biomaterial-induced capsule was measured (in mm) Furthermore the amount of

positive immunolabelled cells ie murine macrophages adherent to the material

surfaces was manually counted for each section In order to compare the material-

adherent macrophages the following formula was used numbers of macrophages in

relation to the biomaterial surfaces (macrophages per mm2) The data were

statistically analysed by a Studentacutes t-test using the SPSS 1601 software (SPSS

Inc Chicago IL USA) Statistically significant differences were considered if P-

values were less than 005 ( P lt 005) and highly significant if P-values less than

001 ( P lt 001) or less than 0001 ( P lt 0001) Finally the

histomorphometrical data were displayed as means plusmn standard deviations (SD) using

the GraphPad Prism 60c software (GraphPad Software Inc La Jolla USA)

74

Chapter 4

Investigation of bacterial

interactions on nano and micro-

structured titanium surfaces

75

41 Overview

Titanium and its alloys have been widely utilised as implant material in the

biomaterial industry (Rack amp Qazi 2006 Niinomi 2008 Valiev et al 2008

Biesiekierski et al 2012 Mahapatro 2012 Minagar et al 2013 Lugovskoy amp

Lugovskoy 2014 Damodaran et al 2015 Liu et al 2015a Nair amp Elizabeth 2015)

Despite excellent biotechnological properties including biocompatibility and

corrosion-resistance unfavourable mechanical behaviours of commercially pure Ti

including insufficient mechanical strength and low fatigue strength have limitations

in heavy-load applications for examples dental implantation and hip joint

replacement (Niinomi 2008 Valiev et al 2008 Niinomi et al 2012) The technique

of equal channel angular pressing (ECAP) technique was employed to enhance the

mechanical properties of bulk metallic materials by refining the bulk crystalline

grain structure (Nakashima et al 2000 Furukawa et al 2001 Chen et al 2010

Dheda amp Mohamed 2011) In this study the effects of the surface nanostructure of

the ultrafine grain titanium to the attachment of two types of pathogenic bacteria

including Gram-positive cocci Staphylococcus aureus and Gram-negative rod-

shaped Pseudomonas aeruginosa were investigated As-received titanium with

polished surfaces were used as control surfaces for all experiments Different

techniques were performed to characterise the surface topography and architecture of

the as-received and modified Ti including X-ray photoelectron spectroscopy (XPS)

contact angle goniometry X-ray diffractometry (XRD) transmission electron

microscopy (TEM) optical profilometry and atomic force microscopy (AFM) The

attachment of bacterial cells and subsequent biofilm formation on the titanium

surfaces were assessed using scanning electron microscopy (SEM) and confocal

laser scanning microscopy (CLSM) The results presented in this chapter were

published with the title ldquoSelf-organised nanoarchitecture of titanium surfaces

influences the attachment of Staphylococcus aureus and Pseudomonas aeruginosa

bacteriardquo in the journal Applied of Microbiology and Biotechnology (refer to List of

publications)

42 Surface characterisation of ECAP modified titanium

Surface elemental composition of ECAP-modified Ti characterised by XPS

were shown in Table 41 Ti elements and their oxidation were detected in similar

76

amounts among all 4 types of specimens The quantities of other organic

contamination such as carbon silica and sodium were also insignificantly different

Table 42 Titanium surfaces elemental composition inferred from XPS analysis

Below the detection limit lt01

The surface wettability and surface free energy of the titanium surfaces were

assessed by the contact angle measurements of three diagnostic liquids including

water formamide and diiodomethane (Table 42) The surface energy of a material is

defined as the amount of energy per area required to reversibly create an

infinitesimally small unit surface estimated using the Lifshitz-van der WaalsLewis

acid-base approach (Van Oss et al 1985 1988b) The surface free energy presented

in table 42 was calculated using the mean value of the contact angle of each liquid


modified titanium surfaces

Grade 2 Grade 4

As-received ECAP As-received ECAP

Contact anglea (degree)

θW 739 plusmn 75 781 plusmn 98 828 plusmn 17 788 plusmn 70 θF 550 plusmn 21 559 plusmn 22 582 plusmn 14 574 plusmn 20 θD 386 plusmn 22 365 plusmn 28 382 plusmn 14 361 plusmn 13

Surface free energyb (mJm2)

γLW 403 413 405 415 γAB 12 10 10 04 γ+ 004 004 006 006 γ 99 65 39 66 γTOT 416 424 415 419

a θW θF θD water formamide and diidomethane contact angles respectively b Surface free energies components Lifshitz-van der Waals (γLW) acidbase (γAB)

electron acceptor (γ+) electron donor (γ) and total surface free energy (γTOT)

components

Peak Position BE (eV) Atomic fractions ()

Grade 2 Grade 4 As-received ECAP As-received ECAP

O 1s 530 539 552 555 532 C 1s 285 156 178 177 179 N 1s 401 03 03 04 02 Ti 2p 459 235 206 211 227 Na 1s 1072 39 37 37 38 Si 2p 102 15 07 ndash ndash Cu 2p 932 ndash 02 ndash 03

77

Statistical analysis has shown that the hydrophobicity as well as surface free

energies of four types of Ti surfaces shown in Table 42 are insignificantly different

Their surfaces exhibited water contact angles in the range of 70deg to 80deg Surface free

energy was observed to remain similar after ECAP modification with a value of

approximately 42 mJm2

Surface crystallinity was examined with XRD (Fig 43) which indicated a

significant change from polished titanium to ECAP modified materials Diffraction

spectra of as-received Ti grade 2 and grade 4 were compared with Crystallographic

Information Files (CIF) available from the Inorganic Crystal Structure Database

(ICSD) XRD results indicated significant changes of titanium crystallinity after

ECAP processing The spectra indicated that all 4 types of materials possess α-

titanium hexagonal close packed crystal structure but with different peak intensities

ECAP Ti exhibited a significant drop of reflection peak intensities compared to the

original crystal structures due to the severe deformation of ECAP modification The

major peaks of grade 2 ECAP titanium decreased 3 times while in grade 4 the peak

intensity of ECAP samples were halved compared to as-received titanium The

reduction of the peak height also demonstrates the ultrafine crystallites in the

modified bulk titanium

Figure 41 X-ray diffractogram of as-received and ECAP modified Ti

78

The grain structures of as-received and ECAP modified titanium were

visualized with a Philips CM20 transmission electron microscope (TEM) operating

at 200 kV Thin-foil specimens of both grades were prepared by slicing the

processed billets in a direction perpendicular to the pressing axis with a low-speed

saw Slices of ~200 μm thickness were subsequently dimpled to around 50 μm

thickness through ion-beam milling using a Gatan PIPSTM system at an anode

voltage of 5 kV and a milling angle of 4deg The resulted slices were imaged under

TEM and presented in Fig 44


4 (C amp D) Scale bar 100 nm

Grade 2 ECAP titanium exhibited greater grain size of approximately 150 nm

to over 200 nm with some large grains divided into sub-grain structure due to

dislocations forming low angle grain boundaries (Fig 44B) Grade 4 ECAP

modified titanium appeared to have smaller nanograins in the range of 50 nm to 100

nm with the ldquoswirledrdquo architecture (Fig 44C) some heavily dislocated grains

existed in a significant proportion accompanied with ultrafine sub-grain structure

(Fig 44D) The smaller grain size of grade 4 ECAP Ti resulted in a higher density

79

of grain boundaries which afforded the sample its particular surface morphology

consistently with previous reports (Assender et al 2002 Aicheler et al 2011)

Detailed surface morphology was further characterized with optical profilometry and

atomic force microscopy (AFM)

An overview of surface topography were first visualised under times50 objective

lens of a Bruker optical profilometer resulted in a large scanning areas of

approximately 78 microm times 104 microm Details in surface structures were then revealed

under AFM which is capable to perform nanoscale imaging (1 microm times 1 microm) operated

with tapping mode (Fig 43) Different surface topographical characteristics were

statistically analysed as shown in Table 43 The large scale imaging indicated that

titanium surfaces of 4 types exhibited heterogeneous characteristics however the

visualization of surface topography can vary significantly depending on the scale of

analysis At the 10 microm times 10 microm AFM scanning areas titanium grade 2 appeared to

be rougher than titanium grade 4 in the respect of as-received and ECAP-processed

materials with higher respective values of average roughness (Sa) root-mean-

squared (RMS) roughness (Sq) and maximum roughness (Smax) In terms of the

changes derived from ECAP modification the ultrafine grain titanium in both

grades exhibited significant smoother surfaces compared to their original state after

ECAP modification (Table 43) However at the nanoscale range (1 microm times 1 microm)

there was no significant difference between these three roughness values with Sa and

Sq being 021 nm and 029 nm for ECAP Ti grade 2 and 016 nm and 023 nm for

ECAP Ti grade 4 respectively Since both of ECAP processed Ti exhibited surface

roughness below 05 nm these surfaces were classified as molecularly smooth

surfaces (Crawford et al 2012 Webb et al 2012 Siegismund et al 2014)

80

Figure 43 Surface topography of as-received and ECAP modified titanium grade 2 and 4 analysed by optical profiling (top) and AFM

(middle) with corresponding surface line profile Typical AFM scanning areas are shown in 1 microm times 1 microm

81


titanium surfaces on two nanoscale scanning areas

Scanning areas (microm)

Grade 2 Grade 4

As-received ECAP As-received ECAP 10 times 10 Sq

250 plusmn 110 127 plusmn 092 085 plusmn 018 051 plusmn 014

Sa 151 plusmn 024 042 plusmn 019 058 plusmn 013 026 plusmn 006

Smax 5215 plusmn 112 6462 plusmn 3908 2982 plusmn 1207 3434 plusmn 969

Sskw 574 plusmn 090 1573 plusmn 1170 186 plusmn 132 1345 plusmn 729 Skur 817 plusmn 126 56289 plusmn 32761 5574 plusmn 3309 61456 plusmn 22046 1 times 1 Sq 035 plusmn 014 029 plusmn 010 027 plusmn 010 023 plusmn 004



Sskw -007 plusmn 024 -086 plusmn 048 -040 plusmn 037 -117 plusmn 073 Skur 403 plusmn 162 793 plusmn 159 586 plusmn 153 1193 plusmn 586

Surface topography represented by the conventional parameters Sa Sq and

Smax describe only one dimension of the surface structure reflecting the height

variations of the surface features and consequently two surfaces that are identical in

this aspect may in fact possess a vastly different overall surface structure or

architecture (Webb et al 2011b Klymov et al 2013) The average and RMS

roughness give an indication of the typical height of the features present on a

surface however they give no indications of the shape or spatial distribution of the

peaks In this study skewness (Sskw) and kurtosis (Skur) are additional parameters that

were used to describe the distribution of titanium surface nanopatterns Skewness is

a description of the symmetry and the shape of the peak distribution across the

surface while kurtosis is used to measure the peakedness of the surface

(Gadelmawla et al 2002) Typically surface with skewness value of 0 exhibits a

perfect symmetric height distribution while a positive or negative values

discriminates between wide valleys with narrow sharp peaks and high plateau with

sharp deep valleys Surface with a Gaussian height distribution has kurtosis value of

3 surface with narrow height distribution has Skur greater than 3 while well spread

height distribution has a kurtosis value less than 3 (Tayebi amp Polycarpou 2004

Webb et al 2012)

82

Grade 2 ECAP titanium surface exhibited an average skewness value of -086

while this value is lower for ECAP Ti grade 4 being -117 The higher the negative

values the higher frequency of high plateaus and sharp deep valleys present on the

surfaces The kurtosis was also found to be appreciably higher for the grade 4 ECAP

Ti surface compared to grade 2 with the values of 1193 compared with 793

respectively indicating that the surface of grade 4 ECAP Ti substrate possess a

narrower height distribution resulted in two distinct different surface architecture In

order to visualise the details of these discreted surface architecture tapping phase

imaging was conducted simultaneously with conventinal surface height tapping

during AFM scan as shown in Fig 44







(httpyoutubeHlwcTV4DXmk)

In the height tapping images the surface of grade 2 ECAP Ti exhibited a

number of broad valleys and peaks appearing in highly contrasting colours that

83

highlighted a distinction between the peaks and the valleys (peaks are in orange

vallyes are in blue) while grade 4 materials appeared to be uniformly flat with few

sharp peaks protruding off the surfaces which were reflected in higher kurtosis

(Table 43) The complimentary phase tapping allows the detection of variable

surface properties thus allows the mapping of the material nanograins and grain

boundary structures These phase images demonstrated that grade 2 ECAP modified

surfaces possess well-defined grain boundaries while grade 4 specimens exhibited

poorly defined curly shaped closely-spaced grain boundaries with complex sub-

grain dislocations (Fig 44) These observations are consistent with the ultrafine

grain nanostructure observed under TEM (Fig 42)

The protrusions of the investiged surfaces were further analysed on the 1 microm

times 1 microm AFM scanning images using ImageJ software (Fig 45) Statistical

distribution analysis showed that grade 4 ECAP titanium surfaces have an average

protrusion diameter of 20 nm with sharper peaks compared with those present on the

grade 2 ECAP surfaces which presented an average diameter of 55 nm The average

spacing (d) between these nanoprotrusions was found to be 620 nm and 350 nm for

grade 2 and 4 ECAP materials respectively




distribution analysis

0

10

20

30

40

50

0 20 40 60 80

Po

pu

lati

on

Diameter (nm)

ECAP grade 2

ECAP grade 4

(a) (b)

(c) (d)

(e)

Ti EG2Ti EG4

84

In conclusion the two distinct nanoarchitecture differences of these two

surfaces are first the nanoprotrusions on grade 4 specimens are 15 times sharper

than those on the grade 2 ECAP Ti and second the spacing between the

nanoprotrusions on the grade 2 ECAP titanium substrates is approximately two time

larger than those present on the grade 4 ultrafine grained substrates

43 Interactions of bacteria on ultrafine grain titanium surfaces

Bacterial responses on 4 types of titanium surfaces were analysed using

Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus CIP 658T

Visualization of cell attachment was performed by SEM while bacterial cell

viability and biofilm production were assessed using confocal laser scanning

microscopy (CLSM) as shown in Fig 46 and 47 S aureus cells appeared to

successfully colonise all types of titanium surfaces after 18 h incubation The

average number of retained cells in as-received titanium grade 2 was found to be

134 times 104 per mm2 and those on titanium grade 4 was similarly found to be 152 times

104 per mm2 The S aureus cell density increased up to 281 times 104 per mm2 on ECAP

modified titanium grade 2 and 302 times 104 cells per mm2 on the modified grade 4 Ti

substrate

In contrast P aeruginosa cells appeared to be poor colonisers with 009 times

104 and 02 times 104 cells per mm2 found on as-received grade 2 and grade 4

respectively This cell number was found to be 5 times higher on grade 4 ECAP

modified Ti (255 times 104 cells per mm2) compared to the population of cells on

modified grade 2 specimens (054 times 104 cells per mm2)

85

Figure 46 The responses of Staphylococcus aureus on the as-received and ECAP modified titanium surfaces after 18 h incubation

SEM images (top) represent the typical cell attachment and morphology Three-dimensional CLSM images (middle) represent cell

viability and EPS production (live cells were stained green dead cells were stained red EPS were stained blue) The CLSM images

were used for further analysis of biofilm performed by COMSTAT software

86

Figure 47 The responses of Pseudomonas aeruginosa on the as-received and ECAP modified titanium surfaces after 18 h incubation




87

The statistical quantification of bacterial cell viability was shown in Fig

410 More than 80 of the attached bacteria of both types were found to be viable

on all surfaces There were no significant differences in cell viability found between

the investigated specimens

Figure 48 Statistical quantification of bacterial viability on titanium surfaces

To investigate the bacterial biofilm produced on the surfaces COMSTAT

software was used to quantify the extracellular polysaccharide substances (EPS)

detected by CLSM (Fig 46 amp 47) Two parameters including the biovolume and the

average thickness were statistically analysed as shown in Fig 49 Both P

aeruginosa and S aureus biofilm were found to be higher on ECAP surfaces than on

polished titanium Also while S aureus exhibited silimilar amount of biofilm on

grade 2 and 4 ECAP modified titanium P aeruginosa appeared to produce

significant more EPS on grade 4 than compared to grade 2 ECAP Ti surface (Fig

49) This was expected due to the similar S aureus cell attachment on both ECAP

modified materials while the number of P aeruginosa cells on grade 4 ECAP Ti was

significantly higher than the those observed on grade 2 ECAP Ti

88



COMSTAT (Heydorn et al 2000)

44 The effects of topographical parameters on bacterial attachment

In order to investigate the influence of surface nanostructure to two different

types of bacteria the correlation of surface topography and architecture with

bacterial colonisation were plotted in Fig 410 Average roughness is the most

conventional and commonly used to describe the surface topography (Gadelmawla et

al 2002 Whitehead et al 2006 Crawford et al 2012 Webb et al 2012) Previous

studies have shown that nanometrically smooth surfaces with Sa less than 05 nm are

not favorable for rod-shaped P aeruginosa however have no restriction to coccoid S

aureus due to their differences in turgor pressure as a results of their corresponding

morphology (Ivanova et al 2011 Webb et al 2013) Spherical S aureus cell

membrane has higher turgor pressure (Whatmore amp Reed 1990 Arnoldi et al 2000)

leading to the higher ability to stretch their membrane and increase the contact area

with the smoothest surfaces while the rod shape of P aeruginosa has limited

thermal fluctuation capability therefore restricting their adaptation with

nanometrically smooth surface (Marrink amp Mark 2001 Ivanova et al 2011 Webb et

al 2013)

89

Figure 410 Statistical analysis showing the relationship between the average roughness and kurtosis of titanium surfaces and the

amount of attached bacteria cells There was no clear correlation between the attachments of both S aureus and P aeruginosa to the Sa

values within the sub-nanometric range while the Skur appeared to be proportional with the number of the adherent cells

90

However as can be seen in Fig 410 within the roughnes range of 01 nm ndash

03 nm there was no clear function between the number of attached cells and the

surface roughness Meanwhile kurtosis value which reflects the peak distribution

showed a proportional relationship with bacterial attachment It was shown that the

higher the kurtosis the higher the capability of bacterial cells to adhere to the

surface indicated by the high number of retained P aeruginosa with grade 4 ECAP

titanium surface which possess the highes kurtosis value of 1193 This results

suggested that even within the nanometrically smooth roughness P aeruginosa cells

were still able to lsquoanchorrsquo to the surface and maintain their subsequent growth if

sharp nanoprotrusions are available with appropriate peak distribution This is in

agreement with a recent report which suggested that the interactions of bacterial cells

is equally sensitive to amplitudinal and spatial parameters of the substrates

particularly the spacing-sensitive was recognized with respect to average roughness

below 70 nm (Siegismund et al 2014)

A computational model proposed by Pogodin et al take into account the

different membrane structure of Gram-positive and Gram-negative bacteria In this

model cell wall is considered as an elastic layer of stiffness k while the free energy

associated upon contact of this layer with nanoprotrusion decreases by an amount ε

which favours local adsorption (Pogodin et al 2013) Equilibrium of a bacterial cell

wall in contact with a surface with nanoprotrusions results from an interplay between

these two competing effects which is controlled by a dimensionless interaction

parameter 120577 = minus120576119899119896 where n is the number density of nanoprotrusions per unit

area Thus the higher flexibility (lower stiffness k) of Gram-negative bacterial cell

walls results in greater stretching ability than that experienced by the significantly

more rigid cell walls of Gram-positive bacteria (Pogodin et al 2013) Furthermore

the stretching of bacterial membrane retained between nanoprotrusions is inversely

proportional in the square of their spacing d which means a two-fold increase of

peak spacing should result in a four-fold increase in the stretching of interacted cell

wall This could explain greater propensity for attachment of P aeruginosa on the

grade 4 ECAP modified titanium surfaces with higher kurtosis and skewness values

than on the grade 2 substrates

91

45 Conclusion

The studies of interactions between surface nanostructures and bacteria cells

often focus on the effects of vertical amplitude-related roughness parameters

Meanwhile the surface architecture of a substrate such as spatial distribution or

sharpness of peaks may significantly contribute to discriminative bacterial

attachment at the same extent of average surface roughness In this study we found

that at molecularly smooth level (Sa below 05 nm) the attachment of coccoid Gram-

positive S aureus was similar on the titanium surfaces of which surface

morphologies were different However at the same surface roughness range rod-

shaped Gram-negative P aeruginosa cells poorly colonised unless sharp

nanoprotrusions were available It is suggested that the presence of sharp

nanoprotrusions could facilitate the stretching of P aeruginosa cell membrane to

anchor and maintain attachment to the nanosmooth surfaces followed by a

subsequent large amount of biofilm formation

92

Chapter 5

The bactericidal effects of

graphene nanosheets

93

51 Overview

The family of graphene materials have been used in a wide variety of

applications since it was first discovered in 2004 (Novoselov et al 2004) A number

of reports have demonstrated the antibacterial activity of graphene in its various

forms such as graphene oxide reduced graphene oxide and graphene composite

The mechanisms responsible for this bactericidal activity are however not fully

understood nor comprehensively investigated

In this chapter multilayer graphene films with two different surface

structures were fabricated using a liquid exfoliation technique A number of

analytical techniques were used to characterise the physico-chemical properties of

graphene surfaces that present on both sides of the film The exfoliation process was

monitored using Ultraviolet-Visible (UV-Vis) spectroscopy the purity and the

number of graphene layers were confirmed by Raman spectroscopy X-ray

diffractometry (XRD) and energy dispersive X-ray spectroscopy (EDS) The surface

topographies of the graphene film were expansively analysed by SEM and AFM

Various surface parameters including feature size shape edge length and interactive

angle of the surface micro and nano-patterns were studied with respect to their

influences to the behaviours of P aeruginosa ATCC 9027 and S aureus CIP 658T

Single chain main field (SCMF) simulations of the interactions taking place between

the lipid bilayer membrane of the bacterial cells and graphene surfaces were also

performed to explain the mechanisms responsible for the destructive effects of the

graphene surfaces The results presented in this chapter were published with the title

ldquoGraphene induces formation of pores that kill spherical and rod-shaped bacteriardquo in

the journal ACS Nano (refer to List of publications) The computational modelling

was contributed by Dr Vladimir Baulin and his team

52 Characterisation of graphene film

Graphite powder was exfoliated using cetyltrimethylammonium bromide

(CTAB) for 6 hours with continuous sonication During the exfoliation process the

formation of single graphene layers was monitored using the UV-visible

spectroscopy (Fig 51)

94


during the 6 hour sonication-assisted exfoliating process

The increasing absorption of UV-Vis light at a λmax of 270 nm indicated the

presence of the π rarr π transition of the C-C bonds in exfoliated graphene sheets

(Punith Kumar et al 2015) The exfoliation process was limited to a maximum

period of 6 hours to avoid further breakage of the graphene single layers After

dialysis the final suspension was vacuum filtered through an alumina membrane

which resulted in the formation of two different surface topographies on the top and

the underside sections of the film The film topside was designated as ldquographene ndash

rough siderdquo (GN-R) and the underside was designated as ldquographene ndash smooth siderdquo

(GN-S) based on their distinctly different surface properties

The purity of the graphene film was confirmed using Raman spectroscopy

and EDS against a graphite block which was used as the negative control (Fig 52)

Raman spectra of the graphene surfaces showed the D G and 2D peaks at 1350 cm-1

1582 cm-1 and 2700 cm-1 indicating the presence of graphene on both sides of the

film surfaces (Lotya et al 2009 Wang et al 2010 Liu et al 2011a Li et al 2013a

Punith Kumar et al 2015) The relative height of the D peak in comparison to the G

peak is characteristic of the edge defects and the single symmetric 2D peak

confirming the presence of atomically thin graphene sheets According to the

literature graphene thickness is estimated from the ratio between the Raman

intensity of the 2D band (2700 cm-1) and that of the G band (1582 cm-1) (Ni et al

2008 Zhu et al 2013) The graphene sheets produced here for both the GN-R and

95

GN-S surfaces were estimated to be about 4 layers thick (I2DIG ~ 03) with a total

thickness of 4 nm




films and the absence of bromine from the CTAB used in the manufacture process

An elemental analysis performed using EDS showed that no traces of

elemental bromine confirming the complete removal of the CTAB surfactant using

in the graphene manufacturing process (after dialysis) It is important to ensure that

no toxic compounds remain in the exfoliated graphene samples if they are to be used

in biological applications The crystallinity of the fabricated films was also examined

using X-ray diffractometry (XRD) The diffractograms presented in Fig 53

highlight that a significant reduction in the characteristic peak of graphene reflection

(002) at 27deg was present compared to that found for graphite surfaces (Lu et al

2012 Tang et al 2012)

96



respectively)

The surface morphology of both sides of the filtered graphene films were

visualized using SEM (Fig 54) Both surfaces appeared to contain nanosized

exfoliated sheets with different dimensions and degrees of organisation The

nanosheets on the GN-R surfaces exhibited a sheet size in the range of 05 μm ndash 15

μm while the average sizes of graphene sheets on GN-S surfaces were between 200

nm - 500 nm Further analysis of the size of the nanosheets involved the

quantification of edge length using ImageJ softaware The edges of the nanosheets

could be exposed by enhancing the contrast of the SEM images and determining the

distribution of edge lengths present on both surfaces The frequency of the edge

lengths were plotted as a function of length and presented in Fig 54 The graphene

sheets present on the GN-R surfaces possessed edge lengths ranging between 100 nm

ndash 250 nm whereas those present on the GN-S surfaces were in the range between 40

nm ndash 100 nm (Fig 54)

97




smooth (GN-S) graphene surfaces to be determined

The average edge lengths of the graphene sheets present on the rough and

smooth surfaces was statistically calculated to be 137 nm and 80 nm respectively as

shown in Table 51 The topographical analysis of graphite and graphene films were

performed using AFM and the results were summarised in Table 51


smooth (GN-S) graphene surfaces

Scanning area (microm)

Roughness parameter

GT GN-R GN-S

2 times 2 Sq (nm) 02 plusmn 01 589 plusmn 97 240 plusmn 14 Sa (nm) 01 plusmn 003 441 plusmn 84 185 plusmn 09 Smax (nm) 20 plusmn 06 6180 plusmn 1434 2156 plusmn 297 Sskw (nm) 07 plusmn 01 010 plusmn 005 -07 plusmn 02 Skur (nm) 56 plusmn 14 49 plusmn 19 41 plusmn 06

Length of edge (nm) (LGN) na 1373 plusmn 939 797 plusmn 567

Density of edge length (μmμm2) (dedge)

0 77 108

Angle of GN sheet () (GN) 0 621 372

Molecularly smooth surfaces used as the reference surface without exposed edges GT surface used as the reference plane to measure the orientation angle of graphene sheet

(a)

98

The graphene nanosheets present on the GN-R and GN-S surfaces exhibited a

distinctive orientation and geometry AFM and Raman spectroscopy were used to

characterise the graphite (GT) and the graphene surfaces and were comparably

presented in Fig 55 The GT surface was used as the control providing a reference

surface containing an average roughness (Sa) of 02 plusmn 01 nm which is considered

nanoscopically smooth The graphite surface contained layers of graphite of

approximately 15 nm ndash 2 nm in thickness as seen in the cross section line profile

given in Fig 55

The GN-R surface was found to be significantly rougher than the GN-S

surface with Sa being 589 nm plusmn 97 nm and 240 nm plusmn 14 nm for the GN-R and

GN-S surfaces respectively It was also observed using AFM and Raman mapping

that the flakes present on the GN-R surface are larger with sharper edges than those

on the GN-S surface The orientation angle of the flakes present on each of the

surfaces was determined using the AFM cross section line profile with the graphite

surface being used as the reference plane From the data presented in Table 51 it

was shown that the graphene sheets present on the GN-R and GN-S were oriented at

angles of 621 and 372 respectively confirming the higher degrees of sharpness of

the graphene flakes on the GN-R surface

99

Figure 55 Surface topographies of GT GN-R and GN-S films visualized by SEM AFM and Raman spectroscopy illustrating the

typical geometry size and thickness of graphite layers and graphene flakes on both the upper and lower sides of the film This reflects

the different dimensions in the arrangement of the flakes AFM images were taken over scanning areas of 5 microm times 5 microm with the

corresponding surface line profile representing the thickness of graphite layers and graphene flakes

100

Other topographical surface roughness parameters including skewness and

kurtosis did not highlight any significant difference between two sides of the

graphene film The same graphene suspension was used to create a single film but

with two significantly different surface structures This difference has been referred

as the ldquoBrazil nut effectrdquo (Shinbrot amp Muzzio 1998 Hong et al 2001) This

phenomenon involved a percolation effect where the graphene nanosheets were able

to pass through the gaps created by graphene microsheets causing a geometrical

reorganization through which small graphene sheets could readily fill gaps present

below the larger graphene sheets

53 Bactericidal effects of graphene nanosheet films

The response of S aureus and P aeruginosa bacteria to the surfaces of the

graphene and graphite films was examined The pyrolytic graphite (GT) was found

to be highly compatible with both types of bacteria with preserved cell morphology

being achieved on the surface and more than 95 viability of both strains being

recorded after 18 hours of contact with the surface Conversely the graphene

surfaces appeared to adversely affect the viability of the bacteria coming into contact

with the surface The morphology of the cells was significantly altered with both

types of bacteria appearing to be severely damaged by the action of both graphene

surfaces A greater number of P aeruginosa cells attached to the rougher GN-R

surface than the smooth GN-S surface with the number of S aureus cells attaching

to the two surfaces being approximately equivalent as detected using SEM (Fig 56)

101




intact cells observed on the surface of the GT

Bacterial cell viability was examined using confocal laser scanning

microscopy (CLSM) Analysis of the CLSM images clearly confirmed the

detrimental effects of exposure of the pathogenic organisms under investigation to

the graphene surfaces used in this study It was found that exposure of the P

aeruginosa bacteria to the GN-R and GN-S film surfaces resulted in 876 and

714 inactivation respectively whereas a 95 viability of these bacteria occurred

after exposure to the GT substrate Exposure of the S aureus bacteria to the GN-R

and GN-S film surfaces resulted in 531 and 771 inactivation respectively (Fig

57)

102

Figure 57 Typical (A) CLSM images and (B) quantification of viable vs non-viable cells and (C) total number of attached cells present on the

surfaces of GT GN-R and GN-S Live cells were stained green dead cells were stained red (scale bars are 10 μm)

103

Based on the evidence presented it could be seen that the geometry of the

graphene flakes profoundly influences the bacterial responses to contact with the

graphene surfaces It is believed that the strong attraction that takes place between

the graphene and the cell membrane lipids on the bacteria is largely derived from the

unique two-dimensional structure of graphene with all sp2 carbons facilitating the

exceptionally strong dispersion interactions taking place with the lipid molecules

The variable bactericidal efficiency of the sharp edges of the graphene micro- and

nano-sheet stacks formed on the GN-R and GN-S films warranted further discussion

A schematic diagram that describes the biointerface between the surface topography

of the graphene and the attaching bacteria was presented in Fig 58





morphologies

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)

Scanning length (μm)Scanning length (μm)

000

20000

40000

60000

80000

100000

000 100 200 300 400 500

GN-R GN-S

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


GN-R GN-S - -

- -

104

The physico-chemical characteristics of the GN surfaces were similar due to

the fact that both surfaces originated from the same exfoliated graphene suspension

The main parameters that were found to distinguish between GN-R and GN-S

surfaces include the edge length (LGN) the angle of orientation of the graphene

stacks (GN) and the density of the graphene edge length (dedge) GN-R surfaces with

a LGN of 1373 nm dedge of 77 μmμm2 and GN of 621ordm were found to be highly

lethal to the P aeruginosa cells (876 killing rate) and less lethal towards the S

aureus cells (531 efficiency) GN-S surfaces (which are five times smoother than

the GN-R surface) with a LGN value of 797 nm dedge of 108 μmμm2 and GN of

372ordm were found to be lethal to both types of bacterial cells (with up to 70 cells

being killed) with an overall lower number cells attaching to the surface (Fig 57)

Longer edges and higher orientation angles for the graphene sheets on the

GN-R surfaces were shown to be capable of inactivating the Gram-negative P

aeruginosa cells (Fig 58A) which is in agreement with previous reports that have

demonstrated the microbial action of a comparable surface with a maximum killing

efficiency being obtained when the exposed graphene edges are at 90deg to the

bacterial cell membrane (Akhavan amp Ghaderi 2010 Hu et al 2010a Liu et al

2011a Liu et al 2012) S aureus cells however were found to be less affected by

the action of the GN-R surface Microcavities formed by the graphene microsheets

on the substrate films were found to be of comparable size to the Gram-positive

cocci affording them some degree of protection during their colonisation of the

surface (Fig58C) In case of the GN-S surface it was found that graphene

nanosheets with a 37deg orientation were effective for inactivating attaching bacteria

Thus the key parameters contributing into the antibacterial activity of this surface

structure are very likely due to the higher density of the graphene edges (dedge 108

μmμm2 Table 51) which resulted in larger contact areas causing more local

damaging points possibly leading to phospholipid cell membrane destruction (Fig

58 B amp D)

54 Mechanism of antibacterial effects of graphene nanoflakes

There have been a number of studies investigating the antibacterial effects of

materials in the graphene family The mechanism by which they achieve their

105

antimicrobial action continues to be the subject of debate A few mechanisms have

been proposed to explain the antibacterial mode of action of such surfaces These

include the production of reactive oxygen species (Krishnamoorthy et al 2012)

oxidative stress (Liu et al 2011a Gurunathan et al 2012) or the direct extraction of

phospholipid membranes (Tu et al 2013 Hui et al 2014) These proposals are

mainly focused on two possible mechanisms one accentuates the sharp edges of

graphene micro- or nano-sheets which act as ldquobladesrdquo to cut through the cell

membrane causing the leakage of intercellular substances and eventually cell death

(Akhavan amp Ghaderi 2010 Akhavan et al 2011 Li et al 2013b Dallavalle et al

2015 Yi amp Gao 2015) The second theory suggested that the antimicrobial effect of

the graphene layers arises mainly from the physico-chemical properties of the

graphene basal plane (Hui et al 2014 Mangadlao et al 2015) More details of these

mechanisms were described in chapter 2 section 2232

The results obtained in this study strongly supports the hypothesis that the

bactericidal efficiency of the substrates depends on the lateral size shape and the

interactive angle of exposed graphene nanoflakes which are likely to puncture the

bacterial cell membranes with their sharp edges This is in agreement with the first

theory mentioned above which is also known as the ldquoinsertion moderdquo To further

understand the mechanism of this insertion process a series of single chain main

field (SCMF) simulations of the interactions taking place between cell lipid bilayer

and hydrophobic graphene surface were performed taking into account the variable

distances between the edges of the graphene flakes and perpendicularly oriented

lipid bilayer plane To implement such a system a simulation box containing the

graphene surface was replicated with periodic boundary conditions The structural

rearrangement of the lipids and the free energy cost associated with the insertion of

the attractive graphene surface was plotted as a function of the distance from the

bilayer center within SCMF theory and was shown in Fig 59 The minimum

penetration energy is at half-insertion ie when the edge of the surface reaches the

centre of the hydrophobic core at a distance of 0 This is a result of the balance

between a gain due to insertion of a hydrophobic object into the core of the bilayer

and the exposure of the edge to the solvent The pattern on the surface consists of

flakes which were treated as attractive truncated flakes of equal size and height as

shown in Fig 59 The widththickness of the cuboid was 119908 = 33 Å The flakes

106

represent a forbidden zone for lipids but the tails of the lipids can strongly interact

with the flakes with an interaction parameter 120576119900119887119895 = minus5 minus6 119896119879 and minus 75 kT at

distances shorter than 81 Aring This parameter was determined by comparison the

graphenendashbilayer interaction energy calculated from all-atom molecular dynamic

simulations Hence a periodic structure of identical graphene layers oriented

perpendicularly to the bilayer and the free energy obtained from such calculation was

considered as the minimum threshold

107

Figure 59 Free energy difference ΔF between phospholipid bilayer and inserted graphene sheets with varying hydrophobicity

(interaction parameter (120576119900119887119895) of (a) -5 (b) -6 and (c) -75 kT) as a function of the distance from the bilayer centre to the edge of the

surface Distance 40 corresponds to the unperturbed bilayer before it has made contact with the surface (zero energy reference state)

the blue stripe corresponds to the solution of insertion of the surface into the bilayer with no change in the bilayer configuration the

orange stripe corresponds to the solution with a pore in the bilayer (positive energy) Selected density profiles correspond to different

positions of graphene surface the colours of the bilayer represent the volume fraction of tails and heads from 0 to 1

108

These results are in agreement with those recently reported by Li et al who

demonstrated the spontaneous penetration of single and few-layer graphene

microsheets into cell lipid bilayers Their simulation revealed that the penetration

begins with Brownian motion including the rotation vibration and migration of GN

flakes to the bilayers followed by localized spontaneous piercing of the flake corner

to the tail groups of the lipids by attractive forces to complete subsequent full

penetration (Li et al 2013b) By simulating different penetrating angles the authors

have shown that the sharper corner of GN flakes has the lower energy barrier and is

hence the more preferable pathway (Li et al 2013b)

The simulation present in the current study was also supported by the

experimental data The surface of the bilayer could lift in order to attach to the GN

sheet increasing the area of contact between the GN and the hydrocarbon tails

present on the lipids Full insertion of the GN sheet into the membrane leads to the

formation of pores the energy of which together with the structure strongly

depends on the lipophilicity of the GN (Akhavan amp Ghaderi 2010)

In the most lipophobic case the bilayer core is separated from the GN by the

layer of head groups while in the most lipophilic case εT = minus75 kT the tails interact

with the surface of the GN The results of this simulation indicate that the surface of

the graphene nano-sheets do not act as a simple blade cutting through the cell

membrane but rather act to induce the formation of pores within the cell membrane

altering the osmotic pressure in the bacterial cell causing them to swell and

eventually die This possible scenario was confirmed experimentally using confocal

microscopy the non-viable S aureus cells (red) present on the surface appeared to

be much larger in size than the corresponding viable cells (green) (see Fig 92

presented in chapter 9)

55 Conclusion

In conclusion a simple fabrication process was carried out to fabricate

graphene films with different bactericidal efficiencies against two pathogenic

bacteria P aeruginosa and S aureus The bactericidal efficiency was found to be

due to various complex surface parameters including size shape edge length edge

density and interactive angle of graphene micro and nanosheets This appears to be

the first study that has provided both experimental and theoretical evidence that the

109

antibacterial behaviour of graphene nano-sheets arises from the formation of pores in

the bacterial cell wall causing a subsequent osmotic imbalance and eventual cell

death

110

Chapter 6

The response of eukaryotic cells on

black silicon

111

61 Overview

The biocompatibility of nanostructured surfaces has been a focus of

biomedical research for a number of years particularly in the development of

powerful tools for biological applications These applications range from cell

guidance biomolecular probes to biosensors and drug delivery systems The

physical and chemical parameters of nanostructured surfaces can be precisely

controlled to enable the manipulation of complex cellular functions including cell

adhesion migration proliferation and differentiation This study evaluated the

biocompatibility of black silicon (bSi) a high aspect ratio nanostructured surface by

investigating the in vitro responses of different cell types and the corresponding in

vivo tissue responses The nanopillar structure of bSi was fabricated by reactive ion

etching using a mixture of SF6 and O2 gas (Ivanova et al 2013) The in vitro study

involved the investigation of the cellular responses of a fibroblast-like cell line

(COS-7) which was used as the model cell type The extent of cell attachment

proliferation and metabolic activities were analysed using scanning electron

microscopy (SEM) immunocytochemistry and spectrophotometric assays

The growth behaviours of other cell lines including human and mouse

fibroblasts osteoblasts epithelial and endothelial cells were also examined to

determine the way in which these cells interact with the surface of bSi In addition

the pHF cells were co-cultured with endothelial cells to form microcapillary

structures on the surface of bSi In the in vivo study the inflammatory responses to

implanted bSi samples were investigated by means of an established subcutaneous

implantation model using CD-1 mice together with a study of the tailored

histological performance involving qualitative and quantitative histomorphometrical

analyses This work has been published under the title ldquoRace for the surfacerdquo

eukaryotic cells can winrdquo in the journal ACS Applied Materials amp Interfaces (refer to

List of publications)The study using COS-7 cells and primary human fibroblast cells

in section 62 was performed in Swinburne University of Technology by the

candidate while the responses of other cell types to bSi surfaces in-vitro and in-vivo

(section 63 - 65) were performed by Dr Shahram Ghanaati and his team

112

62 The response of fibroblast cells to black silicon surfaces

The attachment and morphology of the fibroblast-like COS-7 cells and

primary human fibroblast (pHF) cells on the bSi surface were observed using SEM

(Fig 61) Flat non-structured silicon wafers were used as a control surface It was

observed that both cell types were able to attach to the bSi after day 1 then

proliferated on the bSi as the incubation time increased The cell morphologies

appeared to be typical on both surfaces however on the bSi surfaces the pHF cells

were slightly more elongated than those on the control surfaces The COS-7 cells

also appeared to have a larger cell cytoskeleton on the bSi surface than those on the

control surfaces After 7 days the pHF and COS-7 cells on the bSi surface appeared

to be 90 ˗ 100 confluent respectively

The development of the cell cytoskeleton during 7-day incubation period was

further visualised using immunocytochemistry (Fig 62) Cells were fluorescently

labelled for vinculin (red) and actin filaments (green) which are part of the focal

adhesion network that is responsible for transmitting the regulatory signals and

mechanical forces of a cell in response to adhesion (Burridge amp Chrzanowska-

Wodnicka 1996 Amano et al 1997 Geiger et al 2001 Humphries et al 2007) As

can be seen from the confocal images the cell edges appeared to be stretched

extending the cytoskeletal membranes to an extent that was not observed on the

control surfaces

113

114

Figure 61 SEM images of primary human fibroblast (pHF) cells cultured on the bSi Si and plastic control surfaces compared to the

growth of fibroblast-like cell lines over incubation periods of 1 3 and 7 days

115





were stained with DAPI (blue)

116

The extension of finger-like filaments known as filopodia was more visible

in the cells that were attached to the bSi surface This resulted in a larger extent of

cell coverage on the surface (Fig 62) This phenomenon was also observed in

previous studies which suggested that fibroblast cells generate extended filopodia in

order to create more anchoring points when adhering to surfaces that contain a

complex geometry (Kim et al 2008 Im et al 2012 Dorkhan et al 2014 Jahed et al

2014) This result is indicative of the fact that surfaces containing micro and nano-

scale hierarchical structure can significantly affect the extent of cellular adhesion and

proliferation such as that of bSi (Lu et al 2012 Bonde et al 2014 Kim et al 2014

Starke et al 2014 Beckwith et al 2015) To visualize the cell-surface interface the

real time interactions of a single cell with the surface of bSi was sequentially

recorded using CLSM In addition a freeze fracture of the cross section of cell-

surface interface was also visualised using SEM (Fig 63)

It can be seen that the COS-7 cells began to form filaments after 20 minutes

of contact with the surface After 3 hours the cells appeared to be immobilized at a

local contact point with the well-defined finger-like membrane protrusions ie

filopodia being clearly seen as shown in Fig 63A The formation of the finger-like

membrane protrusions has been reported as being the cue parameter in the initial

attachment of cells to the nano-structured substrata (Albuschies amp Vogel 2013 Kim

amp Yang 2013 Beckwith et al 2015 Leijnse et al 2015) SEM imaging of the freeze

fractured samples of COS-7 cells attached to the bSi was shown in Fig 63C It

appeared that at the interface the cell membrane was deformed and stretched around

the nanopillars without any apparent detrimental effects to the cell morphology The

cell-surface contact plane was focused under CLSM where the cell membrane was

observed to be deformed wrapping around the nanopillars allowing them to be

engulfed within the cell membrane (Fig 63C red arrows) A similar phenomenon

was also reported in a study of embryonic rat neurons interacting with nanopillar

substrata (Hanson et al 2012) Using transmission electron microscopy (TEM) it

was demonstrated that at the local point of contact with the nanopillars the cell

membrane was stretched and distorted to adopt with the shape of the pillar

eventually engulfing the entire pillar into the cell body without disrupting the

cytosolic materials inside the cell (Hanson et al 2012)

117







CMFDA (green)

The effect of the nanopillar surface to the mammalian cellular functions was

assessed using the BCA (Fig 64) and MTT (Fig 65) assays The analysis of both

spectrophotometric assays showed that the COS-7 cells gradually grew over the bSi

surface whilst exhibiting normal cellular functions which was indicated by the

regular increases in metabolic products The growth was approximately 35 slower

than that observed on the plastic or control silicon wafer surfaces (Fig 64 amp 65)

118

The amount of intracellular proteins secreted by the COS-7 cells growing over the

bSi surfaces was found to be significantly lower than that produced by the cells

growing over the control surfaces after day one (the present amount was below the

detection limit) The values however appeared to gradually increase from day three

to day seven (Fig 2c)



different if P = 1 (t gt 005) insignificantly different if P = 0 (t lt 005))

A statistical analysis showed that there was an insignificant difference in the

amount of protein being produced by cells growing over the control and bSi surfaces

which is consistent with the lower proliferation rate of the COS-7 cells on the bSi

surfaces after a seven day growth period

119




63 The response of epithelial osteoblast fibroblast and endothelial cells to

the bSi surface

To investigate the biocompatibility of the bSi surface to a wider range of cell

types four different cell types were examined for their interaction with bSi These

cells included epithelial cells (A549) primary human fibroblasts osteoblast cells

(MG63) and primary human endothelial cells Cell growth was observed at day 1 and

day 4 using fluorescent microscopy to assess the attachment and proliferation of each

cell type on the bSi and this was compared with that observed for the control

surfaces As can be seen in Fig 65 after one day of growth on the plastic control

surfaces each of the cell types successfully attached to the surface exhibiting their

typical attachment morphology On the bSi surfaces however the epithelial and

osteoblast cells appeared to adopt a slightly reduced extent of attachment and

spreading whereas the fibroblast and endothelial cells were present in much lower

numbers exhibiting a largely rounded phenotype After four days the epithelial and

osteoblast cells formed an approximately 95 confluent monolayer on both the

plastic and bSi surfaces with similar respective cell phenotypes The fibroblasts

120

formed a completely confluent monolayer on the plastic control surface whereas on

the bSi surface these cells were found to be growing but covered only

approximately 60 of the available surface area at day four The endothelial cells on

the plastic control surface exhibited a nearly confluent monolayer with cells in

contact with one another beginning to show the typical endothelial cell cobblestone

morphology In contrast few endothelial cells were observed to be present on the bSi

surface and these exhibited little indication of attachment or spreading although a

few non-rounded attached cells were observed (arrowhead) Only very few of the

initially added endothelial cells remained viable after four days

These results indicated that epithelial and osteoblast cells were able to attach

spread and proliferate on the bSi and plastic surfaces with a typical cell morphology

and growth rate Epithelial cell lines have been reported to be successful colonisers

of nanostructured ZnO surfaces which is consistent with the results of the current

study (Li et al 2008b) The attachment response of osteoblast cells was reported to

be variable on nanopillared surfaces with the response being dependent on different

surface parameters For example Singh et al showed that surfaces containing

surface features of 20 nm height enhanced the attachment and proliferation of

osteoblast cells (Singh et al 2012a) Lim and co-workers confirmed a positive

adhesion response to surface nano-features as high as 85 nm (Lim et al 2005) More

recently Fiedler et al suggested that not only the pillar height but also the geometric

parameters such as the pillar size shape and interspacing between pillars may affect

specific cell behaviours (Fiedler et al 2013)

121

Figure 66 Monocultures of human epithelial (A549) osteoblast cells (MG63) fibroblast and endothelial cells growing on the surfaces

of plastic and bSi after 24 h and 96 h of incubation Cells were stained with Calcein-AM After a 24 hr growth period on the bSi

surfaces the epithelial and osteoblast cells exhibited a slightly reduced attachment and spreading whereas the fibroblast and endothelial

cells were present on the surface in much fewer numbers and exhibited a mostly rounded-up phenotype After 96 h the epithelial and

osteoblast cells on both the plastic and bSi surfaces had formed a nearly confluent monolayer Only very few of the initially added

endothelial cells remained viable after 96 h

122

In the first 24 hours the primary human fibroblast cells did not appear to

attach and spread over the bSi surface as quickly as observed for the plastic control

surface but after 96 h these cells were showing definite signs of growth and

spreading across the bSi surface This finding is consistent with earlier studies

which have reported the slower attachment and proliferation capability of primary

human fibroblasts on high aspect ratio surfaces compared to that of non-structured

substrates (Persson et al 2013) Very few endothelial cells attach to the bSi after 24

hour with even fewer remaining after 96 h It is noteworthy that enhanced levels of

endothelial cell growth were observed on different nanostructured surface types

(Hwang et al 2010 Loya et al 2010 Teo et al 2012 Leszczak amp Popat 2014) For

example Teo et al demonstrated that polydimethylsiloxane (PDMS) substrates

containing a 250 nm pillar structure supported the attachment of bovine corneal

endothelial cells with a higher density of microvilli being produced (Teo et al

2012) This attachment induced the up-regulation of Na+K+-ATPase expression and

activity indicating that the nanopillar surface patterns could promote the growth of a

healthy native corneal endothelium Nanopillar structured surfaces were also shown

to be a promising substrate for cardiovascular implants due to their induced

endothelialisation and reduced level of oxidative stress in primary bovine aortic

endothelial cells (BAECs) (Loya et al 2010) The authors suggested that because the

metallic surfaces containing a nanopillar structure enhanced the growth of

endothelial cells these surfaces could mitigate late stent thrombosis and could be

used for construction of other medical implants

64 Co-culture of endothelial and fibroblast cells

Co-cultures of primary human endothelial and fibroblast cells were studied

on the bSi surfaces to determine whether both cell types could survive and whether

the endothelial cells would migrate to form capillary-like structures After 10 days of

incubation cells were fixed and stained for endothelial-cell specific PECAM-1 As

can be seen in Fig 67 the endothelial cells migrated to form long fairly

homogeneous interconnected microcapillary-like structures (as indicated by arrows)

The microcapillary-like structures were observed on both the bSi and plastic

surfaces

123




specific PECAM-1 and the nuclei were stained with DAPI (blue)

The microcapillary-like structures were generated on the bSi surfaces

however they were not as well organized and fully developed as those formed on the

plastic control surfaces (Fig 67) This is in contrast to the single cell culture

experiments where the endothelial cells were not able to survive on the

nanostructured surfaces probably because of the absence of matrix attachment

factors Fibroblast cells produce extracellular matrix proteins such as collagens that

provide cell support in tissues and matrix proteins which have been shown to

increase the in vitro adherence of cells to surfaces (El-Amin et al 2003) Thus the

co-cultures of pHF and endothelial cells were able to grow over the nanostructured

bSi surfaces with microcapillary-like structures being formed by the endothelial

cells but to a lesser extent and less degree of homogeneity than that observed on the

control surfaces (Fig 67)

65 Inflammatory responses of black silicon surface

The histological analysis showed that both materials were found within the

subcutaneous connective tissue without severe inflammatory reactions (Fig 68) A

thin layer of cells was found to be present on the bSi surface (Fig 68A and B)

while a thicker layer of cells was found to be present on the silicon control (Fig 68C

and D) All of the material-adherent cells were found to be mononucleated with no

124

multinucleated giant cells being observed in any of the implantation beds of both

materials Within the surrounding tissue of both materials slightly increased

numbers of mononuclear cells were found compared to the unaffected tissue regions

(data not shown)

The immunohistochemical detection of murine macrophages showed that

only low numbers of macrophages were found within the cell layer adherent to the

bSi (Fig 68B) while the majority of the cells adherent to the surfaces of the silicon

implants were macrophages (Fig 68D) Most of the cells within the surrounding

tissue of both materials were also identified as macrophages without visible

differences being observed between both groups (Fig 68B and D)









125






scale bar are 10 microm

The histomorphometrical measurements of material-adherent macrophages

revealed that significantly more macrophages ( P lt 001) were found at the

material surfaces of the silicon control (2061 plusmn 108 macrophagesmm) as compared

to that of the bSi (821 plusmn 187 macrophagesmm) (Fig 69)




number of material-adherent macrophages as compared to black silicon ( P lt

001)

Overall the in vivo results showed that both materials induced tissue

reactions with the involvement of only mononuclear cells and did not cause any

severe inflammatory tissue reactions Thereby the histological observations showed

126

that the non-structured surfaces of the Si implants seemed to induce a larger extent of

a foreign body response as higher numbers of material-associated macrophages were

found while only small numbers of macrophages were found at the surfaces of the

nanostructured bSi implants These observations were additionally confirmed by the

histomorphometrical measurements which revealed that bSi induced significantly

lower material-adherent macrophages compared to the amount of macrophages

detected on non-structured Si surfaces

In summary the nano-structured surfaces of bSi implants induced a lower

level of an inflammatory tissue reaction These results are in line with previous

studies that have demonstrated that nanostructured surfaces are able to decrease the

level of inflammation caused by application of a biomaterial and can contribute to

reduce the extent of the foreign body response to different materials (Unger et al

2002 Andersson et al 2003 Ainslie et al 2009 Zaveri et al 2010) Zaveri et al

analysed the reaction of macrophages to nanostructured ZnO (Zaveri et al 2010)

The results showed that the number of adherent macrophages on ZnO nanorods was

reduced compared to flat substrate as observed in the present study Since the

macrophages have been identified as ldquokey playersrdquo of the foreign body response to

biomaterials it is of a considerable interest to consider how the nanostructure of

material surfaces influences this cascade of the metabolic reactions (Unger et al

2002) It was suggested that the physicochemical characteristics of biomaterial

surfaces cause a unique pattern of protein absorption to the material surface that

mediate subsequent cell and tissue responses (Unger et al 2002) Unfortunately

until now little is known about the effects of nanostructured material surfaces on the

host response on the molecular level

66 Conclusion

This study demonstrated that bSi surfaces with a specific nanopillar structure

are biocompatible with the mammalian biological system The in vitro results

showed that the surface structure present on the bSi supports the growth of COS-7

fibroblast cells and three human cell types including epithelial fibroblast and

osteoblast cells Endothelial cells when cultivated alone were not able to survive on

the nanostructured surface of bSi probably due to the absence of matrix attachment

factors however when co-cultured with primary human fibroblasts these endothelial

127

cells were able to sustain growth forming microcapillary-like structures An in vivo

study revealed that bSi does not cause a harmful inflammatory response which

strongly suggests that this surface structure could be applicable for the design of

implantable biomaterials

128

Chapter 7

The response of erythrocytes on

black silicon surfaces

129

71 Overview

In this chapter the physical interactions taking place when red blood cells

(RBCs) or erythrocytes come into contact with the nanostructured surface of black

silicon (bSi) were investigated Optical and scanning electron microscopic studies

were used to examine the time-dependent interactions of RBCs upon contact with the

bSi nanopillars The results indicated that this contact results in a rupturing effect to

the erythrocytes

Confocal laser scanning microscopy (CLSM) and Raman imaging were

performed under liquid state conditions to visualise the initial stages of the RBC

attachment to the surface and their subsequent rupture In order to explain the RBC

rupturing mechanism an analysis of the bSi surface using scanning electron

microscopy (SEM) was performed This analysis was combined with a

reconstruction of an atomic force microscopic (AFM) image of the RBC cell

membrane These complimentary techniques allowed the intercorrelation between

substratum surface nanostructure and the RBC membrane microstructure to be

determined In addition computational modelling using Single Chain Mean Field

(SCMF) theory was used to demonstrate the interaction between the nanopillars and

the unanchored lipid bilayers present on the RBC membrane The modelling data

confirmed that it was possible to rupture the RBC membrane when the sharp

nanopillars on the bSi surface could pierce through the phospholipid bilayer

membrane of the RBCs As such the interaction of RBCs with the nanostructured

black silicon material represents the upper boundary of an invasive physical

interaction brought by the congruence of the two surface topologies ie the

nanopillar array present on the bSi surface and the erythrocyte cytoskeleton present

on the RBCs The results presented in this chapter were published with the title

ldquoNanotopography as a trigger for the microscale autogenous and passive lysis of

erythrocytesrdquo in the Journal of Materials Chemistry B (refer to List of publications)

The computational modelling was conducted by the group of Dr Vladimir Baulin

72 Time-dependent interactions of erythrocytes with nanopillar surfaces

Three different control surfaces were used in this study including glass glass

covered with gelatin (1 wv) to enhance the cell attachment and silicon wafer

These control surfaces were used to determine that under optimal conditions RBCs

130

can maintain their integrity for up to three hours after being separated from blood

plasma Therefore in all experiments RBCs were not used over the period longer

than three hours It was also observed that after three hours of contact the surfaces

appeared to become saturated with attached cells cultured under physiological

conditions

The attachment of erythrocytes onto the bSi substratum were first visualised

under SEM at different time interval during three hours of contact The images

presented in Fig 71 demonstrated that RBCs appeared to be damaged after being

exposed to bSi surfaces The RBCs which remained intact preserving their

biconcave discoid shape could be differentiated from their ruptured counterparts

where the lsquofoot printrsquo of the damaged cell membrane could be observed remaining

on the uppermost layer of the nanopillars (Fig 71) This rupturing phenomenon

appeared to be time-dependent As the cell population increased when the incubation

time increased the number of deformed and ruptured cells was also seen to increase

These cells can be compared to those attaching onto the surface of the glass gelatin-

glass and silicon wafer control surfaces (Fig 72) where adhered cells could remain

intact for up to 3 hours

131

Figure 71 SEM images showing an overview of the time-dependent erythrocyte interactions with bSi nanopillar-arrayed surfaces

Images were taken at different time intervals for up to three hours of contact Scale bars are 20 microm

132




independent experiments Scale bars are 20 microm

133

The number of intact and ruptured cells was quantified according to their

distinct morphology in the SEM images (Fig 73) The total number of cells

attaching to the bSi nanopillar array increased as a function of incubation time and

was comparable with the total number of cells adhering to the control surfaces (Fig

73a) indicating a system that was dominated by gravitational sedimentation

without the effect of the bSi nanopillars

Changes in the number of intact and damaged cells that were observed on bSi

surface over time were also quantified In the first 5 minutes the number of damaged

cells appeared to be equal to the number of healthy cells on the nanostructured

surface (Fig 73b) As the time increased more cells were attached to the bSi

surface with the number of ruptured cells also proportionally increasing After 60

minutes the number of ruptured cells on the bSi substrates continued to increase

exceeding the number of intact cells (Fig 73b) After three hours of contact cells

that maintained intact morphology were remained at minimal amount while the

surface was dominated with the lsquofoot printrsquo of rupture cells The proportion of

ruptured cells occupied approximately 87 of the total number of cells that had

attached to the surface which was then saturated with a monolayer of RBCs The

maximum surface attachment density observed on the nanopillar array in this system

was sim15 times 104 cells per mm2 where whole blood diluted to a haematocrit of 2

provides approximately 1 times 109 cells per mL Such domination of damaged RBCs

was not observed in any of the control surfaces

134





ruptured RBCs which appeared like lsquocell printsrsquo on the bSi surfaces

Top and side-on SEM imaging of the interface of a single erythrocyte and the

nanopillar structure of bSi was performed allowing different stages of cell

deformation to be distinguished (Fig 74) It can be seen that after initial contact

with the surface the natural biconcave morphology of the RBC started to deform A

decreased cellular volume was observed accompanied with an engulfment at the cell

135

center and a slight stretch appearing at the edge of cell membrane at the points where

it contacts the tip of the pillars At the end of the interaction process most of cell

cytoplasm appeared to have leaked out of the cell leaving only some traces of cell

membrane on the nanopillars which were then referred to as the cell ldquofoot printrdquo



cell as a result of the action of the nanopillars

The estimated reduction in cell contact area represents a linear strain (l l0)

of approximately 186 prior to the loss of membrane integrity engulfment and

lysis The actual time of the deformation process was recorded using optical

microscopy (Fig 75) The time taken for the cells to be immobilised at the interface

of the bSi substrate to their complete disappearance due to the rupturing effects was

found to be approximately 3 min



136



httpwwwrscorgsuppdatatbc4c4tb00239cc4tb00239c2mpg

The interactions of RBCs with the bSi were also examined using CLSM

Confocal images of RBCs were taken under liquid conditions after 5 15 and 30 min

of contact with the bSi surface (Figure 76) At the first 5 minutes of incubation

most of the cells were observed to possess the typical biconcave shape of the RBCs

which started to deform after 15 minutes A majority of the cell population then

appeared to be completely deformed lacking the biconcave shape and fading in

fluorescence after 30 minutes of interaction This could be compared with the intact

typical morphology of RBCs on all of the control surfaces after 30 minutes of

incubation (Fig 76b)




ruptured cell membrane can be seen which may be regarded as the lsquocell footprintrsquo

137

Raman spectroscopic analysis was performed to obtain an insight into the

impact of real time nanopillar contact with erythrocytes also under liquid conditions

(Fig 77) Excitation at 532 nm was used to provide Raman resonance conditions for

both the bSi and erythrocyte components (Brazhe et al 2009 Brazhe et al 2013

Parshina et al 2013) The information provided in Fig 77 allowed further

visualisation of the stages of erythrocyte attachment and disruption when imaged

with the integrated RBC Raman active range of 1100 cm-1 to 3500 cm-1 The

transition from a normal biconcave discoid RBC (area marked as lsquoBrsquo) to a that of a

deformed cell morphology (area lsquoCrsquo) is clearly seen in the Raman shift image whilst

the corresponding spectra shows the onset of a Raman peak at 2700 cm-1 for cell lsquoCrsquo

undergoing cell rupture which may be due to an enhanced nanopillar resonance

which is not present in the undeformed cell lsquoBrsquo







138


the bSi nanopillar resonance peak at 480 cm-1

The results obtained from three complimentary techniques listed above

including SEM CLSM and Raman spectroscopy consistently demonstrated that the

nanopillars on the bSi tend to bend towards erythrocytes indicating a significant

level of cell affinity for the surface Other studies of the interaction between

nanostructured surfaces with different mammalian cell types such as embryonic

stem cells (Kim et al 2007b Brammer et al 2011) and hippocampal neurons

(Haumlllstroumlm et al 2007 Qi et al 2009 Xu et al 2013) highlighted that high aspect-

ratio surface structures may lead to increased adhesion strength decreased cell

mobility and high cell retention which is similar to our observations in the case of

erythrocytes In contrast to the destructive effects observed in our case however no

biocidal activities of such surfaces was reported for attached cells in these previous

studies rather it was shown that these nanostructured surfaces were compatible with

the reported cell types Moreover the enhanced cell attachment was seen to improve

communication with the cell interior facilitating the delivery of biomolecules into

cells or improving the extent of electrical signalling within neurons

73 Modelling of RBC membrane ndash nanopillar interactions

In order to explain the rupturing effects of bSi nanopillars to RBC the

surface of both bSi and erythrocyte cell membrane were analysed to gain an insight

into the mechanism driving this interaction The SEM images of the bSi showed that

bSi surface possesses a disordered array of hierarchical structure arising from

clustering of pillar tips (Fig 78a) The subsequent image analysis demonstrated that

the area population distribution of the nanopillar system reached a maximum when

the pillars were in the range between approximately 49 nm to 100 nm in diameter

the latter representing the magnitude of the nanopillar tip clusters (dimers trimers)

(Fig 78b) Fast Fourier Transform (FFT) analysis of the SEM images resulted in

images that exhibited an intense ring extending to four broad orthogonal lobes from

this secondary structure from which a grey scale intensity profile analysis allowed

an average frequency distance between adjacent nanopillars of 185 nm to be

determined (Fig 78c d) and without preferential orientation A typical side view

139

SEM image generated by prior fracturing (Fig 78e f) highlighted a characteristic

protrusion shape that exhibited widths between approximately 38 nm and 72 nm and

lengths of approximately 616 nm as diagrammatically represented in Fig 78f

140

Figure 78 Characterisation of the bSi nanopillar arrayed surfaces (a) Top view SEM image of bSi (scale bar 500 nm) (b) Area distribution

of the pillars quantified at widest cross-section showing a maximum at 49 nm in area at the widest pillar width aggregation represented by

the shoulder and tailing in the distribution extending to ~100 nm (c) Fast Fourier 2D Transform of SEM image (a) yields an intense ring

extended to four broad orthogonal lobes from this secondary structure (d) Radial grey scale intensity (0-255) profile showing the intense sharp

ing in the centre peaks at a frequency distance of 185 nm characteristic of the average distance between pillars with extended shoulders

representing secondary pillar ordering (e) Side view of bSi nanopillars and (f) schematic representation showing dimensions calculated from

average plusmn variance of 50 measurements of five SEM images

141

A deeper investigation of RBC membrane structures was conducted to

explain the high affinity of RBCs to the surface of bSi A reconstruction of the

spectrinndashactin polygon network of the RBC membrane skeleton that attached to the

bSi nanopillars was presented in Fig 79











was anchored to the spectrin network of the RBCs

It has been well established that there is a correlation between the

viscoelasticity of erythrocytes and the cytoskeleton structure that reinforces the

surface membrane (Tsubota amp Wada 2010) This skeletal network allows

erythrocytes to undergo significant extensional deformation whilst maintaining their

structural integrity (Hansen et al 1997) This network has a thickness of

approximately 79 nm and is anchored to the phospholipid bilayer which results in

142

membrane spaces of approximately 162 nm times 65 nm according to a study of Liu and

co-workers (Liu et al 2003) A reversible physical deformation of erythrocytes from

their natural biconcave discoid shape can occur under relatively small force gradients

of the order of 1 nN μmminus1 in shear flow The shear elastic modulus has been

determined experimentally to be in the range of 4ndash10 μN mminus1 (micropipette

technique) and sim25 μN mminus1 (optical tweezers technique) while the area expansion

modulus was found to be 300ndash500 mN mminus1 (Heacutenon et al 1999 Lenormand et al

2001) The schematic representation shown in Fig 79 allows the interface between

the microstructure of the erythrocyte lipid bilayer membrane (with its underlying and

reinforcing spectrinndashactin network situated on the inner cytoplasmic surface having

both junctional nodes anchoring transmembrane protein nodes) and the bSi

nanopillar surface to be examined A reconstruction of an AFM image of Liu et al

(Liu et al 2003) of the cytoplasmic side of a lectin immobilised erythrocyte was also

provided in Fig 79 which had been processed to provide comparable image

parameters to that of the nanopillar array given in Fig 78 The area distribution of

the nanopillars quantified at a distance of 20 nm from the pillar tip was given in

Fig 78b The data indicate an average diameter of approximately 12 nm while the

corresponding area distribution of the freestanding lipid bilayer within the network

mesh size displayed an average distance distribution of approximately 52 nm Hence

on average 3 to 4 nanopillar contact points may interact with each unanchored lipid

bilayer region on the erythrocyte subjecting it to a deformational strain both

between the nanopillars and the spectrin anchored bilayer

Within these unanchored lipid bilayer areas the interaction between a

nanopillar and the lipids was modelled using a Single Chain Mean Field theory

(SCMF) simulation where the lipid is represented by two hydrophobic and one

hydrophilic freely jointed spherical beads connected by rigid bonds (Fig 710) The

driving force for insertion and pinching into the bilayer arises from an attraction

between parts of the lipid to the hydrophilic bSi nanopillar (Pogodin et al 2013)

Fig 710 illustrated the changes that take place in the lipid bilayer density profile as

a cell approaches a single nanopillar and its corresponding change in free energy

143

Figure 710 Single Chain Mean Field density profile of a lipid bilayer in contact with regularly distributed nanopillars (A) General view of the

lipid bilayer and the tips of the pillars and the simulation box representing the mesh of the 3D periodic structure The box size represents the

spacing between nanopillar tips (B) A sequence of solutions corresponding to relative positions of the bilayer with respect to the nanopillar The

distances are given in Angstrom while the colours of the bilayer represent the volume fraction of tails and heads from 0 to 1 (below)

144

Within the SCMF theory structural rearrangements of lipids in the bilayer

induced by interaction with an attractive lsquoconersquo are reflected in the density profiles of

tails and heads of lipids inside the bilayer They are obtained through the solution of

SCMF equations which gives the distribution of lipids around the cone as well as the

free energy of such distribution for each position of the bilayer with respect to the cone

(Fig 710)

The difference in free energy between the unperturbed bilayer the bilayer in

contact with the nanopillar (deforming it but not piercing it) and the nanopillar piercing

the bilayer to produce a pore in which it resides was given in Fig 711 Here the initial

reduction in free energy is seen on the approach of the attractive surfaces most likely

arising from the loss of a solvation layer followed by the deformation of the bilayer

prior to the formation of a pore at approximately minus20 nm which is consistent with the

parameters used in modelling the interfacial topologies given above Insertion of the

pillar which leads to the rupture of the RBC appeared to reduce the free energy per

nanopillar by about 200 kT over the 2 nm distance (Fig 711) or by a change in force of

about 400 pN

There are basically three solutions that correspond to the different energy of the

system while the transitions between them can result in a change in the topology of the

membrane and thus the transitions are discontinuous and can therefore in principle co-

exist The free energy cost of the insertion of the attractive cone as a function of the

distance from bilayer centre is shown in Fig 711

145


between unperturbed bilayer and the bilayer with inserted attractive cone as a function

of the distance from the centre of the bilayer to the tip of the cone The red stripe

corresponds to the solution of an unperturbed bilayer and a cone before contact

(reference state zero energy) the grey stripe corresponds to a cone touching the bilayer

without piercing the bilayer the green stripe corresponds to a cone having induced the

formation of a pore in the bilayer

The three solutions are designated as red grey and green (the patterned area

corresponds to the error bar of each solution) The red curve corresponds to an

unperturbed bilayer which does not make contact with the cone (Fig 711a) This

solution could be referred as a reference state to which the free energies of the other

states can be compared The black curve corresponds to an unbroken bilayer in contact

with the attractive cone (Fig 711b) This solution has a lower free energy than the

scenario where an unperturbed bilayer does not make contact with the cone but for deep

insertion of the cone into the bilayer it co-exists with the solution corresponding to the

membrane containing a pore green curve (Fig 711c d e) The membrane containing a

146

pore is the lowest energy state for this attractive cone thus it is stable and therefore the

pore will not lsquohealrsquo upon removal of the cone This insertion-removal hysteresis (Fig

711c d e f) arises due to the lipids that are left on the surface of the cone that was in

contact with the membrane A similar behaviour was suggested for a carbon nanotube

interacting with a lipid bilayer (Wallace amp Sansom 2008) The dashed line in Fig 711

depicts a possible energy path but jumps at different points are also possible

74 Conclusion

In this study the physical interactions taking place between the nanopillars

present on the surface of bSi and erythrocytes derived from mouse were

comprehensively investigated It was demonstrated that the nanopillars present on bSi

surfaces can cause stress-induced cell deformation rupture and eventually complete cell

lysis The rupturing process was studied using multiple microscopic techniques to

examine the cell-surface interactions taking place in both dry and liquid conditions It

was found that erythrocyte rupture occurred via a process of initial surface adhesion

followed by the strain and deformation of intact cells by about 18 prior to their

rupture where the elapsed time between cell immobilisation and rupture was

approximately 3 min Experimental analysis allowed the determination that

approximately 3 to 4 nanopillars on the surface of bSi would be interacting with the

unanchored lipid bilayer region on the RBC membrane within the spectrin-actin

network Finally these interactions were modelled using Single Chain Mean Field

theory in terms of a free energy driving force which indicated that the spontaneous

rupture of the lipid membrane occurred through the direct piercing of the RBC

membrane by the nanopillars This study provides an insight into the hemocompatibility

of nanostructured surfaces which are important for further biomedical applications

147

Chapter 8

Competitive colonisation of bacteria

and eukaryotic cells onto the surface

of bactericidal black silicon

148

81 Overview

With the increasing demand for medical implants managing bacterial infections

associated with implant surgeries remains a global challenge Despite there being

numerous research investigations reporting new antibacterial bio-surfaces there appears

to be a paucity of data pertaining to how host cells can compete with bacteria that may

be present on an implant material for their effective surface integration This was

initially described as ldquothe race for the surfacerdquo by Anthony Gristina (Gristina 1987) If

the race is won by the host tissue the implant becomes protected from invading

pathogens allowing normal tissue integration of the implant to take place In contrast if

the race is won by the pathogenic bacteria severe inflammatory responses often occur

leading to unsuccessful tissue integration In the later scenario bacteria that were

successfully colonized onto implant surfaces can further develop into bacterial biofilm

which affords them the ability to resist multiple antibiotic treatments leading to failure

of implant and even mortality (Donlan 2001 Zimmerli 2006 Del Pozo amp Patel 2009

Levent et al 2010 Busscher et al 2012 Daşbaşı amp Oumlztuumlrk 2016 Ranghino et al 2016

Rasamiravaka amp El Jaziri 2016) For these reasons appropriate understandings on how

newly designed biomaterial surfaces can affect the competitive colonisation between

eukaryotic cells and bacteria onto the surfaces are essential so that effective

antibacterial biocompatible surfaces can be designed

Black silicon (bSi) was previously reported to possess broad spectrum

bactericidal activity (Ivanova et al 2013) It was also demonstrated in previous chapters

that the nanopillar surface structure of bSi can selectively support the growth of various

mammalian cells In this chapter the growth of the model eukaryotic cells COS-7 was

on the bSi surface that was previously infected with pathogenic bacteria to mimic the

typical post-infection scenario of implanted biomaterials To conduct the experiments

black Si and the Si wafer control surfaces were infected with Staphylococcus aureus

CIP 658T and Pseudomonas aeruginosa ATCC 9027 bacteria at their infective doses as

given by the FDA USA for 6 hours The infected surfaces were then exposed to COS-7

cells with the co-culturing of both species being examined for up to 7 days using SEM

and CLSM It was found that the COS-7 cells successfully attached and proliferated

149

over the infected bSi while the bacteria appeared to be completely eliminated from the

bSi surfaces Meanwhile the COS-7 cells on the non-structured Si surfaces were

observed to be poorly attached with a limited number of proliferated cells due to the

domination of the bacterial contaminants The results presented in this chapter were

published with the title ldquoRace for the surface eukaryotic cells can winrdquo in the journal

ACS Applied Materials amp Interfaces (refer to List of publications)

82 Real time antibacterial activity of bSi

The antibacterial effects of bSi were evaluated using Pseudomonas aeruginosa

and Staphylococcus aureus bacterial cells at their respective infective doses as indicated

by the FDA (Schmid-Hempel amp Frank 2007 FDA 2012) The results obtained from

SEM and CLSM images showed that both types of microorganisms appeared to be

damaged after 6 hours of contact with the nanopillars with more than 90 of bacterial

population appeared to be dead (Fig 81) Meanwhile there was no such rupturing that

was observed on the flat non-structured silicon wafer control surfaces This is consistent

with the previous findings of Ivanova et al who demonstrated that bSi exhibited highly

efficient bactericidal activity in a mechano-responsive manner in which the mechanism

is based on the rupturing effects of the sharp tips of bSi nanopillars to bacterial cell

membrane (Ivanova et al 2013) This resulted in a deforming stress being applied to the

contact areas of the cell membranes leading to membrane disruption causing cell

cytoplasmic fluid leakage and eventually cell death (Ivanova et al 2013 Pogodin et al

2013)

150

Figure 81 SEM images of the damaged bacterial cells on the nanopillar structured surface of bSi (a amp b) and intact bacterial

cells on non-structured silicon wafer control surfaces (c amp d) scale bars are 2 microm Sequential time lapse confocal microscopic

images showing the dynamic bactericidal activities of bSi interacting with P aeruginosa (e) and S aureus (f) over 6 hours

scale bars are 5 microm

151

The first 6 hours of contact between bacteria and an implant surface has been

recognised as the most critical period for the initiation of infection this stage is

referred to as the ldquodecisive periodrdquo It has been reported that during this stage the

host immune system can potentially be effective in neutralizing invading pathogenic

bacteria with the aid of prophylactic antibiotics (Poelstra et al 2002 Hetrick amp

Schoenfisch 2006) Therefore the pathogenic bacteria were allowed to interact with

the bSi surface for 6 hours to evaluate whether this period would be sufficient for the

bSi surface to passively eliminate the bacterial cells Time-lapse sequential confocal

imaging showed that initially more than 80 of the bacterial population was viable

(Fig 81 shown in green colour) These cells were maintained in a humidified 37degC

chamber to ensure that optimal growth could be achieved during the entire imaging

time It was observed that the cell viability progressively reduced with the number

of dead cells increasing with time (shown in red) After 6 hours less than 10 of

both cell types were found to be still viable on the nanopillar surface (Fig 81 eampf)

This is in consistent with the previous study which reported the broad spectrum

antibacterial property of bSi (Ivanova et al 2013) In order to address how the bSi

surface nanostructure can affect the colonisation of host cells in the presence of

bacteria the infected bSi surfaces were cultured with COS-7 cells to examine the

effect of the surface to both cell types

83 Competitive colonisation of pathogenic bacteria and COS-7 on bSi

The colonization of COS-7 cells on pre-infected silicon surfaces was

observed over a seven day incubation period As can been seen from the SEM

images given in Fig 82 the COS-7 cells that had attached to the infected

nanostructured bSi appeared to maintain their typical morphology with extended

filopodia being observed within the first 24 hours of adhesion There were no signs

of bacterial contamination on the surfaces from day one to day seven suggested all

the S aureus and P aeruginosa bacterial cells had been killed by the action of the

surface on the first day After this time only bacterial cell debris was detected on the

bSi surfaces This was confirmed by examining the bSi surfaces using SEM (Fig

82) and confocal microscopy (Fig 83) These results are consistent with the

previous study that highlighted the bactericidal efficiency of the bSi surfaces

(Ivanova et al 2013) The COS-7 cells that had been seeded onto the infected bSi

surfaces appeared to be viable after one day of incubation with a significant increase

152

in cell numbers being apparent after three days of incubation and 100 confluency

being reached after seven days These results confirmed that the fibroblasts were

able to successfully colonize the infected nanostructured bSi surfaces Notably

traces of the bacterial debris that had been detected one day after the initial seeding

were not observed after three and seven days indicating that the dead bacterial

debris had detached from the surface thereby not interfering with the growth of the

COS-7 cells (Fig 82)

In contrast both the P aeruginosa and S aureus cells were observed to form

biofilms on the silicon wafer control surfaces These cells inhibited the growth of the

the inoculated fibroblasts It can be seen that after 7 days of incubation the P

aeruginosa cells had completely overgrown the COS-7 cells such that no COS-7

cells could be detected (Fig 82 amp 83) The fibroblast cells were however able to

maintain their viability in the presence of S aureus cells and co-exist for up to 7

days on the silicon wafer control surfaces This is likely because the S aureus

colonisation of the surface was partially inhibited by the presence of antibiotics (1

penicillin-streptomycin) present as supplements in the Dulbeccos Modified Eagles

medium (DMEM) used for the cultivation of the COS-7 fibroblast cells while the P

aeruginosa cells appeared to be resistant to this antibiotic supplement

153

Figure 82 SEM images of COS-7 cell growth onto the infected bSi surface and Si wafer control surfaces after 1 3 and 7 days of

incubation Both surfaces were infected with P aeruginosa and S aureus cells for 6 hours at their respective infective doses prior to

the surfaces being exposed to the COS-7 cells

154


silicon wafer control surfaces Live COS-7 cells were stained with Calcein AM

(green) dead COS-7 cells were stained with Ethidium homodimer-1 (red) bacteria

were stained SYTOreg 9 (blue)

The numbers of viable COS-7 cells on the pre-infected bSi and Si surfaces

were plotted as a function of incubation time for comparison (Fig 84) Starting at

the same seeding density of 5000 COS-7 cells per cm2 for all substrate surfaces both

of the groups that were seeded onto the infected bSi exhibited a similar growth rate

155

reaching a population of approximately 9 times 105 cells per cm2 which covered more

than 90 of the surface area


and silicon wafer control surfaces

The Si wafer control surfaces however showed a selective growth of COS-7

cells on surfaces infected with S aureus at a constant rate reaching approximately

34 times 105 cells per cm2 after one week In case of growth on surfaces infected with P

aeruginosa cells an initial attachment of COS-7 cells was observed after day one

however this mammalian cells failed to maintain long-term viability with no growth

being detected at day three and day seven These results most likely represent the in-

vitro scenarios taking place when implant materials contain microorganism

infections Even with aid of antibiotics the nanostructured biomaterials would be a

critical factor that contributes to successful cell attachment and subsequent tissue

integration protecting the implant material from infections

156

84 Conclusion

The surface nanostructure of black silicon with its particular nanopillar

geometry was shown to effectively eliminate bacterial colonisation while at the

same time being able to support the growth of mammalian cells with no apparent

negative effects With the challenge of increasing clinical infection being induced by

the presence of antibiotic-resistant microorganisms the nanostructure of bSi

represents a model surface in the design of safe biocompatible smart nanomaterials

that are able to physically prevent bacterial contamination These results offer a

promising surface topology for the fabrication of newly antibacterial biomedical

devices

157

Chapter 9

General discussion

158

91 Overview

The interactions that take place between cells and substrate surfaces with

which they interact have long been a focus of research These interactions have been

known to play critical role in determining whether or not a biomaterial or device can

resist or prevent the formation of a biofilm which will in turn determine the ultimate

success of the biomaterial or device This research has focused on the physical

chemical and biological aspects of cellndashsurface interactions mainly at the micro and

nano length scales It is now recognised that the fate of the cell is determined by the

various complex cellular events that happen initially over nano- and molecular size

scales These fundamental discoveries have opened a new era for nanotechnology in

which the surface structure of a material can be precisely controlled to manipulate

some specific cell functionalities on a nanometric scale A thorough understanding of

the mechanisms taking place as well as the parameters affecting these cell-surface

behaviours have not yet been attained and hence further investigation was

warranted

Recently a new approach for dealing with biomaterial-associated infections

has been proposed This involves modulating the nanostructure of a material surface

providing the surface an ability to mechanically kill bacteria or prevent bacterial

colonisation simply through physical contact These surface nanotopographies are

inspired by the antibacterial self-cleaning properties of natural surfaces such as

those of insect wings lotus leaves or shark skin (Bhushan amp Jung 2010 Reddy et al

2011 Webb et al 2011a Ivanova et al 2012 Truong et al 2012 Hasan et al

2013b Ivanova et al 2013 Mann et al 2014 Falde et al 2016 Waugh et al 2016)

The synthetic antibacterial surfaces can be constructed on biomaterials affording

them the advantage of being chemical free and hence are potentially a solution for

the bacterial resistance problems that have arisen as a result of increasing levels of

chemical-based infection treatments The mechanisms driving the effects of these

synthetic surfaces to host cells including the question of biocompatibility and the

cytotoxicity of these materials to the human system however remain unknown

Furthermore the ability of a material surface to support the overgrowth of host cells

in the presence of pathogenic bacteria affording the surface the ability to prevent

infection whilst at the same time ensuring proper tissue integration is highly

desired Prior to the current work being undertaken there has not been a surface

159

capable of exhibiting these dual properties reported in the literature Fortunately

advances in nanotechnology have allowed new surfaces to be synthesised that may

provide new hope in facing these challenging problems

This chapter will provide an overview of the new experimental results

presented in the previous chapters discussing the significant effects that different

surface nanostructures have on bacterial colonisation While surface roughness can

be used as one indicator of surface topography it was found in this research that this

parameter alone is unable to predict the complex processes associated with bacterial

attachment at the nanoscale level the process involves other spatial and geometrical

parameters that can play vital roles in determining whether bacterial colonisation

will take place on a surface Also the in vitro and in vivo responses of host cells to

one potential antibacterial surface black silicon were demonstrated using a range of

different mammalian cell types including red blood cell fibroblast osteoblast

epithelial endothelial cells (in-vitro) and macrophages (in-vivo) The novel ability of

the bSi surface to be able to support mammalian cell growth over pathogenic

bacteria in an infection event known as the ldquorace for the surfacerdquo will also be

discussed


and rough surfaces with distinct surface architecture

It is known that the attachment and colonisation of bacterial cells cannot be

adequately explained and predicted by the accepted theories based on cell surface

charge hydrophobicity Van der Waals gravitational and electrostatic forces

(Costerton et al 1999 Donlan amp Costerton 2002 Costerton et al 2005) It is now

known that the attachment of bacterial cells is greatly related to surfaces containing

micro nano and molecular scale topography which may affect the bacterial viability

and subsequent biofilm formation (Whitehead et al 2005 Diacuteaz et al 2007 Park et

al 2008 Anselme et al 2010 Decuzzi amp Ferrari 2010 Puckett et al 2010) The

mechanisms and the parameters involved in the interactions between bacterial cells

and surface nanostructures however are not fully understood In this study various

bacterial cells were found to exhibit distinctive responses to smooth and rough

substrate surfaces These responses were dependent on the various surface

parameters present on the substrates at the nanoscale other than surface roughness

160

Comparison of the behaviours of the same bacterial strains to different surface

topographies and architecture provided some striking observations regarding the

effects of these surface structures to bacterial colonisation

As reported in chapter 4 two molecularly smooth titanium surfaces with

similar surface roughness properties were found to result in different extents of

attachment of P aeruginosa cells A higher number of P aeruginosa cells were

found to attach onto a titanium surface that possessed nanoprotrusions of

approximately 20 nm high and 35 nm spacing between each other compared to the

unmodified titanium substrate (see section 43) These nanoprotrusions act to provide

a greater number of anchoring points to the P aeruginosa cells causing the cell

membrane to stretch and therefore allow the rod-shaped P aeruginosa to attach to

the smoothest surface compared to that obtained on other similar smooth surfaces but

without the nanoprotrusions (Mitik-Dineva et al 2008 Anselme et al 2010 Truong

et al 2010 Almaguer-Flores et al 2012) The presence and distribution of these

nanoprotrusions can be determined by analysing AFM spatial surface parameters

such as skewness and kurtosis (Gadelmawla et al 2002 Whitehead et al 2006

Crawford et al 2012 Webb et al 2012) (refer to Table 43) Transmission electron

micrographs of the substrate surfaces clearly revealed the different sizes shapes and

distribution of the ultrafine grains between the two titanium surface structures where

those possessing the nanoprotrusions were shown to display significantly enhanced

levels of bacterial attachment (Fig 91) Previously Ivanova et al reported that the

attachment of P aeruginosa cells was highly restricted on the molecularly smooth

titanium thin film surfaces (Ivanova et al 2011) They suggested that the rod shape

of P aeruginosa cells maintained a low turgor pressure which generates a repulsive

force that is sufficiently large so that the cells exhibited the ability to unbind and

slide off the nanosmooth surface The kurtosis and skewness values shown for these

surfaces were however extremely low (approximately 001 nm for both Skur and

SSkw) indicating the absence of anchoring points for rod-shaped P aeruginosa cells

leading to the inability of these cells to remain attached to such smooth surfaces

161




angular pressing (ECAP) modification process

Some earlier studies suggested a similar mechanism of attachment when

describing bacterial attachment onto micro-patterned surfaces For example P

aeruginosa and S aureus cells were found to attach onto surfaces containing

regularly spaced pits of 1 microm and 2 microm in size yet not onto surfaces containing

irregularly spaced pits of 02 microm and 05 microm in size while both surfaces exhibited

highly similar physico-chemical properties (Whitehead et al 2005) E coli cells

were also shown to attach to surfaces containing micro-scale patterns but were

aligned along the microgrooves that were 13 μm wide and 130 nm deep (Diacuteaz et al

2007) In a later study these bacteria were however unable to attach onto surfaces

with a groove height of 50 nm and period of 16 μm (Ploux et al 2009) These

observations were explained in light of the ldquoattachment point theoryrdquo in which

bacteria favourably respond to the surfaces containing micron scale features which

afford the bacteria shelter from the external environment (Scardino et al 2008

Mitik-Dineva et al 2009 Truong et al 2012)

Not all surfaces that contain nano and micro-features favour the colonisation

of bacteria Other parameters such as the geometry and orientation of a specific

surface pattern can also greatly affect bacterial responses This was demonstrated

162

using the nanoflake structure of graphene surfaces which exhibited variable

antibacterial activities towards bacterial cells (see Chapter 5) Graphene surfaces are

rougher than titanium surfaces exhibiting Sa values from 219 nm to 119 nm The

bactericidal activities of graphene surfaces were found to be induced by the sharp

edges of the graphene nanoflakes present on the surface This result is consistent

with one of the proposed mechanisms reported in recent research stating that the

sharp edges of two-dimensional graphene sheets can act as ldquoknivesrdquo to cut through

the cell membrane causing the leakage of intercellular substances and eventually

cell death (Dallavalle et al 2015 Luan et al 2015 Mangadlao et al 2015 Yi amp Gao

2015 Zou et al 2016) In this study the geometry and orientation of the graphene

nanoflakes were identified for the first time as the critical parameters that directly

influence the antibacterial efficiency It was found that long dimension and high

orientation angles of graphene edges (62ordm) can effectively cut through Gram-negative

P aeruginosa cells but not coccoid S aureus cells The presence of microcavities

formed by the graphene microsheets may act as lsquosheltersrsquo for S aureus colonisation

(refer to Chapter 5 section 53) Graphene nanosheets with a lower orientation (37ordm)

but present in a higher density would result in a larger number of contact points for

the coccoid S aureus cells causing membrane destruction and therefore cell death

A mechanism was thus proposed based on the simulation and experimental data that

the bactericidal activities of the graphene nanoflakes arise from the sharp nanoflake

edges causing pores to form within the phospholipid membrane of bacterial cells

This leads to an osmotic imbalance in the bacterial cells eventually resulting in cell

death (Fig 92)

163






membrane resulting in pore formation

Graphene nanosheets possess antibacterial properties that do not rely on any

chemical interactions with bacteria and therefore represent a prospective coating

material for biomaterial surfaces A similar mechano-responsive bactericidal effect

was previously reported for black silicon (bSi) (Ivanova et al 2013) Black silicon

contains an array of nanopillars on its surface similar to that found on the wings of

some species of dragonflies The bactericidal activity of bSi can reach up to

~450000 and ~360000 killed cells min-1 cm-2 over the first 3 hours of contact with

respect to S aureus and P aeruginosa cells respectively This antibacterial property

was shown to arise from a mechanical process that was not a function of the

chemical characteristics of the bSi surface This makes the bSi nanotopology also

suitable for the design of biomedical implants The identification of this surface

prompted the further investigations in this current study into the eukaryotic cell

(a)

(b)

164

responses to the bSi surface and investigations into the ability with which such a

surface can support host cell integration including situations where pathogenic

bacteria are present on this surface


structured black silicon surface

The nanopillar structure that was found to be responsible for the broad

spectrum antibacterial properties of bSi were tested for its biocompatibility using a

range of different mammalian cell types The in-vitro analyses showed that bSi

surfaces were able to promote the attachment and proliferation of fibroblasts

osteoblasts and epithelial cells (see Chapter 6) Endothelial cells did not sufficiently

attach to the bSi surface however they appeared to form interconnected

microcapillary-like structures after 10 days of being co-cultured with fibroblast cells

These results confirm the biocompatibility of high aspect ratio surfaces that have

been well-documented in the literature (Anandan et al 2006 Nomura et al 2006

Haumlllstroumlm et al 2007 Kim et al 2007b Bettinger et al 2009 Brammer et al 2011

Hanson et al 2012) Additionally a single cell analysis of COS-7 cells has shown

that the nanopillar array on the bSi surface can enhance the formation of filopodia

which significantly contributes to the focal adhesion network promoting cell-cell

intercommunication and the subsequent bacterial adhesion process (Burridge amp

Chrzanowska-Wodnicka 1996 Sniadecki et al 2006 Hanson et al 2012

Albuschies amp Vogel 2013)

Erythrocytes or red blood cells (RBC) are a critical component of blood

These cells plays a major role in determining the haemolytic activity and blood

clotting associated with biomaterial surfaces (Weber et al 2002) It was found that

the nanopillars present on the bSi surface can trigger the autogenous lysis of RBCs

after only five minute of contact (see Chapter 7) It is believed that this phenomenon

arises from a combination of the high aspect ratio surface structure and the geometry

of nanopillar tips which were sufficient to disrupt the spectrin-actin network present

in the lipid bilayer of RBCs resulting in the lysis of the RBC interior components

Haematological toxicity studies have to date predominately focused on the effect of

nanoparticles on blood cells (Choi et al 2011 Love et al 2012 Nemmar et al 2012

Shah et al 2012 Wang et al 2012b Baumann et al 2013 Joglekar et al 2013)

165

where it has been found that haemolysis is dependent on the size shape

concentration and chemical nature of the nanostructured materials (Sohaebuddin et

al 2010 Love et al 2012 Shah et al 2012 Wang et al 2012b Joglekar et al

2013) It should be noted that the lysis of RBCs were observed when the first

monolayer of RBCs had come into contact with the bSi surface (within 3 hours)

while the accepted hemolysis level for blood is 2 (Allison et al 2007 Nemani et

al 2013) Therefore long term exposure of RBCs to bSi as well as the responses of

the other blood components such as platelets and monocytes should be further

studied to determine the complete hemocompatibility of bSi

In the in-vivo analysis where bSi materials were inserted into the

subcutaneous connective tissue of mice the animals did not exhibit a severe

inflammatory reaction with a low number of macrophages being observed to be

present in the layer adherent to bSi surface (see Section 65 Chapter 6) This positive

histological analysis has provided evidence that the bSi surface exhibits

biocompatibility characteristics with regard to mammalian cells Another piece of

work that focussed on determining the in vivo toxicity of silicon nanowires

demonstrated that lung injury and inflammation caused by exposure to silicon

nanorods could be resolved over time in a dose-dependent manner (Roberts et al

2012) These authors observed that more than 70 of deposited silicon nanowires

were able to be cleared from the lungs after 28 days with none being detected after

91 days in the lung tissue (Roberts et al 2012) The authors also pointed out that

collagen might have been deposited after long term exposure leading to fibrosis

when very high aspect ratio (25 nm in diameter 15 microm in length) fibres were

present which is not the case of bSi (25 nm in diameter 600 nm in length) Overall

the surface of the bSi is both antibacterial and biocompatible The remaining

question is whether or not the advantages afforded by the combination of these two

properties could allow the mammalian cells to win the ldquorace for the surfacerdquo when

pathological bacteria are also present

94 Competitive colonisation of bacteria and mammalian cells onto the

surface of black silicon

The study presented in Chapter 8 was performed in order to obtain an insight

into whether a biomaterial that had been contaminated with pathogenic bacteria

166

during handling or transport could be designed to exhibit antibacterial properties

whilst also being able to sustain the normal attachment and proliferation of

mammalian cells Staphylococcus aureus and Pseudomonas aeruginosa bacterial

cells were chosen as representative pathogenic bacteria based on a number of

medical research projects that have reported these species as two of the most

frequently encountered Gram-positive and Gram-negative infection-related

pathogens (Fig 93) (Zimmerli et al 1982 Murdoch et al 2001 Zimmerli 2006 Del

Pozo amp Patel 2009 Montanaro et al 2011 Sendi et al 2011)





device Adapted with permission from (Montanaro et al 2011)

The ldquorace for the surfacerdquo between COS-7 fibroblast-like cells and the

bacteria under investigation onto the bSi surface was studied by pre-infecting the bSi

surfaces with these two strains prior to allowing the COS-7 cells to come into

contact with the surface This experimental design mimics the common post-

infection situation in which infection may occur in a foreign body despite the use of

a perioperative antimicrobial prophylaxis since fewer than 100 cfu of

167

microorganisms can induce infection (Zimmerli et al 1982) Murdoch et al

observed that during S aureus bacteraemia an implant-associated infection

developed in 15 out of 44 patients with prosthetic joints (Murdoch et al 2001) Thus

infection can occur not only during surgery by pre-adherent bacteria but can also

occur during the entire lifetime of the implant

Under the co-culture conditions the nanopillar surface structure of the black

silicon was shown to be able to effectively maintain the attachment and growth of

COS-7 cells with no signs of infection after 7 days Similar results were observed

regardless of bacterial type indicating a dual efficiency of the surface which not

only exhibits bactericidal properties but also has the ability to selectively eliminate

only the bacterial cells whilst promoting the growth and proliferation of the

eukaryotic cells Given that the nanotopology demonstrated by this bSi topology has

now been shown to exhibit substantial biocompatibility and a lack of an

inflammatory response together with its ability to eliminate bacterial contamination

without the need for antimicrobial agents this topology represents a significant

prospect for smart antibacterial nanomaterials especially in an era of increasing

concern for antibiotic resistance

It should be noted that the results presented in this study demonstrate the

initial interactions between bacteria and host cell to the nanostructured bSi surfaces

The event of host cell integration involves various other processes including protein

adsorption blood coagulation cell differentiation and tissue integration The effects

of these biological activities to the functions of nanostructured surfaces as well as

the question whether or not the presence of different biological components would

attenuate the antibacterial properties of this surface topology require further research

168

Chapter 10

Conclusions and future directions

169

101 Summary and conclusions

The study of the activity of biological organisms at substrate surfaces is

necessary to allow a greater fundamental knowledge of the factors that influence cell

behaviours so that biomaterials and other biological devices can be effectively

designed The nanostructure of material surfaces has been shown to correlate with a

number of complex cellular processes however this relationship remains poorly

understood In this project the effects of substrates having different micro- and

nanoscale level surface structures were compared to the corresponding behaviours of

various bacterial and mammalian cells

Titanium substrates possessing 20 nm tall nanoprotrusions with an average

distance of 35 nm were shown to enhance the attachment of P aeruginosa bacterial

cells It was previously reported that molecularly smooth surfaces restrict the

adhesion of P aeruginosa cells This study however demonstrated that if the

surfaces possess nano-features that could act as anchoring points for bacteria at an

appropriate size and distribution bacteria could adhere to the smoothest surfaces In

contrast rough surfaces that contained sharp features at different orientation angles

could cause variable destructive effects to bacterial cells as were shown with the

graphene surfaces The extent of bactericidal activity of graphene films is sensitive

to the morphology of the bacteria and the geometry of the graphene nanoflakes that

are present on the film surfaces including the dimension orientation and the edge

length of the flakes A mechanism was proposed that the graphene nanosheets were

able to puncture the cell membrane via the sharp edges of the graphene nanoflakes

inducing the formation of pores in the cell membrane causing the osmotic imbalance

inside the cells eventually resulting in cell death

The nanostructure of black silicon being known for its broad spectrum

mechano-responsive antibacterial properties was investigated to determine the

responses of other mammalian cell types to the bSi surface It was found that black

silicon was compatible and non-damaging to various mammalian cells in-vitro

including epithelial cells primary human fibroblasts osteoblast cells and COS-7

fibroblast-like cells Whilst endothelial cells when seeded alone were not able to

survive on the bSi nanostructured surfaces they were able to sustain their growth

forming microcapillary-like structures when co-cultured with primary human

170

fibroblasts When applied to erythrocytes contact with the bSi surface resulted in

highly active autogenous lysis The physical interaction brought about by the spatial

convergence of the nanopillar array present on the bSi and the erythrocyte

cytoskeleton present on the red blood cell membranes provided sufficient force to

spontaneously induce rupture of the cells leading to passive lysis In the in vivo

environment bSi showed a reduced inflammatory response compared to its non-

nanostructured equivalent

The positive attachment response of the mammalian cells on the black silicon

surface together with the destructive effects caused to pathogenic bacterial cells

was confirmed when each cell types were allowed to interact separately to the

surface The ldquorace for the surfacerdquo in which both mammalian and bacterial cells had

to compete for the effective colonisation of the surface was experimentally studied

by investigating the behaviours of COS-7 cells on the bSi surface that had been

previously infected with live bacteria at their infective doses It was found that bSi

surface was able to eliminate the bacterial cells whilst simultaneously promoting the

growth of the mammalian cells After seven days of interaction the surface was fully

confluent with fibroblast cells with no signs of bacterial contamination being

evident

This work provides the first demonstration of the dual behaviour of a surface

nanostructure which not only possesses bactericidal properties but also has the

ability to selectively eliminate only bacterial cells whilst supporting the growth and

proliferation of eukaryotic cells

102 Future directions

While the current work has generated useful knowledge regarding the effects

of nanostructured surfaces on bacterial and mammalian cells coming into contact the

interactions of these surfaces with other biological components would require further

investigation to understand the complex host responses to antibacterial surfaces One

of the important events that occur on implant surfaces is the adsorption of plasma

proteins Gaining an insight into how essential plasma proteins such as fibronectin

fibrinogen vitronectin and collagen behave on the nanostructured materials would

contribute to the body of knowledge regarding the biological response properties of

bSi These adhesive proteins are known to mediate the adhesion of cells thus

171

determining the extent of subsequent tissue integration The bactericidal efficiency

of bSi as well as the role played by the bSi nanostructure with an adsorbed protein

layer in the race for the surface should also be determined The possible long-term

toxicity of the nanopillar structure in vivo could also be a subject of future research

The nanoflake structure of graphene films is another prospective design for

antibacterial surfaces thus the interaction of these surfaces with mammalian cells

would be of interest in further studies Recent reports have shown that graphene and

graphene derivatives can be used as a coating and functionalised material for implant

materials to prevent bacterial infection (Kulshrestha et al 2014 Zhang et al 2014

He et al 2015 Jung et al 2016) The nanostructure of antibacterial surfaces such as

bSi and graphene could be used as models to be replicated on other materials that are

used in biomedical and implant applications such as metal and polymer substrates

The surface micro- and nano-structures that were fabricated on the two sides of the

single graphene film could be applied to the generation of other double-sided

antibacterial film with dual effects

103 Final remarks

Generating compatible long-term efficient antibacterial surfaces for

biomaterials has been one of the challenging goals in life sciences for decades

Clinical issues associated with biomaterial infection include a severe inflammatory

responses antibiotic resistance failure of implantation and even mortality

accompanied with increased health care costs Researchers have been seeking

alternatives that could prevent bacterial infection without the use of antimicrobial

chemicals or additives Several antibacterial surfaces have been introduced that

contain a surface structure that is capable of exhibiting antimicrobial behaviour

based on the physical interactions between the surface nanostructure and the

bacterial cells At the same time it is important to understand the behaviours of host

cells on such antibacterial surface structures especially when bacteria are also

present on the surface The results of this competitive event would determine the

success of an implant however an in-depth knowledge of this phenomenon still

needs to be achieved

The results presented in this thesis contribute to the body of knowledge of the

complex biological activities taking place at material surface interfaces Various

172

surface parameters have been identified for their effects to the behaviours of cells A

novel experimental design has been shown to be very useful in studying the cell-

material interactions in an infection event The nanostructured surface of black

silicon with a dual effect in promoting host cell response while eliminating bacteria

marks a milestone in the search for an effective surface structure that acts against

bacterial contamination

173

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174

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Abragravemoff MD Magalhatildees PJ amp Ram SJ 2004 Image processing with ImageJ Biophotonics International vol 11 no 7 36-41

Aicheler M Sgobba S Arnau-Izquierdo G Taborelli M Calatroni S Neupert H amp Wuensch W 2011 Evolution of surface topography in dependence on the grain orientation during surface thermal fatigue of polycrystalline copper International Journal of Fatigue vol 33 no 3 396-402 Ainslie KM Tao SL Popat KC Daniels H Hardev V Grimes CA amp Desai TA 2009 In-vitro inflammatory response of nanostructured titania silicon oxide and polycaprolactone Journal of Biomedical Materials Research Part A vol 91 no 3 647-55

Akhavan O amp Ghaderi E 2010 Toxicity of graphene and graphene oxide nanowalls against bacteria ACS Nano vol 4 no 10 5731-5736 Akhavan O amp Ghaderi E 2012 Escherichia coli bacteria reduce graphene oxide to bactericidal graphene in a self-limiting manner Carbon vol 50 no 5 1853-1860

Akhavan O Ghaderi E amp Esfandiar A 2011 Wrapping bacteria by graphene nanosheets for isolation from environment reactivation by sonication and inactivation by near-infrared irradiation The journal of Physical Chemistry B vol 115 no 19 6279-88 Akhtar MJ Ahamed M Fareed M Alrokayan SA amp Kumar S 2012 Protective effect of sulphoraphane against oxidative stress mediated toxicity induced by CuO nanoparticles in mouse embryonic fibroblasts BALB 3T3 Journal of Toxicological Sciences vol 37 no 1 139-148

Akhtar MJ Ahamed M Kumar S Siddiqui H Patil G Ashquin M amp Ahmad I 2010 Nanotoxicity of pure silica mediated through oxidant generation rather than glutathione depletion in human lung epithelial cells Toxicology vol 276 no 2 95-102 Albrektsson TO Johansson CB amp Sennerby L 1994 Biological aspects of implant dentistry osseointegration Periodontology 2000 vol 4 58-73

Albuschies J amp Vogel V 2013 The role of filopodia in the recognition of nanotopographies Scientific Reports vol 3 1658

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Alexander KE Donggyoon H Philseok K amp Joanna A 2013 Biofilm attachment reduction on bioinspired dynamic micro-wrinkling surfaces New Journal of Physics vol 15 no 9 095018

Allison BC Applegate BM amp Youngblood JP 2007 Hemocompatibility of hydrophilic antimicrobial copolymers of alkylated 4-vinylpyridine Biomacromolecules vol 8 no 10 2995-2999 Almaguer-Flores A Olivares-Navarrete R Wieland M Ximeacutenez-Fyvie LA Schwartz Z amp Boyan BD 2012 Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro Clinical Oral Implants Research vol 23 no 3 301-307

Amano M Chihara K Kimura K Fukata Y Nakamura N Matsuura Y amp Kaibuchi K 1997 Formation of actin stress fibers and focal adhesions enhanced by rho-kinase Science vol 275 no 5304 1308-1311

Anandan V Rao YL amp Zhang G 2006 Nanopillar array structures for enhancing biosensing performance International Journal of Nanomedicine vol 1 no 1 73-79

Andersson AS Backhed F von Euler A Richter-Dahlfors A Sutherland D amp Kasemo B 2003 Nanoscale features influence epithelial cell morphology and cytokine production Biomaterials vol 24 no 20 3427-36

Anselme K 2011 Biomaterials and interface with bone Osteoporosis International vol 22 no 6 2037-2042 Anselme K Davidson P Popa AM Giazzon M Liley M amp Ploux L 2010 The interaction of cells and bacteria with surfaces structured at the nanometre scale Acta Biomaterialia vol 6 no 10 3824-3846 Arciola CR Bustanji Y Conti M Campoccia D Baldassarri L Samorigrave B amp Montanaro L 2003 Staphylococcus epidermidis-fibronectin binding and its inhibition by heparin Biomaterials vol 24 no 18 3013-3019 Arciola CR Campoccia D Speziale P Montanaro L amp Costerton JW 2012 Biofilm formation in Staphylococcus implant infections A review of molecular mechanisms and implications for biofilm-resistant materials Biomaterials vol 33 no 26 5967-5982

Arciola CR Radin L Alvergna P Cenni E amp Pizzoferrato A 1993 Heparin surface treatment of poly(methylmethacrylate) alters adhesion of a Staphylococcus aureus strain Utility of bacterial fatty acid analysis Biomaterials vol 14 no 15 1161-1164

176

Arecchi FT Bertani D amp Ciliberto S 1979 A fast versatile optical profilometer Optics Communications vol 31 no 3 263-266

Arnoldi M Fritz M Baumluerlein E Radmacher M Sackmann E amp Boulbitch A 2000 Bacterial turgor pressure can be measured by atomic force microscopy Physical Review E - Statistical Physics Plasmas Fluids and Related Interdisciplinary Topics vol 62 no 1 B 1034-1044

Assender H Bliznyuk V amp Porfyrakis K 2002 How surface topography relates to materials properties Science vol 297 no 5583 973-976 Bacakova L Filova E Parizek M Ruml T amp Svorcik V 2011 Modulation of cell adhesion proliferation and differentiation on materials designed for body implants Biotechnology Advances vol 29 no 6 739-767 Bai C amp Liu M 2012 Implantation of nanomaterials and nanostructures on surface and their applications Nano Today vol 7 no 4 258-281

Bar GK amp Meyers GF 2004 The application of atomic force microscopy to the characterization of industrial polymer materials MRS Bulletin vol 29 no 7 464-470

Barbeck M Lorenz J Grosse Holthaus M Raetscho N Kubesch A Booms P Sader R Kirkpatrick CJ amp Ghanaati S 2014a Porcine dermis and pericardium-based non cross-linked materials induce multinucleated giant cells after their in vivo implantation A physiological reaction The Journal of Oral Implantology 20141112

Barbeck M Lorenz J Kubesch A Booms P Boehm N Choukroun J Sader R Kirkpatrick CJ amp Ghanaati S 2014b Porcine dermis-derived collagen membranes induce implantation bed vascularization via multinucleated giant cells a physiological reaction The Journal of Oral Implantology 20141230 Barbeck M Udeabor S Lorenz J Schlee M Grosse Holthaus M Raetscho N Choukroun J Sader R Kirkpatrick CJ amp Ghanaati S 2014c High-temperature sintering of xenogeneic bone substitutes leads to increased multinucleated giant cell formation In vivo and preliminary clinical results The Journal of Oral Implantology 20140812 Barber SC Mead RJ amp Shaw PJ 2006 Oxidative stress in ALS A mechanism of neurodegeneration and a therapeutic target Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease vol 1762 no 11ndash12 1051-1067

177

Barhate RS amp Ramakrishna S 2007 Nanofibrous filtering media Filtration problems and solutions from tiny materials Journal of Membrane Science vol 296 no 1ndash2 1-8

Baumann D Hofmann D Nullmeier S Panther P Dietze C Musyanovych A Ritz S Landfester K amp Mailaumlnder V 2013 Complex encounters Nanoparticles in whole blood and their uptake into different types of white blood cells Nanomedicine vol 8 no 5 699-713

Bayston R Vera L Mills A Ashraf W Stevenson O amp Howdle SM 2010 In vitro antimicrobial activity of silver-processed catheters for neurosurgery Journal of Antimicrobial Chemotherapy vol 65 no 2 258-265

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He J Zhu X Qi Z Wang C Mao X Zhu C He Z Li M amp Tang Z 2015 Killing dental pathogens using antibacterial graphene oxide ACS Applied Materials amp Interfaces vol 7 no 9 5605-5611

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Howarth M Takao K Hayashi Y amp Ting AY 2005 Targeting quantum dots to surface proteins in living cells with biotin ligase Proceedings of the National Academy of Sciences of The United States of America vol 102 no 21 7583-7588

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Whitehead KA Colligon J amp Verran J 2005 Retention of microbial cells in substratum surface features of micrometer and sub-micrometer dimensions Colloids and Surfaces B Biointerfaces vol 41 no 2-3 129-138

Whitehead KA Rogers D Colligon J Wright C amp Verran J 2006 Use of the atomic force microscope to determine the effect of substratum surface topography on the ease of bacterial removal Colloids and Surfaces B Biointerfaces vol 51 no 1 44-53 Whitehouse JD Deborah Friedman N Kirkland KB Richardson WJ amp Sexton DJ 2002 The impact of surgical-site infections following orthopedic surgery at a community hospital and a university hospital Adverse quality of life excess length of stay and extra cost Infection Control and Hospital Epidemiology vol 23 no 4 183-189 Williams DF 2008 On the mechanisms of biocompatibility Biomaterials vol 29 no 20 2941-2953

Williams JF amp Worley SD 2000 Infection-resistant nonleachable materials for urologic devices Journal of Endourology vol 14 no 5 395-400 Witkin DB amp Lavernia EJ 2006 Synthesis and mechanical behavior of nanostructured materials via cryomilling Progress in Materials Science vol 51 no 1 1-60 Wozniak MA Modzelewska K Kwong L amp Keely PJ 2004 Focal adhesion regulation of cell behavior Biochimica et Biophysica Acta (BBA) - Molecular Cell Research vol 1692 no 2ndash3 103-119

223

Wu B Kumar A amp Pamarthy S 2010 High aspect ratio silicon etch A review Journal of Applied Physics vol 108 no 5 051101

Wu M-C Deokar AR Liao J-H Shih P-Y amp Ling Y-C 2013 Graphene-based photothermal agent for rapid and effective killing of bacteria ACS Nano vol 7 no 2 1281-1290

Wu S Zuber F Brugger J Maniura-Weber K amp Ren Q 2016a Antibacterial Au nanostructured surfaces Nanoscale vol 8 no 5 2620-2625 Wu Y Zitelli JP TenHuisen KS Yu X amp Libera MR 2011 Differential response of Staphylococci and osteoblasts to varying titanium surface roughness Biomaterials vol 32 no 4 951-960

Wu Z Fu Q Yu S Sheng L Xu M Yao C Xiao W Li X amp Tang Y 2016b PtAuNPs integrated quantitative capillary-based biosensors for point-of-care testing application Biosensors and Bioelectronics vol 85 657-663 Xia Q Yin JJ Cherng SH Wamer WG Boudreau M Howard PC amp Fu PP 2006 UVA photoirradiation of retinyl palmitate - Formation of singlet oxygen and superoxide and their role in induction of lipid peroxidation Toxicology Letters vol 163 no 1 30-43

Xia T Kovochich M Liong M Maumldler L Gilbert B Shi H Yeh JI Zink JI amp Nel AE 2008 Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties ACS Nano vol 2 no 10 2121-2134 Xiao B Pradhan SK Santiago KC Rutherford GN amp Pradhan AK 2016 Topographically engineered large scale nanostructures for plasmonic biosensing Scientific Reports vol 6 Xie C Hanson L Cui Y amp Cui B 2011 Vertical nanopillars for highly localized fluorescence imaging Proceedings of the National Academy of Sciences vol 108 no 10 3894-3899

Xie C Hanson L Xie W Lin Z Cui B amp Cui Y 2010 Noninvasive neuron pinning with nanopillar arrays Nano Letters vol 10 no 10 4020-4024 Xie C Lin Z Hanson L Cui Y amp Cui B 2012 Intracellular recording of action potentials by nanopillar electroporation Nature Nanotechnology vol 7 no 3 185-190 Xu M Liang T Shi M amp Chen H 2013 Graphene-like two-dimensional materials Chemical Reviews vol 113 no 5 3766-3798

224

Yamashita K Yoshioka Y Higashisaka K Morishita Y Yoshida T Fujimura M Kayamuro H Nabeshi H Yamashita T Nagano K Abe Y Kamada H Kawai Y Mayumi T Yoshikawa T Itoh N Tsunoda S-i amp Tsutsumi Y 2010 Carbon nanotubes elicit DNA damage and inflammatory response relative to their size and shape Inflammation vol 33 no 4 276-280 Yi X amp Gao H 2015 Cell interaction with graphene microsheets near-orthogonal cutting versus parallel attachment Nanoscale vol 7 no 12 5457-5467

Yildirimer L Thanh NTK Loizidou M amp Seifalian AM 2011 Toxicological considerations of clinically applicable nanoparticles Nano Today vol 6 no 6 585-607

Yin JJ Lao F Fu PP Wamer WG Zhao Y Wang PC Qiu Y Sun B Xing G Dong J Liang XJ amp Chen C 2009 The scavenging of reactive oxygen species and the potential for cell protection by functionalized fullerene materials Biomaterials vol 30 no 4 611-621 Yin JJ Liu J Ehrenshaft M Roberts JE Fu PP Mason RP amp Zhao B 2012 Phototoxicity of nano titanium dioxides in HaCaT keratinocytes-Generation of reactive oxygen species and cell damage Toxicology and Applied Pharmacology vol 263 no 1 81-88

Yu L Zhang Y Zhang B amp Liu J 2014a Enhanced antibacterial activity of silver nanoparticleshalloysite nanotubesgraphene nanocomposites with sandwich-like structure Scientific Reports vol 4 4551

Yu Q Liu H amp Chen H 2014b Vertical SiNWAs for biomedical and biotechnology applications Journal of Materials Chemistry B vol 2 no 45 7849-7860

Yue C Kuijer R Kaper HJ van der Mei HC amp Busscher HJ 2014 Simultaneous interaction of bacteria and tissue cells with photocatalytically activated anodized titanium surfaces Biomaterials vol 35 no 9 2580-2587

Zaveri TD Dolgova NV Chu BH Lee J Wong J Lele TP Ren F amp Keselowsky BG 2010 Contributions of surface topography and cytotoxicity to the macrophage response to zinc oxide nanorods Biomaterials vol 31 no 11 2999-3007 Zhang L amp Webster TJ 2009 Nanotechnology and nanomaterials Promises for improved tissue regeneration Nano Today vol 4 no 1 66-80

Zhang L Zheng W Tang R Wang N Zhang W amp Jiang X 2016 Gene regulation with carbon-based siRNA conjugates for cancer therapy Biomaterials vol 104 269-278

225

Zhang W Lee S McNear KL Chung TF Lee S Lee K Crist SA Ratliff TL Zhong Z Chen YP amp Yang C 2014 Use of graphene as protection film in biological environments Scientific Reports vol 4 4097

Zhang W Li Y Niu J amp Chen Y 2013 Photogeneration of reactive oxygen species on uncoated silver gold nickel and silicon nanoparticles and their antibacterial effects Langmuir vol 29 no 15 4647-4651

Zhang Y Figueiredo RB Alhajeri SN Wang JT Gao N amp Langdon TG 2011 Structure and mechanical properties of commercial purity titanium processed by ECAP at room temperature Materials Science and Engineering A vol 528 no 25-26 7708-7714 Zhao L Chu PK Zhang Y amp Wu Z 2009 Antibacterial coatings on titanium implants Journal of Biomedical Materials Research - Part B Applied Biomaterials vol 91 no 1 470-480 Zhao L Hu L Huo K Zhang Y Wu Z amp Chu PK 2010 Mechanism of cell repellence on quasi-aligned nanowire arrays on Ti alloy Biomaterials vol 31 no 32 8341-9 Zhao L Wang H Huo K Cui L Zhang W Ni H Zhang Y Wu Z amp Chu PK 2011 Antibacterial nano-structured titania coating incorporated with silver nanoparticles Biomaterials vol 32 no 24 5706-5716 Zhao R Torley P amp Halley P 2008 Emerging biodegradable materials starch- and protein-based bio-nanocomposites Journal of Materials Science vol 43 no 9 3058-3071 Zheng CY Nie FL Zheng YF Cheng Y Wei SC amp Valiev RZ 2011 Enhanced in vitro biocompatibility of ultrafine-grained titanium with hierarchical porous surface Applied Surface Science vol 257 no 13 5634-5640 Zhu L Zhao X Li Y Yu X Li C amp Zhang Q 2013 High-quality production of graphene by liquid-phase exfoliation of expanded graphite Materials Chemistry and Physics vol 137 no 3 984-990 Zimmerli W 2006 Prosthetic-joint-associated infections Best Practice and Research Clinical Rheumatology vol 20 no 6 1045-1063

Zimmerli W Waldvogel FA Vaudaux P amp Nydegger UE 1982 Pathogenesis of foreign body infection Description and characteristics of an animal model Journal of Infectious Diseases vol 146 no 4 487-497

226

Zou X Zhang L Wang Z amp Luo Y 2016 Mechanisms of the antimicrobial activities of graphene materials Journal of the American Chemical Society vol 138 no 7 2064-2077

227

Appendix

Below is the bio-safety clearance email from the secretary of Swinburne Biosafety

Committee (SBC) Sheila Hamilton-Brown obtained on 17th March 2014 allowing

this research project to be conducted under the regulation of Swinburne Ethics

To Professor Elena Ivanova FSET

Ms Thi Hong Vy Pham

Dear Elena and Vy

Biosafety Project 2014SBC01 ndash Competitive colonisation of biomaterial surfaces by bacterial and eukaryotic cells

Professor Elena Ivanova Thi Hong Vy Pham FSET

Date Approved 17032014 to 17032017

I refer to the review of the above project application for biosafety clearance

undertaken by the Swinburne Biosafety Committee (SBC)

I am pleased to advise that as submitted to date the project has approval to

proceed with standard on-going conditions here outlined

- All teaching and research activity undertaken under Swinburne auspices

must conform to Swinburne and external regulatory standards including the

current National Framework of Ethical Principles in Gene Technology 2012

and with respect to secure data use retention and disposal

- The named Swinburne Chief InvestigatorSupervisor remains responsible

for any personnel appointed to or associated with the project being made

aware of clearance conditions Any change in chief investigatorsupervisor

requires timely notification and SBC endorsement

228

- The above project has been approved as submitted for ethical review by or

on behalf of SBC Amendments to approved procedures ordinarily require

prior appraisal clearance Separate to any Swinburne OHS reporting the

SBC must be notified immediately or as soon as possible thereafter of (a)

any serious or unexpected adverse events and any redress measures (b)

proposed changes in protocols

- A duly authorised external or internal audit of the project may be

undertaken at any time

- Please also note that an annual progress report is required before the end

of each fiscal year (30 June 2014) Approval for continuation per annum is

subject to annual progress reporting

Copies of clearance emails should be retained as part of project record-

keeping Please contact the Research Ethics Office if you have any queries

about the SBC process citing the Biosafety Project number

Best wishes for the project

Yours sincerely

Sheila

Secretary SBC

229

Since the bio-safety clearance had been granted all conditions pertaining to

the clearance were properly met and the annual reports were submitted as required

The first pages of the annual and final reports which were submitted during the

period from 2014 to 2016 were shown as below

230

231

232

233

The exemption from Swinburne Animal Ethics was also granted for the

project as stated in the email below The exemption was based on the animal ethics

approval number MARP2011076 granted from Monash University The evidence

of the exemption and the animal ethics approval are shown as below

From Ann Gaeth

Sent Monday 17 March 2014 454 PM

To Pauline Doran

Cc Wendy Zeng Vy Pham RES Ethics Elena Ivanova

Subject Exempt from SAEC review - MARP2011076

Dear Pauline T ank you for t e notification of Vy P amrsquos involvement in t e animal work being conducted at Monash The Chair of the Swinburne Animal Ethics Committee has been consulted and the Committee will be informed at the next meeting As no live animal material is being brought to Swinburne no further documentation is required It is understood that all live animal work is being conducted at Monash under the MARP approved protocol 2011076 The approval for MARP2011076 expires on the 31 December 2014 To continue beyond this date please email our office the new Monash MARP approval document Please note if your research involves the use of genetically modified organisms andor biohazardous materialsagents you will need approval from the Swinburne Biosafety Committee prior to commencing any work at Swinburne University Please do not hesitate to contact me if you have any queries Regards

Ann

_____________________________________

Dr Ann Gaeth

Secretary SAEC

Swinburne Research (H68) Swinburne University of Technology P O Box 218 HAWTHORN VIC 3122

Ph +61 3 9214 8356

234

235

236

237

THE END

ii

Abstract

Recent developments in nanotechnology have opened a new era for

nanostructured materials due to their unique physical chemical and biological

properties The surface of certain nanostructured materials can be manipulated to

impose certain metabolic activities onto cells coming in contact with these

substrates Implantable materials with a particular surface micro- andor

nanostructure often promote human cell attachment and tissue integration however

these structures can also stimulate the attachment of pathogenic bacteria which may

come in contact with the substrate prior to or during surgical processes If

biomaterial surfaces become infected with pathogenic bacteria it is likely that the

implantation of such surface will result in an infection requiring the removal of the

device and treatment of the infection With the increase in the use of medical

implants an in-depth investigation into the events taking place at the interface when

nanostructured materials come into contact with biological systems is of

considerable importance

This project investigated the surface properties of different nanostructured

surfaces derived from titanium graphene and black silicon and their effects to

different types of cells The nano-smooth titanium surfaces were fabricated by using

an equal channel angular pressing technique Two bacterial strains namely

Staphylococcus aureus and Pseudomonas aeruginosa exhibited different attachment

affinities towards these substrates It was found that Gram-positive S aureus

attachment was not restricted on surfaces that possessed an average roughness less

than 05 nm In contrast P aeruginosa cells were found to be unable to colonise

surfaces possessing an average roughness below 1 nm unless sharp nanoprotrusions

of approximately 20 nm in diameter were present It is postulated that the attachment

of P aeruginosa cells onto surfaces possessing these nanoprotrusions was facilitated

by the ability of the flexible cell membrane to stretch over the tips of the

nanoprotrusions

Two types of graphene films containing variable edge lengths and different

angles of orientation between the graphene sheets were fabricated It was found that

these graphene surfaces exhibited substantial bactericidal activity towards S aureus

and P aeruginosa bacteria The density of the edges was found to be one of the most

iii





death




















iv

Acknowledgement




























Australia

v








around













manuals









vi

Declaration









Signature

________________________________________________________________

vii



Book chapters


























viii















Other publications














ix




4 no 1 p 3




vol 896 pp 259-262








18 no 1

x


Acknowledgement iv

Declaration vi





1 Chapter 1

Introduction 1

11 Overview 2


6 Chapter 2

Literature review 6

21 Overview 7













xi









the surfacerdquo 52



56 Chapter 3


31 Overview 57














xii













354 BCA assay 71

355 MTT assay 71



74 Chapter 4


74

41 Overview 75




45 Conclusion 91

92 Chapter 5


51 Overview 93

xiii




55 Conclusion 108

110 Chapter 6


61 Overview 111



bSi surface 119



66 Conclusion 126

128 Chapter 7


71 Overview 129



74 Conclusion 146

147 Chapter 8



81 Overview 148



84 Conclusion 156

157 Chapter 9


xiv

91 Overview 158







168 Chapter 10





Bibliography 173

Appendix 227

xv




AR As-received




bSi Black silicon
















GT Graphite






xvi






RBC Red blood cell








Si Silicon






xvii

List of Figures
































xviii

































xix

































xx































xxi
































xxii

































xxiii
































xxiv

































xxv
































xxvi




























xxvii

List of Tables









1

Chapter 1

Introduction

2

11 Overview

































3

































4


































5








6

Chapter 2

Literature review

7

21 Overview









al 2013)



















Publications

8

































9


































10









Types of

nanostructures

Base

materials

Research and


References

Nanoparticles





2007 Boisselier amp


2016 Wu et al 2016b

Zhang et al 2016)


2003 Mei et al 2005


2005 Wang et al 2008)


care products


(Tsuang et al 2008

Simchi et al 2011

Zhao et al 2011)


cotton fabrics


food additives

(Fan amp Lu 2005



2012)



fabrics


2004 El-Rafie et al

2012)

11

Types of

nanostructures

Base

materials

Research and


References


GaAs




sensors

(Lodahl et al 2004


al 2005)



guns and cathodes



(Huang et al 2003

Minoux et al 2005


2007 Liu et al 2007

Ezzati Nazhad


Yu et al 2014b)


bone implant

(Ercan et al 2011

Minagar et al 2013




air filters

(Huang et al 2003


2007)


et al 2008a)


composites



engineering bone

disease treatments

(Wang et al 2007


Nanoplatelets

nanoflakes

Graphite and

graphene

composites



production

(Potts et al 2011


Graphene

oxide and its

composite



thin films


Prinz et al 2008

Akhavan amp Ghaderi

12

Types of

nanostructures

Base

materials

Research and


References


Luan et al 2015)

Nanoclay Polymer

composites

Improved plastic



resistance


et al 2008)

Nanopillars

Nanowires



solar cells

(Hu amp Chen 2007






platforms for cell

guiding signalling


and biomolecule

delivering tools

(Stevens amp George

2005 Pimenta et al

2007 Qi et al 2007


Yeon amp Eun Gyeong

2013 Pan et al 2014

Prinz 2015)



et al 2014)


optoelectronic and

sensing devices


Lo et al 2011)

Gallium

phosphide



neurite growth and

neurotransmission


13



































14























15














16

































17


































18
































19

















20


































21























Raja et al 2010)










22
































23




























24























25















2232 Graphene film


















26















graphene thin film




27



































28





























2233 Black silicon




29

























30






















- - - - - - - -

- -

31

































32



















33
















34

































2005)

35




Society)
















infections

36


































37
































(Fig 28)

38




















39














pathogenic bacteria



















40


































41





















42























43





























et al 2015)





44
























2011 Tay et al 2011)










45

































46































2013)




47



































48
































al 2010)

49








2014)


























50




investigation





























51





























52




























53










of Chemistry






Neoh et al 2012)








54


































55






56

Chapter 3


57

31 Overview

























equiaxed grains






58










322 Graphene films
























59




graphene sheets




























60















Tomita et al 2012)














software)

61
































62
































63



software















et al 2005)

64



















height image



65













2012)




















66





2011 Webb et al 2013)























Appendix)




67




















cell type













68
































69






experiment



























70




(Invitrogen)











imaging under CLSM



















71











Truong et al 2010)

354 BCA assay






355 MTT assay













72






























microphotographs



73


















74

Chapter 4




75

41 Overview




























publications)




76














Grade 2 Grade 4





γLW 403 413 405 415 γAB 12 10 10 04 γ+ 004 004 006 006 γ 99 65 39 66 γTOT 416 424 415 419



components




77




















78


















79

























80



81




Grade 2 Grade 4


























Webb et al 2012)

82




















83






















0

10

20

30

40

50

0 20 40 60 80

Po

pu

lati

on

Diameter (nm)

ECAP grade 2

ECAP grade 4

(a) (b)

(c) (d)

(e)

Ti EG2Ti EG4

84

















substrate






85





86





87

















88



















al 2013)

89




90
































91

45 Conclusion














92

Chapter 5


graphene nanosheets

93

51 Overview






























94























95


thickness of 4 nm













2012 Tang et al 2012)

96



respectively)














97












Roughness parameter

GT GN-R GN-S




0 77 108



(a)

98








given in Fig 55











99





100






















101













57)

102



103















morphologies

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


000

20000

40000

60000

80000

100000

000 100 200 300 400 500

GN-R GN-S

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


GN-R GN-S - -

- -

104




























58 B amp D)




105


































106








107







108


























55 Conclusion







109



death

110

Chapter 6


black silicon

111

61 Overview






























112




















control surfaces

113

114



115






116

































117







CMFDA (green)







118













119





the bSi surface















120























121







122































surfaces

123























124




(data not shown)















125















001)




126

























66 Conclusion








127





128

Chapter 7



129

71 Overview






the erythrocytes


























130





conditions













131



132





133






















134











135
















136

















137



















138
































139




140








141






















142































143





144






(Fig 710)










about 400 pN






145

















146







74 Conclusion


















147

Chapter 8




148

81 Overview






























149





















2013)

150





151


































152



















153




154









155















156

84 Conclusion









devices

157

Chapter 9

General discussion

158

91 Overview














warranted



















159

















discussed
















160






























161






















162























death (Fig 92)

163



















(a)

(b)

164

































165

































166




















167

























168

Chapter 10


169

































170


















evident















171
















103 Final remarks

















172







173

Bibliography

174








175








176







177










178








179









180








181










182







183







184








185










186




187











188







189






190







191







192










193




194








195








196





197











198







199





200








201






202







203










204








205





206






207








208









209







210







211










212






213








214







215





216







217






218









219






220








221








222






223







224









225





226


227

Appendix





Ms Thi Hong Vy Pham

Dear Elena and Vy
















228
















Yours sincerely

Sheila

Secretary SBC

229





230

231

232

233





From Ann Gaeth


To Pauline Doran




Ann

_____________________________________

Dr Ann Gaeth

Secretary SAEC


Ph +61 3 9214 8356

234

235

236

237

THE END

iii





death




















iv

Acknowledgement




























Australia

v








around













manuals









vi

Declaration









Signature

________________________________________________________________

vii



Book chapters


























viii















Other publications














ix




4 no 1 p 3




vol 896 pp 259-262








18 no 1

x


Acknowledgement iv

Declaration vi





1 Chapter 1

Introduction 1

11 Overview 2


6 Chapter 2

Literature review 6

21 Overview 7













xi









the surfacerdquo 52



56 Chapter 3


31 Overview 57














xii













354 BCA assay 71

355 MTT assay 71



74 Chapter 4


74

41 Overview 75




45 Conclusion 91

92 Chapter 5


51 Overview 93

xiii




55 Conclusion 108

110 Chapter 6


61 Overview 111



bSi surface 119



66 Conclusion 126

128 Chapter 7


71 Overview 129



74 Conclusion 146

147 Chapter 8



81 Overview 148



84 Conclusion 156

157 Chapter 9


xiv

91 Overview 158







168 Chapter 10





Bibliography 173

Appendix 227

xv




AR As-received




bSi Black silicon
















GT Graphite






xvi






RBC Red blood cell








Si Silicon






xvii

List of Figures
































xviii

































xix

































xx































xxi
































xxii

































xxiii
































xxiv

































xxv
































xxvi




























xxvii

List of Tables









1

Chapter 1

Introduction

2

11 Overview

































3

































4


































5








6

Chapter 2

Literature review

7

21 Overview









al 2013)



















Publications

8

































9


































10









Types of

nanostructures

Base

materials

Research and


References

Nanoparticles





2007 Boisselier amp


2016 Wu et al 2016b

Zhang et al 2016)


2003 Mei et al 2005


2005 Wang et al 2008)


care products


(Tsuang et al 2008

Simchi et al 2011

Zhao et al 2011)


cotton fabrics


food additives

(Fan amp Lu 2005



2012)



fabrics


2004 El-Rafie et al

2012)

11

Types of

nanostructures

Base

materials

Research and


References


GaAs




sensors

(Lodahl et al 2004


al 2005)



guns and cathodes



(Huang et al 2003

Minoux et al 2005


2007 Liu et al 2007

Ezzati Nazhad


Yu et al 2014b)


bone implant

(Ercan et al 2011

Minagar et al 2013




air filters

(Huang et al 2003


2007)


et al 2008a)


composites



engineering bone

disease treatments

(Wang et al 2007


Nanoplatelets

nanoflakes

Graphite and

graphene

composites



production

(Potts et al 2011


Graphene

oxide and its

composite



thin films


Prinz et al 2008

Akhavan amp Ghaderi

12

Types of

nanostructures

Base

materials

Research and


References


Luan et al 2015)

Nanoclay Polymer

composites

Improved plastic



resistance


et al 2008)

Nanopillars

Nanowires



solar cells

(Hu amp Chen 2007






platforms for cell

guiding signalling


and biomolecule

delivering tools

(Stevens amp George

2005 Pimenta et al

2007 Qi et al 2007


Yeon amp Eun Gyeong

2013 Pan et al 2014

Prinz 2015)



et al 2014)


optoelectronic and

sensing devices


Lo et al 2011)

Gallium

phosphide



neurite growth and

neurotransmission


13



































14























15














16

































17


































18
































19

















20


































21























Raja et al 2010)










22
































23




























24























25















2232 Graphene film


















26















graphene thin film




27



































28





























2233 Black silicon




29

























30






















- - - - - - - -

- -

31

































32



















33
















34

































2005)

35




Society)
















infections

36


































37
































(Fig 28)

38




















39














pathogenic bacteria



















40


































41





















42























43





























et al 2015)





44
























2011 Tay et al 2011)










45

































46































2013)




47



































48
































al 2010)

49








2014)


























50




investigation





























51





























52




























53










of Chemistry






Neoh et al 2012)








54


































55






56

Chapter 3


57

31 Overview

























equiaxed grains






58










322 Graphene films
























59




graphene sheets




























60















Tomita et al 2012)














software)

61
































62
































63



software















et al 2005)

64



















height image



65













2012)




















66





2011 Webb et al 2013)























Appendix)




67




















cell type













68
































69






experiment



























70




(Invitrogen)











imaging under CLSM



















71











Truong et al 2010)

354 BCA assay






355 MTT assay













72






























microphotographs



73


















74

Chapter 4




75

41 Overview




























publications)




76














Grade 2 Grade 4





γLW 403 413 405 415 γAB 12 10 10 04 γ+ 004 004 006 006 γ 99 65 39 66 γTOT 416 424 415 419



components




77




















78


















79

























80



81




Grade 2 Grade 4


























Webb et al 2012)

82




















83






















0

10

20

30

40

50

0 20 40 60 80

Po

pu

lati

on

Diameter (nm)

ECAP grade 2

ECAP grade 4

(a) (b)

(c) (d)

(e)

Ti EG2Ti EG4

84

















substrate






85





86





87

















88



















al 2013)

89




90
































91

45 Conclusion














92

Chapter 5


graphene nanosheets

93

51 Overview






























94























95


thickness of 4 nm













2012 Tang et al 2012)

96



respectively)














97












Roughness parameter

GT GN-R GN-S




0 77 108



(a)

98








given in Fig 55











99





100






















101













57)

102



103















morphologies

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


000

20000

40000

60000

80000

100000

000 100 200 300 400 500

GN-R GN-S

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


GN-R GN-S - -

- -

104




























58 B amp D)




105


































106








107







108


























55 Conclusion







109



death

110

Chapter 6


black silicon

111

61 Overview






























112




















control surfaces

113

114



115






116

































117







CMFDA (green)







118













119





the bSi surface















120























121







122































surfaces

123























124




(data not shown)















125















001)




126

























66 Conclusion








127





128

Chapter 7



129

71 Overview






the erythrocytes


























130





conditions













131



132





133






















134











135
















136

















137



















138
































139




140








141






















142































143





144






(Fig 710)










about 400 pN






145

















146







74 Conclusion


















147

Chapter 8




148

81 Overview






























149





















2013)

150





151


































152



















153




154









155















156

84 Conclusion









devices

157

Chapter 9

General discussion

158

91 Overview














warranted



















159

















discussed
















160






























161






















162























death (Fig 92)

163



















(a)

(b)

164

































165

































166




















167

























168

Chapter 10


169

































170


















evident















171
















103 Final remarks

















172







173

Bibliography

174








175








176







177










178








179









180








181










182







183







184








185










186




187











188







189






190







191







192










193




194








195








196





197











198







199





200








201






202







203










204








205





206






207








208









209







210







211










212






213








214







215





216







217






218









219






220








221








222






223







224









225





226


227

Appendix





Ms Thi Hong Vy Pham

Dear Elena and Vy
















228
















Yours sincerely

Sheila

Secretary SBC

229





230

231

232

233





From Ann Gaeth


To Pauline Doran




Ann

_____________________________________

Dr Ann Gaeth

Secretary SAEC


Ph +61 3 9214 8356

234

235

236

237

THE END

iv

Acknowledgement




























Australia

v








around













manuals









vi

Declaration









Signature

________________________________________________________________

vii



Book chapters


























viii















Other publications














ix




4 no 1 p 3




vol 896 pp 259-262








18 no 1

x


Acknowledgement iv

Declaration vi





1 Chapter 1

Introduction 1

11 Overview 2


6 Chapter 2

Literature review 6

21 Overview 7













xi









the surfacerdquo 52



56 Chapter 3


31 Overview 57














xii













354 BCA assay 71

355 MTT assay 71



74 Chapter 4


74

41 Overview 75




45 Conclusion 91

92 Chapter 5


51 Overview 93

xiii




55 Conclusion 108

110 Chapter 6


61 Overview 111



bSi surface 119



66 Conclusion 126

128 Chapter 7


71 Overview 129



74 Conclusion 146

147 Chapter 8



81 Overview 148



84 Conclusion 156

157 Chapter 9


xiv

91 Overview 158







168 Chapter 10





Bibliography 173

Appendix 227

xv




AR As-received




bSi Black silicon
















GT Graphite






xvi






RBC Red blood cell








Si Silicon






xvii

List of Figures
































xviii

































xix

































xx































xxi
































xxii

































xxiii
































xxiv

































xxv
































xxvi




























xxvii

List of Tables









1

Chapter 1

Introduction

2

11 Overview

































3

































4


































5








6

Chapter 2

Literature review

7

21 Overview









al 2013)



















Publications

8

































9


































10









Types of

nanostructures

Base

materials

Research and


References

Nanoparticles





2007 Boisselier amp


2016 Wu et al 2016b

Zhang et al 2016)


2003 Mei et al 2005


2005 Wang et al 2008)


care products


(Tsuang et al 2008

Simchi et al 2011

Zhao et al 2011)


cotton fabrics


food additives

(Fan amp Lu 2005



2012)



fabrics


2004 El-Rafie et al

2012)

11

Types of

nanostructures

Base

materials

Research and


References


GaAs




sensors

(Lodahl et al 2004


al 2005)



guns and cathodes



(Huang et al 2003

Minoux et al 2005


2007 Liu et al 2007

Ezzati Nazhad


Yu et al 2014b)


bone implant

(Ercan et al 2011

Minagar et al 2013




air filters

(Huang et al 2003


2007)


et al 2008a)


composites



engineering bone

disease treatments

(Wang et al 2007


Nanoplatelets

nanoflakes

Graphite and

graphene

composites



production

(Potts et al 2011


Graphene

oxide and its

composite



thin films


Prinz et al 2008

Akhavan amp Ghaderi

12

Types of

nanostructures

Base

materials

Research and


References


Luan et al 2015)

Nanoclay Polymer

composites

Improved plastic



resistance


et al 2008)

Nanopillars

Nanowires



solar cells

(Hu amp Chen 2007






platforms for cell

guiding signalling


and biomolecule

delivering tools

(Stevens amp George

2005 Pimenta et al

2007 Qi et al 2007


Yeon amp Eun Gyeong

2013 Pan et al 2014

Prinz 2015)



et al 2014)


optoelectronic and

sensing devices


Lo et al 2011)

Gallium

phosphide



neurite growth and

neurotransmission


13



































14























15














16

































17


































18
































19

















20


































21























Raja et al 2010)










22
































23




























24























25















2232 Graphene film


















26















graphene thin film




27



































28





























2233 Black silicon




29

























30






















- - - - - - - -

- -

31

































32



















33
















34

































2005)

35




Society)
















infections

36


































37
































(Fig 28)

38




















39














pathogenic bacteria



















40


































41





















42























43





























et al 2015)





44
























2011 Tay et al 2011)










45

































46































2013)




47



































48
































al 2010)

49








2014)


























50




investigation





























51





























52




























53










of Chemistry






Neoh et al 2012)








54


































55






56

Chapter 3


57

31 Overview

























equiaxed grains






58










322 Graphene films
























59




graphene sheets




























60















Tomita et al 2012)














software)

61
































62
































63



software















et al 2005)

64



















height image



65













2012)




















66





2011 Webb et al 2013)























Appendix)




67




















cell type













68
































69






experiment



























70




(Invitrogen)











imaging under CLSM



















71











Truong et al 2010)

354 BCA assay






355 MTT assay













72






























microphotographs



73


















74

Chapter 4




75

41 Overview




























publications)




76














Grade 2 Grade 4





γLW 403 413 405 415 γAB 12 10 10 04 γ+ 004 004 006 006 γ 99 65 39 66 γTOT 416 424 415 419



components




77




















78


















79

























80



81




Grade 2 Grade 4


























Webb et al 2012)

82




















83






















0

10

20

30

40

50

0 20 40 60 80

Po

pu

lati

on

Diameter (nm)

ECAP grade 2

ECAP grade 4

(a) (b)

(c) (d)

(e)

Ti EG2Ti EG4

84

















substrate






85





86





87

















88



















al 2013)

89




90
































91

45 Conclusion














92

Chapter 5


graphene nanosheets

93

51 Overview






























94























95


thickness of 4 nm













2012 Tang et al 2012)

96



respectively)














97












Roughness parameter

GT GN-R GN-S




0 77 108



(a)

98








given in Fig 55











99





100






















101













57)

102



103















morphologies

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


000

20000

40000

60000

80000

100000

000 100 200 300 400 500

GN-R GN-S

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


GN-R GN-S - -

- -

104




























58 B amp D)




105


































106








107







108


























55 Conclusion







109



death

110

Chapter 6


black silicon

111

61 Overview






























112




















control surfaces

113

114



115






116

































117







CMFDA (green)







118













119





the bSi surface















120























121







122































surfaces

123























124




(data not shown)















125















001)




126

























66 Conclusion








127





128

Chapter 7



129

71 Overview






the erythrocytes


























130





conditions













131



132





133






















134











135
















136

















137



















138
































139




140








141






















142































143





144






(Fig 710)










about 400 pN






145

















146







74 Conclusion


















147

Chapter 8




148

81 Overview






























149





















2013)

150





151


































152



















153




154









155















156

84 Conclusion









devices

157

Chapter 9

General discussion

158

91 Overview














warranted



















159

















discussed
















160






























161






















162























death (Fig 92)

163



















(a)

(b)

164

































165

































166




















167

























168

Chapter 10


169

































170


















evident















171
















103 Final remarks

















172







173

Bibliography

174








175








176







177










178








179









180








181










182







183







184








185










186




187











188







189






190







191







192










193




194








195








196





197











198







199





200








201






202







203










204








205





206






207








208









209







210







211










212






213








214







215





216







217






218









219






220








221








222






223







224









225





226


227

Appendix





Ms Thi Hong Vy Pham

Dear Elena and Vy
















228
















Yours sincerely

Sheila

Secretary SBC

229





230

231

232

233





From Ann Gaeth


To Pauline Doran




Ann

_____________________________________

Dr Ann Gaeth

Secretary SAEC


Ph +61 3 9214 8356

234

235

236

237

THE END

v








around













manuals









vi

Declaration









Signature

________________________________________________________________

vii



Book chapters


























viii















Other publications














ix




4 no 1 p 3




vol 896 pp 259-262








18 no 1

x


Acknowledgement iv

Declaration vi





1 Chapter 1

Introduction 1

11 Overview 2


6 Chapter 2

Literature review 6

21 Overview 7













xi









the surfacerdquo 52



56 Chapter 3


31 Overview 57














xii













354 BCA assay 71

355 MTT assay 71



74 Chapter 4


74

41 Overview 75




45 Conclusion 91

92 Chapter 5


51 Overview 93

xiii




55 Conclusion 108

110 Chapter 6


61 Overview 111



bSi surface 119



66 Conclusion 126

128 Chapter 7


71 Overview 129



74 Conclusion 146

147 Chapter 8



81 Overview 148



84 Conclusion 156

157 Chapter 9


xiv

91 Overview 158







168 Chapter 10





Bibliography 173

Appendix 227

xv




AR As-received




bSi Black silicon
















GT Graphite






xvi






RBC Red blood cell








Si Silicon






xvii

List of Figures
































xviii

































xix

































xx































xxi
































xxii

































xxiii
































xxiv

































xxv
































xxvi




























xxvii

List of Tables









1

Chapter 1

Introduction

2

11 Overview

































3

































4


































5








6

Chapter 2

Literature review

7

21 Overview









al 2013)



















Publications

8

































9


































10









Types of

nanostructures

Base

materials

Research and


References

Nanoparticles





2007 Boisselier amp


2016 Wu et al 2016b

Zhang et al 2016)


2003 Mei et al 2005


2005 Wang et al 2008)


care products


(Tsuang et al 2008

Simchi et al 2011

Zhao et al 2011)


cotton fabrics


food additives

(Fan amp Lu 2005



2012)



fabrics


2004 El-Rafie et al

2012)

11

Types of

nanostructures

Base

materials

Research and


References


GaAs




sensors

(Lodahl et al 2004


al 2005)



guns and cathodes



(Huang et al 2003

Minoux et al 2005


2007 Liu et al 2007

Ezzati Nazhad


Yu et al 2014b)


bone implant

(Ercan et al 2011

Minagar et al 2013




air filters

(Huang et al 2003


2007)


et al 2008a)


composites



engineering bone

disease treatments

(Wang et al 2007


Nanoplatelets

nanoflakes

Graphite and

graphene

composites



production

(Potts et al 2011


Graphene

oxide and its

composite



thin films


Prinz et al 2008

Akhavan amp Ghaderi

12

Types of

nanostructures

Base

materials

Research and


References


Luan et al 2015)

Nanoclay Polymer

composites

Improved plastic



resistance


et al 2008)

Nanopillars

Nanowires



solar cells

(Hu amp Chen 2007






platforms for cell

guiding signalling


and biomolecule

delivering tools

(Stevens amp George

2005 Pimenta et al

2007 Qi et al 2007


Yeon amp Eun Gyeong

2013 Pan et al 2014

Prinz 2015)



et al 2014)


optoelectronic and

sensing devices


Lo et al 2011)

Gallium

phosphide



neurite growth and

neurotransmission


13



































14























15














16

































17


































18
































19

















20


































21























Raja et al 2010)










22
































23




























24























25















2232 Graphene film


















26















graphene thin film




27



































28





























2233 Black silicon




29

























30






















- - - - - - - -

- -

31

































32



















33
















34

































2005)

35




Society)
















infections

36


































37
































(Fig 28)

38




















39














pathogenic bacteria



















40


































41





















42























43





























et al 2015)





44
























2011 Tay et al 2011)










45

































46































2013)




47



































48
































al 2010)

49








2014)


























50




investigation





























51





























52




























53










of Chemistry






Neoh et al 2012)








54


































55






56

Chapter 3


57

31 Overview

























equiaxed grains






58










322 Graphene films
























59




graphene sheets




























60















Tomita et al 2012)














software)

61
































62
































63



software















et al 2005)

64



















height image



65













2012)




















66





2011 Webb et al 2013)























Appendix)




67




















cell type













68
































69






experiment



























70




(Invitrogen)











imaging under CLSM



















71











Truong et al 2010)

354 BCA assay






355 MTT assay













72






























microphotographs



73


















74

Chapter 4




75

41 Overview




























publications)




76














Grade 2 Grade 4





γLW 403 413 405 415 γAB 12 10 10 04 γ+ 004 004 006 006 γ 99 65 39 66 γTOT 416 424 415 419



components




77




















78


















79

























80



81




Grade 2 Grade 4


























Webb et al 2012)

82




















83






















0

10

20

30

40

50

0 20 40 60 80

Po

pu

lati

on

Diameter (nm)

ECAP grade 2

ECAP grade 4

(a) (b)

(c) (d)

(e)

Ti EG2Ti EG4

84

















substrate






85





86





87

















88



















al 2013)

89




90
































91

45 Conclusion














92

Chapter 5


graphene nanosheets

93

51 Overview






























94























95


thickness of 4 nm













2012 Tang et al 2012)

96



respectively)














97












Roughness parameter

GT GN-R GN-S




0 77 108



(a)

98








given in Fig 55











99





100






















101













57)

102



103















morphologies

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


000

20000

40000

60000

80000

100000

000 100 200 300 400 500

GN-R GN-S

000

20000

40000

60000

80000

100000

000 100 200 300 400 500000

20000

40000

60000

80000

100000

000 100 200 300 400 500

Hei

ght (

nm)

Hei

ght (

nm)


GN-R GN-S - -

- -

104




























58 B amp D)




105


































106








107







108


























55 Conclusion







109



death

110

Chapter 6


black silicon

111

61 Overview






























112




















control surfaces

113

114



115






116

































117







CMFDA (green)







118













119





the bSi surface















120























121







122































surfaces

123























124




(data not shown)















125















001)




126

























66 Conclusion








127





128

Chapter 7



129

71 Overview






the erythrocytes


























130





conditions













131



132





133






















134











135
















136

















137



















138
































139




140








141






















142































143





144






(Fig 710)










about 400 pN






145

















146







74 Conclusion


















147

Chapter 8




148

81 Overview






























149





















2013)

150





151


































152



















153




154









155















156

84 Conclusion









devices

157

Chapter 9

General discussion

158

91 Overview














warranted



















159

















discussed
















160






























161






















162























death (Fig 92)

163



















(a)

(b)

164

































165

































166




















167

























168

Chapter 10


169

































170


















evident















171
















103 Final remarks

















172







173

Bibliography

174








175








176







177










178








179









180








181










182







183







184








185










186




187











188







189






190







191







192










193




194








195








196





197











198







199





200








201






202







203










204








205





206






207








208









209







210







211










212






213








214







215





216







217






218









219






220








221








222






223







224









225





226


227

Appendix





Ms Thi Hong Vy Pham

Dear Elena and Vy
















228
















Yours sincerely

Sheila

Secretary SBC

229





230

231

232

233





From Ann Gaeth


To Pauline Doran




Ann

_____________________________________

Dr Ann Gaeth

Secretary SAEC


Ph +61 3 9214 8356

234

235

236

237

THE END

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Investigation of the biointerfaces of nanostructured surfaces · 2017. 3. 1. · surfaces...

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