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«Dissertation zur Erlangung des Grades des Doktors der Ingenieurwissenschaften der Naturwissenschaftlich‐Technischen Fakultät III Chemie, ...»

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Topographical Control and Characterization of

Al/Al2O3 Nanowire Coatings for Improved

Osseointegration of Implant Materials

Dissertation

zur Erlangung des Grades des Doktors der Ingenieurwissenschaften

der Naturwissenschaftlich‐Technischen Fakultät III

Chemie, Pharmazie, Bio‐ und Werkstoffwissenschaften

der Universität des Saarlandes

von

Marina Martinez Miró

Saarbrücken, Germany

Tag des Kolloquiums: 22.02.2013

Dekan: Prof. Dr. V. Helms

Vorsitz: Prof. Dr. D. Scheschkewitz Berichterstatter: Prof. Dr. Dr. h.c. M. Veith Prof. Dr. A. Ott Akad. Mitarbeiter: Dr. G. Falk Die vorliegende Arbeit wurde in der Zeit von Oktober 2009 bis Oktober 2012 am Leibniz Institut für Neue Materialien gGmbH unter Anleitung von Prof. Dr. Dr. h.c Michael Veith angefertigt.

Als meus pares Abstract Medical implants are a clinical reality and thousands of patients have been treated increasing in this way his life quality. Although many native materials have been used for bone implants, future biomaterials will include specific topographies to obtain the desired function. In this thesis, Al/Al2O3 nanowires (NWs) are suggested as possible coatings for improved osseointegration. The coatings have been fabricated by chemical vapour deposition (CVD) of the molecular precursor [tBuOAlH2]2. Depending on the deposition time four different densely coated nanostructures have been obtained. A 3D-model has been reconstructed using the Focus Ion Beam/Scanning Electron Microscopy (FIB/SEM) technique and software reconstruction tools and the surface properties have been characterised using well known techniques. Finally, the prepared Al/Al2O3 NWs have been biologically tested. The Al/Al2O3 coatings are here studied as models for a better understanding of the topographic effect of the features on the cells independently from the chemical effect. Human osteoblast cells (HOB) and normal dermal human fibroblast cells (NHDF) were cultured separately on the deposited Al/Al2O3 NWs to investigate a possible selective cell adhesion. Additionally, gene experiments have been performed in order to understand the effect of the different topographies on the osteogenic gene expression. Lastly, the cell monolayer rheology (CMR) has been used to quantify the mechanical behaviour of the cells.

i Kurzfassung Medizinische Implantate sind in der klinischen Praxis eingeführt und die Lebensqualität Tausender behandelter Patienten hat sich damit erhöht. In dieser Arbeit werden Al/Al2O3 Nanodrähte (NWs) als mögliche Beschichtungen für eine verbesserte Osseointegration vorgeschlagen. In diesem Zusammenhang wurden Al/Al2O3 Schichten durch chemische Gasphasenabscheidung (CVD) des molekularen Präkursors [tBuOAlH2]2 hergestellt. Je nach Dauer der Abscheidung wurden vier verschiedene Nanostrukturen gewonnen. 3D-Modelle wurden für jede dieser Nanostrukturen mit Hilfe der Fokus Ion Beam/Scanning Electron Microscopy (FIB/SEM) Technik rekonstruiert. Die Oberflächeneigenschaften wurden unter Verwendung etablierter Techniken charakterisiert. Schließlich wurden die vorbereiteten Al/Al2O3 NWs biologisch getestet. Die Al/Al2O3 Beschichtungen wurden hier als Modelle für ein besseres Verständnis des topographischen Effekts der Oberflächenmerkmale von Implantwerkstoffen auf Zellen unabhängig von der chemischen Wirkung des Werkstoffs selbst untersucht. Menschliche Osteoblasten (HOB) und normale dermale menschliche Fibroblasten (NHDF) wurden separat auf den abgeschiedenen Al/Al2O3 NWs kultiviert, um eine mögliche selektive Zell-Adhäsion zu untersuchen. Genexperimente wurden durchgeführt. Genexpression auf Al/Al2O3 NWs wurde quantifiziert. Schließlich hat die Zellmonoschicht Rheologie (CMR) verwendet worden, um das mechanische Verhalten der Zellen zu quantifizieren.

–  –  –

I would like to mention special thanks to all the people from the INM who helped me during these

years:

 Dr. Cenk Aktas, group leader of the CVD/Life Science group, for helping me improve my career.

 Special thanks to all the colleagues from the CVD/Life Science group; Dieter Anschütz, Awadelkareem Ali, Stefan Brück, Cecile Dufloux, Sandra Litzenburger, Fadime Shain, Dr. Karin Löw, Ina Marsollek and Alexander May, for the nice and friendly working atmosphere.

 Sylvia de Graaf for giving always a solution to my logistic problems.

 Dietmar Serwas and all the workshop: Herbert Beermann, Johannes Berrar, Uwe Mager, Klaus Schmitt and Wermer Schneider for their help with all the devices.

 Melanie Groh and Elke Bubel from the library for their help during my literature search.

 Wolfgang Türk, Ralph Stoltz and Ralph Muth from the EDV.

 The former people from the AK Veith, Inorganic Chemistry department of the Universität des Saarlandes: Dr. Tatjana Kirs, Dr. David Kolano and Dr. Hameed Ullah.

I would like to mention special thanks to my dear colleagues and friends Juseok Lee and Cagri Kaan Akkan; walking our “PhD roads” together during these years built a very strong relationship between us.

Thank you for everything, including KFC sessions and mensa discussions.

Also my sincere thanks to the experts who helped me with the material characterization:

 Thanks to Dr. Falvio Soldera and Federico Miguel (Material Science Department, Saarland University) for their work, suggestions and guidance concerning the FIB analysis.





 Thanks to Johannes Maurer and Prof. Dr. Roland Bennewitz (INM, nanotribologie group) for the roughness measurement using AFM technique.

 Special mention to Dr. Vladimir Zaporojtchenko (TF Kiel Universität – AK Prof. Fappel) who performed the XPS analysis. I had the pleasure to spend with him nice conference moments and the recent news about his unexpected death still shock me. Rest in peace.

I would like to express my thanks to all the cell experts who made this work possible:

 Dr. Wolfgang Metzger, Benedikt Schwab, Lisa Schimmelpfennig, Prof. Dr. Tim Pohleman and PD Dr. Martin Oberringer (Clinic of Trauma, Hand and Reconstructive Surgery, Saarland University, Homburg) for their collaborative works with the osteoblast and fibroblast cells, their suggestions, their interest and their continuous help. Thanks to Prof. med. G. Wennemuth for the SEM imaging of the cells.

 Collaborative Research Centre of Homburg from AO foundation and the AO Reasearch Institute Davos in Switzerland for the gene expression experiments.

 Thanks to Mathias Sander and Prof. Dr. Albrecht Ott (Biophysics Department, Universität des Saarlandes) for his collaborative work with the CMR technique. Thank you for introducing the CMR technique to me, for your suggestions and fruitful discussions.

Thanks to my dear friends here in Saarbrücken: Carla Sofia Amado, Salut Camilleri Rimbau, Stephan Rouven, Hassan Soumsoumani and Jana Kathrin for all the memorable moments we spent together.

Last but not least I would like to thank my family: Andrés Martinez Gil, Conxita Miró Perez, Diana Martinez Miró and Ayman Haidar. I have no words enough to thank you for the unconditional support, the encouragement in the difficult moments and for being the real rocks of my life. Gràcies per estar sempre al meu costat i ser la meva inspiració constant.

iii

Table of contents

Abstract……………………………………………………………...………...…………i Kurzfassung…………………..…………………………………………………….……ii Acknowledgments…………………...……………………………………………….…iii Table of contents……………………………..…………………………………………iv Symbols and abbreviations…………….……………………………………………...viii

1 Introduction

2 State of the art

2.1 1D Nanostructures

2.2 1D Alumina structures

2.3 Use of 1D nanostructures in biomaterials

2.4 Gas Phase Synthesis of 1D Nanostructures: Vapour-liquid-solid (VLS) growth………………………………………………………………………………………………………………….5 2.5.1 Advantages and disadvantages of the CVD technique

2.5 Basics of chemical vapour deposition (CVD) technique

2.5.2 Key steps of the CVD process

2.5.3 CVD of Alumina and precursors

3 Synthesis of Al/Al2O3 Nanostructures:

3.1.1 Single source precursor [tBuOAlH2]2

3.1 Background: MOCVD of [tBuOAlH2]2

3.1.2 Production of stoichiometric bi-phasic composites using molecular precursors

3.1.3 One-dimensional (1D) growth of biphasic Al/Al2O3 NWs by self-catalysis from zero dimensions (0D)

3.1.4 Functional applications of Al/Al2O3 nanostructured surfaces

3.2 Experimental Approach: MOCVD technique

iv 3.2.1 Synthesis of the single source precursor [tBuOAlH2]2

3.2.3 Repeatability and reproducibility of the Al/Al2O3 nanowire coatings

3.2.4 Chemical Vapour Deposition System Design

3.2.4.1 Precursor/gas delivery system

3.2.4.2 Reaction chamber (reactor)

3.2.4.3 Loading/Unloading system

3.2.4.4 Energy system

3.2.4.5 Vacuum system

3.2.4.6 Exhaust gas system

3.2.5 Routine and maintenance of the system

3.2.6 Deposition of different Al/Al2O3 deposited nanostructures

4 Characterization of Al/Al2O3 NWs

4.1 Characterization and modelling of Al/Al2O3 NWs using FIB-nanotomography 4.1.1 Introduction

4.1.2 Experimental approach

4.1.2.1 Sample preparation

4.1.2.1.1 Synthesis of Al/Al2O3 NWs

4.1.2.1.2 Scanning Electron Microscopy (SEM)

4.1.2.1.3 Preparation of the Region of Interest (ROI)

4.1.2.1.4 Serial Sectioning

4.1.2.1.5 Imaging

4.1.2.1.6 Image analysis techniques and 3D reconstruction

4.1.2.1.7 Error during FIB-nanotomography

4.1.3 Results and discussion

4.1.3.1 SEM images/cross sections

4.1.3.2 Reconstruction of 3D models

4.1.3.3 Analysis of reconstructed 3D models

4.1.3.3.1 2D analysis: NW coverage, 2D porosity and aspect ratio

4.1.3.3.2 Analysis of the aspect ratio

4.1.3.3.2 3D analysis

4.1.3.3.2.1 Analysis of the volume density (Vv), porosity (Ф), specific surface area (Sv), and specific surface area to volume ratio (SA/V)

4.1.3.3.2.2 Analysis of the geometric tortuosity (τ)

4.1.3.3.2.3 Analysis of the connectivity: Euler number (χ)

4.2 Surface characterization of the Al/Al2O3 NWs

v 4.2.1 Introduction

4.2.2 Experimental approach

4.2.2.1 CVD deposition on glass

4.2.2.2 Transmission

4.2.2.3 Ultraviolet–visible spectroscopy (UV-VIS)

4.2.2.4 X-ray photoelectron spectroscopy (XPS)

4.2.2.5 Roughness (profilometer and AFM)

4.2.2.6 Contact angle

4.2.3 Results

4.2.3.1 Transmission measurements

4.2.3.2 UV-VIS

4.2.3.3 XPS

4.2.3.4 Roughness analysis with AFM and profilometer

4.2.3.5 Contact angle

5 Interactions of Cells with Al/Al2O3 NWs Prepared by CVD

5.1.1 Interactions between an implant and a host tissue

5.1 Introduction

5.1.2 The interface between the biomaterial and the biological system: mechanisms involved in cell-surface interactions

5.1.3 Protein interaction with a biomaterial

5.1.4 Bone composition and formation

5.1.5 Background of biocompatibility studies on nanostructured alumina

5.1.6 Background of biocompatibility studies on surfaces prepared by CVD of [tBuOAlH2]2

5.2.1 Experimental approach

5.2 Selective adhesion of osteoblast and fibroblast on Al/Al2O3 NWs

5.2.1.1 Synthesis of Al/Al2O3 NWs

5.2.1.2 Cell experiments

5.2.1.3 Microscopic analysis of the cells

5.2.2 Results and discussion

5.2.2.1 Cell fluorescence images after staining

5.2.2.2 Morphology analysis of the cells by SEM

5.2.2.3 Cell growth: cell density and mean cell area

5.2.2.4 Overall metabolic activity, WST-1 test

5.3 Osteogenic differentiation on Al/Al2O3 NWs

vi 5.3.1 Basics of Osteoblast differentitaion

5.3.3 Experimental approach

5.3.3.1 Synthesis of Al/Al2O3 NWs

5.3.3.2 Gene expression analysis

5.3.4 Results and discussion



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