1. Università degli Studi di Padova
Dipartimento di Scienze Chimiche
Corso di Laurea Magistrale in Scienza dei Materiali
Surface Engineering Approaches
for Biomedical Applications
Relatore: Mauro Sambi Laureando: Roberto Rossi
Correlatore: Morten Foss Matricola: 1057256
Anno Accademico 2014/2015

3. Dedico questa tesi a tutti coloro che mi hanno supportato in questo incredibile e
arduo percorso. A mio papà Alessandro, mia mamma Franca, nonna Tarsilla e nonno
Gino. Grazie per aver reso questo possibile.
“La natura utilizza solo i fili più lunghi per tessere i suoi modelli, così ogni piccolo
pezzo del suo tessuto rivela la composizione dell’intero arazzo.”

5. Contents
1. Introduction 11
1.1 Motivation 11
1.2 Outline of the thesis 12
2. Biomaterial science - an Introduction 15
2.1 Understanding Cells and their Microenvironment 19
2.1.1 Cell and their microenvironment 19
2.2 Fundamentals of protein absorption 21
2.3 Tissue and cellular host response 23
3. Orthopaedic applications bioactive coatings 27
3.1 Ti as biomaterial 28
3.2 Bones’ physiology and the role of Hydroxyapatite 30
4. Surface engineering for biomedical applications 33
4.1 Surface roughness modification 35
4.2 Design of 2D nano and microenvironment for cell-
biomaterials studies in vitro 36
5. Experimental techniques and systems 39
5.1 Cleanroom environment 40
5.1.1 Cleanroom construction 40
5.1.2 Cleanroom standards 41
5.2 Fabrication Techniques 42
5.2.1 Electron Beam Physical Vapour Deposition 42
5.2.2 Spin coating (SC) and Photolithography (PL) 44
5.3.3 Nano Imprint Lithography (NIL) 46
5.2.4 Reactive Ion Etching (RIE) 47
5.3 Microscopy and characterization 48
5.3.1 Scanning Electron Microscopy (SEM) 48
5.3.2 Atomic Force Microscopy (AFM) 50
5.3.3 Attenuated Total Reflectance Fourier Transform IR
Spectroscopy (ATR-FTIR) 52

6. 5.3.4 X-ray powder diffraction (XRD) 53
6. Experimental section: 55
Project A: Nano roughness control of Titanium Glancing Angle
Deposited surfaces 55
A.1 General considerations 56
A.2 The root-Mean-Square roughness, power law
scaling 57
A.3 Thin films growth from physical vapour
deposition (PVD) 58
A.4 Glancing angle deposition (GLAD) 60
A.5 Materials and Methods 61
A.5.1 Materials 61
A.5.2 Surface preparation 61
A.5.3 Surface characterisation 63
A.6 Results and discussion 64
A.6.1 Morphology investigation of height
aspect-ratio Ti films 64
A.6.2 Roughness Power law scaling behavior 71
A.6.3 Height correcting factor for roughness
estimation 72
A.6.4 Growth factor of hight aspect ratio
Ti thin films 75
A.7 Conclusions 77
Project B: Biomimetic routes for the synthesis of bone-like apatite 79
B.1 Phosphates and biomineralization 80
B.2 Hydroxyapatite synthesis from aqueous solutions 82
B.3 Materials and Methods 83
B.3.1 Materials 83
B.3.2 Biocompatible Synthesis of bone-like apatite
nanocrystals 83

7. B.3.3 Simulated Body Fluid Preparation 85
B.3.4 Coating procedures 87
B.4 Results and discussion 89
B.4.1 Characterization of bone-like apatite
nanocrystals 89
B.4.2 Spin coating of bone-like HAp 94
B.4.3 Biomimetic coatings from SBF 98
B.5 Conclusions 103
Project C: Preparation of Au/SiO2 micro arrays and nano-lines for
future development of in vitro substrate fabrications 105
C.1 General considerations 106
C.2 Gold surfaces as the starting point for
chemical pattering 107
C.4 Materials and Methods 108
C.4.1 Materials 108
C.4.2 Micro-fabrication of of Au/SiO2 circular
arrays 108
C.4.3 Nano-fabrication of multiple Au/SiO2
pattern lines 112
C.5 Results and discussion 115
C.5.1 Circular gold array fabrication 115
C.6 Conclusions 120
7. Conclusions and outlook of the thesis 121
5.1 General conclusion 121
5.2 Outlook of the thesis 122
Supplementary informations 125
References 129
Acknowledgements 135

9. Abbreviations
PVD: Physical vapour deposition
GLAD: Glancing angle deposition
EB-PVD: Electron beam physical vapour deposition
QCM: Quartz chrystal microbalance
SC: Spin coating
PL: Photolithography
NIL: Nanoimprint lithography
T-NIL: Thermal nanoimprint lithography
RIE: Reactive ion etching
SEM: Scanning electron microscopi
BSE: Back scattering electrons
AFM: Atomic force microscopy
ATR-FTIR: Attenuated total reflectance Fourier transform IR spectroscopy
XRD: X-ray diffraction
TGA: Thermogravimetric analysis
HAp: Hydroxyapatite
SBF: Simulated body fluid
mSBF: Modified simulated body fluid
PBS: Phosphate saline buffer
RMS: Root mean square

11. PART 1
Introduction
1.1 Motivations
Surface engineering refers to a wide range of technologies designed to modify the
surface properties of metallic and non-metallic components for functional purposes.
In this field, micro and nano fabrication tools are used by almost every part of
manufacturing industry to modify surfaces, with the result on having extremely
diverse product and applications. Surface engineering approaches at micro and
nanoscale are motivated by several areas of applied and commercial interest, like
biomaterial science, production and energy storage industry and semiconductor
industry and can be used to modify a wide range of material properties. Some of
these properties are listed below:
• Mechanical properties (e.g., low wear properties, low friction properties)
• Thermal properties (e.g., thermal barrier coatings)
• Chemical properties (e.g., corrosion and oxidation-resistant coatings)
• Functional properties (coatings for electronic, optical and magnetic applications)
In modern society, the demand for novel applications in the area of biomaterials,
bionanotechnology, tissue engineering and medical devices are becoming the core of
health care.

12. In this field, through the development of micro-nanostructured surfaces, the field of
surface engineering is approaching this demand by investigating new materials’
functionalities when an interface with the biological system is involved.
This thesis has been devoted to the investigation of novel techniques of nano and
micro fabrication for development of substrates that can be used for in vitro studies.
In the context of biomaterials science, as will be well explained later, when a
substrate for in vitro applications is developed, properties such as roughness,
topography and chemistry have to be tailored as a function of the final aim in the
application. This can be done by applying surface engineering approaches, and for
this reason, the thesis has been addressed to face the field of biomaterial science from
different points of view.
1.1 Outline of the thesis
In PART 2 an introduction on the field of biomaterials science is given, also focusing
on a briefly description of cell behavior and cell-biomaterial interaction, presenting
some fundamental concepts in protein adsorption, followed by most of the important
reactions involved when cells and biological system interact with a foreign material.
Following, in PART 3, attention is addressed to hard tissue and skeletal applications.
In particular, the role of metal implants and the advantages of using titanium are
presented and related to some basic concepts in bone physiology and on the
importance of calcium phosphates compounds as bioactive coating materials.
PART 4 concludes the background knowledge necessary for a clear understanding of
the work. A broad description of the novel techniques in surface engineering for
roughness modification is given, also explaining the major challenges for the design
of 2D nano and microenvironment for cell-biomaterials studies in vitro.
In PART 5 all experimental techniques used in this work are described. Starting from
the concept of cleanroom environment, where a large chunk of the work has been
processed, the chapter goes through coating techniques (electron beam deposition and
spin coating), etching/cleaning technique (Reactive ion etching) to conclude with the
used imaging and characterization techniques (Scanning electron microscopy, Atomic
Force microscopy, Fourier Transform IR spectroscopy, X-ray diffraction).
PART 6 is dedicated to the experimental section and is divided in three different
project; A, B and C. In these projects, different aspects of surface engineering on
biomaterials is dealt with. Going through novel techniques in roughness modification,
synthesis of bioactive compound focused on the development of coatings, and micro/
nano fabrication aimed on the production of substrate for in vitro in pursued. For each
experimental part a brief introduction is given, followed by materials and methods,
results and discussions, and final conclusions.
A general conclusion of the entire work is given in PART 7, and an outlook on future
developments is given to underline the importance of studies and research in this
field.

13. … have a pleasant journey