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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
different if P = 1 (t gt 005) insignificantly different if P = 0 (t lt 005))helliphelliphellip119
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
biomedical applications
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
Barhate amp Ramakrishna
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
biomedical applications
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
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
16
nanoparticles (GNPs) which selectively bind to and control the expression of a
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
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 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
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 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
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
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
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
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)
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
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 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
Figure 27 Total arthroplasty operations performed and total prosthetic infections
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
Figure 28 Schematic representation of the different strategies currently being used
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
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 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
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
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 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 2009 Subbiahdoss et al 2010a Subbiahdoss et al 2010b
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
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 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
Table 43 Contact angle and surface free energy of the as-received and ECAP
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
Figure 42 TEM images of the ultrafine grains of ECAP grade 2 (A amp B) and grade
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
Table 44 AFM surface roughness analysis of the as-received and ECAP modified
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
Sa 027 plusmn 011 021 plusmn 007 019 plusmn 010 016 plusmn 002
Smax 317 plusmn 076 456 plusmn 128 360 plusmn 077 485 plusmn 185
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
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)
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
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 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
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
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
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)
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
Figure 51 The UV-Visible absorption spectra of aqueous graphene suspension
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
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 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
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)
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
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
edges allowing the size distribution of edge lengths of both the rough (GN-R) and
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
Table 55 Topographical analysis of graphite (GT) together with rough (GN-R) and
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
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 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
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
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)
Scanning length (μm)Scanning length (μm)
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
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)
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
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)
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)
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))
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
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
different if P = 1 (t gt 005) insignificantly different if P = 0 (t lt 005))
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
Figure 67 Formation of interconnected microcapillary-like structures (red arrows)
of co-cultures between primary human fibroblasts and endothelial cells growing on
plastic and black silicon surfaces Cells were fixed and stained with endothelial cell-
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)
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
125
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 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)
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)
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
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 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
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
separated quantitative plotting of intact biconcave RBCs versus deformed and
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
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 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
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
136
bSi surface disappearing after rupture when they moved out of camera focus The
real-time movie can be found at
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)
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 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
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
138
Raman activity from 1100 cm-1 to 3500 cm-1 which provides discrimination from
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
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 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
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 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
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)
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
Figure 84 Quantification of the number of COS-7 cells present on the infected bSi
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
92 Proposed mechanisms of bacterial attachment on nanoscopically smooth
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
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 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
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 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
93 The responses of different mammalian cell types to the nanopillar
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)
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
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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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
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