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

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.”

Contents

1. Introduction ��� 111.1 Motivation ��� 111.2 Outline of the thesis ��� 12

2. Biomaterial science - an Introduction ��� 152.1 Understanding Cells and their Microenvironment ��� 19

2.1.1 Cell and their microenvironment ��� 192.2 Fundamentals of protein absorption ��� 212.3 Tissue and cellular host response ��� 23

3. Orthopaedic applications & bioactive coatings ��� 273.1 Ti as biomaterial ��� 283.2 Bones’ physiology and the role of Hydroxyapatite ��� 30

4. Surface engineering for biomedical applications ��� 334.1 Surface roughness modification ��� 354.2 Design of 2D nano and microenvironment for cell-

biomaterials studies in vitro ��� 36

5. Experimental techniques and systems ��� 395.1 Cleanroom environment ��� 40

5.1.1 Cleanroom construction ��� 405.1.2 Cleanroom standards ��� 41

5.2 Fabrication Techniques ��� 425.2.1 Electron Beam Physical Vapour Deposition ��� 425.2.2 Spin coating (SC) and Photolithography (PL) ��� 445.3.3 Nano Imprint Lithography (NIL) ��� 465.2.4 Reactive Ion Etching (RIE) ��� 47

5.3 Microscopy and characterization ��� 485.3.1 Scanning Electron Microscopy (SEM) ��� 485.3.2 Atomic Force Microscopy (AFM) ��� 505.3.3 Attenuated Total Reflectance Fourier Transform IR ���

Spectroscopy (ATR-FTIR) ��� 52

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 ��� 56A.2 The root-Mean-Square roughness, power law

scaling ��� 57A.3 Thin films growth from physical vapour

deposition (PVD) ��� 58A.4 Glancing angle deposition (GLAD) ��� 60A.5 Materials and Methods ��� 61

A.5.1 Materials ��� 61A.5.2 Surface preparation ��� 61A.5.3 Surface characterisation ��� 63

A.6 Results and discussion ��� 64A.6.1 Morphology investigation of height

aspect-ratio Ti films ��� 64A.6.2 Roughness Power law scaling behavior ��� 71A.6.3 Height correcting factor for roughness

estimation ��� 72A.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 ��� 80B.2 Hydroxyapatite synthesis from aqueous solutions ��� 82B.3 Materials and Methods ��� 83

B.3.1 Materials ��� 83B.3.2 Biocompatible Synthesis of bone-like apatite

nanocrystals ��� 83

B.3.3 Simulated Body Fluid Preparation ��� 85B.3.4 Coating procedures ��� 87

B.4 Results and discussion ��� 89B.4.1 Characterization of bone-like apatite

nanocrystals ��� 89B.4.2 Spin coating of bone-like HAp ��� 94B.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 ��� 106C.2 Gold surfaces as the starting point for chemical pattering ��� 107C.4 Materials and Methods ��� 108

C.4.1 Materials ��� 108C.4.2 Micro-fabrication of of Au/SiO2 circular

arrays ��� 108C.4.3 Nano-fabrication of multiple Au/SiO2

pattern lines 112C.5 Results and discussion ��� 115

C.5.1 Circular gold array fabrication ��� 115C.6 Conclusions ��� 120

7. Conclusions and outlook of the thesis ��� 1215.1 General conclusion ��� 1215.2 Outlook of the thesis ��� 122

Supplementary informations 125

References ��� 129

Acknowledgements ��� 135

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

PART 1

Introduction

1.1 MotivationsSurface 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.

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.

… have a pleasant journey