Bio-Nano Interactions: Synthesis, Functionalization and Characterization of Biomaterial Interfaces

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UNIVERSITATISACTA UPSALIENSIS

UPPSALA 2016

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1345

Bio-Nano Interactions

Synthesis, Functionalization and Characterization of Biomaterial Interfaces

YIXIAO CAI

ISSN 1651-6214 ISBN 978-91-554-9478-0 urn:nbn:se:uu:diva-277121

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Dissertation presented at Uppsala University to be publicly examined in Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, Wednesday, 1 June 2016 at 10:15 for the degree of Doctor of Philosophy. The examination will be conducted in English.

Faculty examiner: Docent, associate professor Martin Andersson (Chalmers University of Technology, Department of Chemistry and Chemical Engineering).

Abstract

Cai, Y. 2016. Bio-Nano Interactions. Synthesis, Functionalization and Characterization of Biomaterial Interfaces. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1345. 37 pp. Uppsala: Acta Universitatis Upsaliensis.

ISBN 978-91-554-9478-0.

Current strategies for designing biomaterials involve creating materials and interfaces that interact with biomolecules, cells and tissues. This thesis aims to investigate several bioactive surfaces, such as nanocrystalline diamond (NCD), hydroxyapatite (HA) and single crystalline titanium dioxide, in terms of material synthesis, surface functionalization and characterization.

Although cochlear implants (CIs) have been proven to be clinically successful, the efficiency of these implants still needs to be improved. A CI typically only has 12-20 electrodes while the ear has approximately 3400 inner hair cells. A type of micro-textured NCD surface that consists of micrometre-sized nail-head-shaped pillars was fabricated. Auditory neurons showed a strong affinity for the surface of the NCD pillars, and the technique could be used for neural guidance and to increase the number of stimulation points, leading to CIs with improved performance.

Typical transparent ceramics are fabricated using pressure-assisted sintering techniques.

However, the development of a simple energy-efficient production method remains a challenge.

A simple approach to fabricating translucent nano-ceramics was developed by controlling the morphology of the starting ceramic particles. Translucent nano-ceramics, including HA and strontium substituted HA, could be produced via a simple filtration process followed by pressure-less sintering. Furthermore, the application of such materials as a window material was investigated. The results show that MC3T3 cells could be observed through the translucent HA ceramic for up to 7 days. The living fluorescent staining confirmed that the MC3T3 cells were visible throughout the culture period.

Single crystalline rutile possesses in vitro bioactivity, and the crystalline direction affects HA formation. The HA growth on (001), (100) and (110) faces was investigated in a simulated body fluid in the presence of fibronectin (FN) via two different processes. The HA layers on each face were analysed using different characterization techniques, revealing that the interfacial energies could be altered by the pre-adsorbed FN, which influenced HA formation.

In summary, micro textured NCD, and translucent HA and FN functionalized single crystalline rutile, and their interactions with cells and biomimetic HA were studied. The results showed that controlled surface properties are important for enhancing a material’s biological performance.

Keywords: Bioactive surfaces, nanocrystalline diamond, hydroxyapatite, protein secondary structure, protein absorption, auditory neurons, single crystalline rutile, nano morphology, surface functionalisation, in vitro biomineralisation, translucent nano-ceramics, bio-window material, material characterisation.

Yixiao Cai, Department of Engineering Sciences, Applied Materials Sciences, Box 534, Uppsala University, SE-75121 Uppsala, Sweden.

urn:nbn:se:uu:diva-277121 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-277121)

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Preface

‘I love those who can smile in trouble, who can gather strength from distress, and grow brave by reflection. ‘Tis the business of little minds to shrink, but they whose heart is firm, and whose conscience ap- proves their conduct, will pursue their principles unto death’.

Leonardo da Vinci

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To My Magnificent Mom

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Yixiao Cai, Fredrik Edin, Zhe Jin, Andrei Alexsson, Olafur Gudjonsson, Wei Liu, Helge Rask-Andersen, Mikael Karlsson, Hao Li. Strategy towards independent electrical stimulation from cochlear implants: Guided auditory neuron growth on topographically modified nanocrystalline diamond. Acta Bio- materialia. 31 (2016) 211–220.

II Yixiao Cai, Song Chen, Kathryn Grandfield, Håkan Engqvist, Wei Xia. Fabrication of translucent nano-ceramics via a simple filtration method. RSC Advances 5 (2015) 99848–99855.

III Yixiao Cai, Shiuli Pujari-Palmer, Le Yu, Håkan Engqvist, Mar- jam Karlsson-Ott, Wei Xia. Utilization of translucent hydroxy- apatite nano-ceramics as a bio-window material. Nano Ad- vances, 1 (2016) 45–49.

IV Yixiao Cai, Hu Li, Mikael Karlsson, Klaus Leifer, Håkan Engqvist, Wei Xia. Biomineralization on Single Crystalline Ru- tile: The Modulated Growth of Hydroxyapatite by Fibronectin in a Simulated Body Fluid. RSC Advances, 6 (2016) 35507- 35516.

Reprints were made with permission from the respective publishers.

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Publications not included in thesis:

I Yixiao Cai, Wei Xia. A method of fabricating translucent nano- ceramics. (PCT/SE2016/050147).

II Yixiao Cai, Christofer Lendel, Lars Österlund, Lars Lannfelt, Martin Ingelsson, Fredrik Nikolajeff, Mikael Karlsson, Joakim Bergström. Changes in secondary structure of -synuclein dur- ing oligomerization induced by reactive aldehydes. Biochemical and Biophysical Research Communications 464 (2015) 336–

341.

III Camilla Russell, Ken Welch, Jonas Jarvius, Yixiao Cai, Rimantas Brucas, Fredrik Nikolajeff, Peter Svedlindh, Mats Nilsson. Gold nanowire based electrical DNA detection using rolling circle amplification. ACS Nano, 8 (2014) 1147–1153.

IV Chen Xia, Baoyuan Wang, Ying Ma, Yixiao Cai, Muhammad Afzal, Yanyan Liu, Yunjuan He, Wei Zhang, Wenjing Dong, Junjiao Li, Bin Zhu. Industrial-grade rare-earth and perovskite oxide for high performance electrolyte layer-free fuel cell.

Journal of Power Sources 307 (2016) 270–279.

V Mikael Malmström, Mikael Karlsson, Pontus Forsberg, Yixiao Cai, Fredrik Nikolajeff, Fredrik Laurell. Waveguides in poly- crystalline diamond for mid-IR sensing. Optical Materials Ex- press, 6 (2016) 1286-1295.

VI Song Chen, Yixiao Cai, Håkan Engqvist, Wei Xia. Enhanced bioactivity of glass ionomer cement by incorporating calcium silicates. Biomatter. 6 (2016) e1123842.

VII Wei Zhang, Yixiao Cai, Baoyuan Wang, Hui Deng, Chu Feng, Wenjing Dong, Junjiao Li and Bin Zhu. The fuel cells studies from ionic electrolyte Ce_0.8Sm_0.05Ca_0.15O_2-δto the mixture layers with semiconductor Ni_0.8Co_0.15Al_0.05LiO_2-δ. International Journal of Hydrogen Energy. 2016 (In press).

VIII Tao Qin, Yixiao Cai, Håkan Engqvist, Wei Xia. Occlusion of open tubules and remineralization of teeth using a paste con- taining calcium phosphate. Journal of Materials Science: Mate- rials in Medicine (under review).

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Author’s contributions to the listed papers:

I. I participated in the planning of the study, the material preparation, characterization and the major writing of the manuscript.

II. I participated in the planning of the study, most of the experimental work, including the material preparation and the material characteri- zations and the final writing of the manuscript.

III. I participated in the planning of the study, parts of the experimental work including the material preparation and the material characteri- zations and the major writing of the manuscript.

IV. I participated in the planning of the study, most of the experimental work including the material functionalization and characterization, and the major writing of the manuscript.

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Abbreviations

AFM Atomic Force Microscopy CI Cochlear Implant

CVD Chemical Vapour Deposition

EDS Energy-dispersive X-ray Spectroscopy FN Fibronectin

HA Hydroxyapatite

ICP Inductively Coupled Plasma NCD Nanocrystalline Diamond PBS Phosphate Buffered Saline SBF Simulated Body Fluid

SEM Scanning Electron Microscopy TEM Transmission Electron Microscopy tHA Translucent Hydroxyapatite

UV Ultraviolet

XPS X-ray Photoelectron Spectroscopy XRD X-ray Diffraction

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Introduction

The term biomaterials refers to materials that can be made into units and devices to partially or completely replace the function of living tissues. A biomaterial’s biocompatibility is one of its most pivotal and decisive characteristics, as the developed materials must be accepted by their biological surroundings, including cells and tissues. The development of bio- nanotechnology by combining modern medical technologies represents a promising path toward creating a new generation of advanced biomaterials.

Figure 1 summarizes some key factors that influence the properties of bio- materials. All of these factors play significant roles in determining whether a biomaterial will be suitable for clinical use. In this thesis the surface properties, mechanical properties, bio-interactions, physical properties and biocompatibility of biomaterials are studied.

Figure 1. Major topics of current biomaterials research divided into the categories of physical properties, chemical properties and biocompatibility.

A ‘bioactive material’ can be described as a material that is capable of inter- acting with body fluid, tissues and its biological surroundings, such as pep- tides, proteins, growth factors or even viruses and bacteria. Several bioactive surfaces, that use different materials, are investigated in this thesis including topological modifications of the surface (paper I), synthesis (paper II), and

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characterization (paper III and IV). In the present thesis, three materials were investigated: nanocrystalline diamond (NCD), hydroxyapatite (HA) and titanium dioxide (single crystalline rutile).

Nanocrystalline diamond

Diamond has excellent thermal, chemical, optical and mechanical properties.

Hardness is an exceptional quality that can make the material resistant to wear. Although harder candidates, such as wurtzite boron nitride and lons- daleite, have been developed recently ¹, synthesis and large-scale commer- cialisation remain to be realised. Diamond is electrically insulating ², but can become a semi-conductor via doping (boron or phosphorus). Diamond also has a relatively high refractive index (approximately 2.4) and is one of the few materials that is transparent in the ultraviolet, visible and infrared wave- lengths. One commercial application of synthetic diamond is as an attenuat- ed total reflection (ATR) crystal for use in Fourier transform infrared spectroscopy (FTIR) ³. Here, the broad optical transmission window, high refractive index, chemical inertness and hardness of diamond are appreciated. Di- amond has also been studied as a biomaterial ⁴.

NCD is polycrystalline diamond with grains smaller than approximately 100 nm. NCD has unique material properties that are similar to those of single crystal synthetic diamond, including extraordinary mechanical properties (a low friction coefficient, extremely hard etc.), chemical stability and biocompatibility ⁵. NCD is particularly favourable for use in biomedical applications, such as dental ⁶, orthopaedic ^7,8 and ophthalmological implants ⁹ and surgical instruments ^10,11. Moreover, NCD is resistant to bacterial coloniza- tion when compared with steel and titanium ¹². NCD is most often grown as a solid thin film on a substrate in a chemical vapour deposition (CVD) reac- tor.

Hydroxyapatite

Hydroxyapatite (HA) is a natural mineral form of calcium phosphate and its formula is written as Ca₅(PO₄)₃(OH). Another chemical formula, Ca10(PO4)6(OH)2, is also used to reveal the crystal unit cell that comprises two entities of Ca₅(PO₄)₃(OH). HA exhibits osteo-conductive, non-toxic and non-immunogenic properties, particularly in the repair of bone and teeth ¹³. The mineral in our teeth and bone (50% by volume and 70% by weight ¹³) is composed of a calcium-deficient carbonated HA. Synthetic HA in different forms, such as; powders, bulks, porous scaffolds, cements and composites, has been widely used as a biomedical material.

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15 The similarity of HA in terms of chemical composition and its good biocompatibility and bioactivity make HA a preferred material in bone repair and regeneration. Bulk HA ceramics are rarely used in orthopaedic and dental applications because of their poor intrinsic mechanical properties. How- ever, bulk HA ceramics can be used as prototype materials for studies of the interactions between bioceramics and cells/tissue on different scales. In this case, the transparency of HA ceramics would allow for the direct observation of cell-material interactions using optical microscopy, which is very difficult to achieve with currently available materials.

To achieve a transparent or translucent HA ceramic, the material should have a dense microstructure, as pores often contribute to opacity via light scattering. However, it is a challenge to achieve the full densification of polycrystalline ceramics ^14,15, which strictly requires a good vacuum level and high temperature and pressure during sintering. Efforts should be made to minimize light scattering to achieve transparency in a ceramic. In the field of optics, transparency is the physical property that describes a material that allows light to pass through the material without being scattered. A translucent material could be defined as letting light pass through to allow objects on the other side to be distinguished. The identification of a simple method of fabricating transparent or translucent HA ceramics represents a technical challenge, as the current employed method requires complicated sintering techniques, such as hot isostatic pressure.

Bioactive Ti implants

Titanium (Ti) is widely used as a load bearing implant material in bone replacement applications ^16–19. The interaction of Ti with the host tissue is of utmost importance for the long-term clinical outcome of the implant. An oxide forms rapidly on Ti implants after exposure to air, which then interacts with the host tissue and determines the biological properties of the material

20. Oxide formation has also been reported to increase the interaction with calcium ions, which are important for extracellular protein adhesion and future bone conduction.

Crystalline titanium oxides have been proven to be bioactive ²¹, due to the presence of hydroxyl groups on the surface, which induces HA formation from body fluids. Titanium oxide coatings have been studied in vitro and in vivo for many years ^22–24. It is worth noting that the surface atomic configura- tion of titanium oxides is pivotal in determining their physicochemical properties, such as adsorption and reactive selectivity ^25,26. For single crystalline rutile, the majority of research efforts have been devoted to the photocatalyt- ic and photo-electrochemical properties of the materials ^27,28. Very few studies have reported on their bioactivity of such materials ^29,30.

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Simulated body fluid (SBF), which is a solution close to that of human blood plasma in terms of concentrations of inorganic ions and the pH value, is used as a standard in vitro method to investigate HA formation on the surface of materials ^31–35. This method is useful for evaluating in vitro bioactivity. To improve the bioactivity of titanium dioxide surfaces, the current strategy is to coat or functionalize the surfaces with bioactive compounds, such as apatite, to improve bone bonding, accelerate bone formation ^36–38, and enhance osteoconductivity in vivo ^39–41.

When used in vivo the implants first encounter body fluids containing not only salts but also proteins. The interaction of proteins with the potential bioactive properties of a material could be a very important addition to current testing methods and could help us to gain a significant new understand- ing of how bioactive biomaterials interact with body fluids in vivo.

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Aims and Goals

The design of biomaterial interfaces is particularly interesting because the interaction between materials and tissues always occurs at the interface.

Such interfaces are thus important for determining the clinical outcome of a material. The aim of this thesis is to establish strategies for the synthesis, surface functionalization and characterization of three materials and thereby possibly improve their biological properties.

In paper I, microstructured NCD was used as a potential nerve-electrode interface to improve CIs, which are used to treat sensorineural hearing loss and deafness. Although currently existing CIs are clinically successful, the patient’s hearing cannot be completely restored. One problem is the number of electrodes; 12–20 electrodes are used to replace the function of 3400 inner hair cells. Via our material design on micrometre-sized NCD pillars, we aimed to develop individual neuron stimulation patterns for regenerated human neurons, which could be used for improved CIs.

HA exhibits osteo-conductive, non-toxic and non-immunogenic properties, particularly in the repair of bone and teeth. However, it is difficult to directly observe HA-cell or HA-tissue interactions with conventional light microscopy methods because HA is typically opaque. Making an HA ceramic transparent will therefore allow for the direct observation of the cell- material interaction. In paper II and III, a simple method of fabricating translucent HA nano-ceramics was studied, and the characteristics of the fabricated materials were applied to cell observation, functioning as a new type of ‘bio-window’ material.

The biomaterial surface is typically exposed to numerous extracellular proteins in biological fluids. The crystal phase can determine the interaction of materials and cell/tissue with proteins and body fluids. Thus the effect of proteins on different crystal planes must be addressed. In paper IV, the ef- fect of fibronectin (FN) on biomineralization on single-crystal rutile sub- strates was studied at time points from 1 week to 4 weeks. The objective of this study was twofold: (i) to investigate the effect of pre-adsorbed FN on different single rutile faces and observe the differences in HA formation by using a pure SBF solution, and (ii) to conduct a comparison study, using a solution that contains both FN and SBF.

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Preparation and characterization of the investigated materials

Micro-fabrication

Micro-fabrication represents a meticulous process of fabricating miniature structures on the micrometre or nanometre scale. This process originates from the need to miniaturise components in the semi-conductor industry, and the micro-electronics industry remains a major driving force behind the development of better processes for the fabrication of even smaller and com- plex structures in a wide range of different materials. However, in medicine and biology, micro-fabrication can have a remarkable impact, as illustrated in paper I. Commonly used techniques in micro-fabrication include lithog- raphy, doping, thin film deposition, etching, bonding and polishing.

This section will give a short overview of the key methods, used in this thesis, for the micro-fabrication of micro-metre sized NCD pillars.

Photolithography

Photolithography is a rather simple and inexpensive technique for defining small features in a thin polymer layer (photoresist) on top of a substrate. The substrate is often a silicon wafer and the technique is highly suitable for mass production with the possibility to define patterns with a size of less than 1 µm. In the photolithography process, a light source is used to transfer an image from a chromium glass mask to a photosensitive layer (photoresist or resist) on a substrate.

There are three main steps in photolithography (Fig. 2). The first step is spin-coating, which means that a photosensitive material (photoresist) is applied to the substrate surface. The second step is exposure; the photoresist is exposed using an ultraviolet (UV) light source. The final step is called developing, which means that the exposed or unexposed photoresist is dissolved with a chemical developer. Before exposure (soft-bake) and after development (hard-bake), the substrate is placed on a hot plate at 100-120 C (normally for 1 minute). In the soft-baking step, nearly all solvents are removed from the photoresist coating while in the hard-baking step the photoresist is made harder and more resistant to etchants. The type of photoresist

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19 determines whether the exposed (positive) or unexposed photoresist (negative) is dissolved.

Figure 2. Flow chart for photolithography.

Plasma Etching

Plasma etching, or dry etching, represents an important micro-fabrication process for integrated circuits. In general, plasma etching is a chemical etching method, which means that a chemical reaction occurs between the solid atoms from the substrate surface and reactive gas atoms formed in the plasma (radicals). Volatile products are formed, which are pumped out of the plasma chamber. Originally, in the late-1960s plasma etching was used to strip resists from silicon wafers ⁴². Later in the 1980s, plasma etching was developed as a mainstream process in the semiconductor industry, primarily due to the possibility of anisotropic etching. The main categories of plasma etching include reactive ion etching (RIE), electron cyclotron resonance (ECR) and inductively coupled plasma (ICP) etching. The latter two systems are so called high density plasma systems and were developed relatively late in the 1990s. High density plasma systems typically offer higher etch rates, better uniformity and lower surface roughness than RIE systems. ICP systems are currently the most widely used systems due the complexity of ECR systems.

For the work in this thesis, ICP etching was used for the fabrication of microtextured NCD surfaces. ICP systems are driven with a coil (radio frequency at 13.56 MHz as power source) that sustains the plasma through a dielectric window. Moreover, an ICP system is equipped with a separate

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capacitively coupled radio frequency (rf) power supply. In principle, this feature is equivalent to a RIE system. In this way, the electrode where the substrate is placed can be biased leading to ion bombardment of the substrate surface.

For instance, in an oxygen plasma discharge, which is used to etch NCD, highly reactive radicals in the form of atomic oxygen (reactive species) are created. The atomic oxygen, in combination with the ion bombardment of the surface, reacts with the outermost carbon atoms on the NCD surface and CO₂ is formed and pumped out of the system. The main steps in the etching process are as follows:

1) Generation of the reactive species (plasma)

2) Diffusion of the reactive species to the substrate surface 3) Adsorption of the reactive species at the substrate surface 4) Diffusion of reactive species on the substrate surface 5) Formation of reaction products

6) Desorption of the volatile reaction products

7) Diffusion of the reaction products to the plasma and removal by the pumping system

To control an ICP etching process in terms of etch rate, geometry of the fabricated microstructures and surface roughness several parameters should be taken into consideration. These parameters are gas mixture, flow rate, ICP power, substrate bias and process pressure.

Fabrication of micro-structured NCD biomaterials

In paper I, we described the application of one type of micro-structured NCD (Fig. 3) as a potential nerve-electrode interface. However, we did not address in detail how the NCD pillars were fabricated. Here the entire procedure is described.

The micro-structured NCD fabrication was conducted in the cleanroom facility of Ångström Laboratory, Uppsala University. There are seven steps in the entire procedure.

1) The NCD wafer (1 µm thick NCD on a 4 inch silicon wafer) was first soaked in a piranha solution to obtain a clean surface (H₂SO₄/H₂O₂).

2) The wafer was subsequently coated with a 1-μm-thick aluminium layer by sputtering (VonArdenne CS 730S).

3) Using standard photolithography (Karl Suss mask aligner), a resist pattern was fabricated on top of the Al-coated wafer.

4) The square pattern was then etched into the Al layer by ICP etching (Plasmatherm SLR) using Cl₂/BCl₃/Ar chemistry.

5) The remaining Al pattern could then be used as a mask for NCD etching. The same ICP system was employed for the NCD plasma etching, using O2/Ar chemistry.

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21 6) By changing the plasma process to the so called Bosch process ⁴³, pillars consisting of silicon with NCD/Al on top were fabricated. The Bosch process is used to fabricate high aspect ratio structures (deep etching of small structures/grooves) in silicon. The process consists of three steps that are repeated several times, depending on the desired depth of etching. The first step is a polymer deposition process, and the second step is a polymer etching step in which all of the deposited polymer is etched away except the polymer on the sidewalls of the etched microstructures. In the third step, the plasma chemistry is chosen to ensure that the silicon is etched without etching the polymer. In this way extremely high aspect ratio structures can be fabricated. The chemistry is a combination of C₄F₈/SF₆/Ar.

In a final etching step, an isotropic silicon etch is used (i.e. using the third step in the Bosch process). In this way, an undercut of the NCD squares can be fabricated.

7) Finally, the Al is etched away using wet etching (phosphoric acid, water, and nitric acid).

Figure 3. (a) SEM images of NCD/silicon pillars fabricated by ICP etching. (b) Close-up image of the pillars.

Fabrication of translucent nano-ceramic biomaterials

In paper II and III, we introduced how we fabricated two types of translu- cent nano-ceramics and made them available for the direct observation of cells via optical microscopy. Here the standard procedure is illustrated. The three main steps are described below:

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Synthesis of HA and SrHA nanoparticles

The first step can be simplified and described in the reaction scheme shown below, which is one of the most standard routes of precipitating HA based on the reaction between calcium nitrate and ammonium dihydrogen phosphate.

5Ca(NO3)2 + 3(NH4)2HPO4 ^ସ୒ୌሱۛۛۛۛሮ^ర^୓ୌ Ca5(PO4)3(OH) + 3H2O + 10NH4NO3

HA nanoparticles were synthesized using a typical precipitation procedure.

Diammonium hydrogen phosphate was individually prepared in deionized water to form a clear solution with a concentration of 0.2 M. Calcium nitrate was prepared in deionised water with a stoichiometric Ca/P molar ratio of 1.67 for the formation of Ca5(PO4)3OH. The initial pH of each solution was adjusted to 10. Similar to the synthesis of HA nanoparticles, the strontium substituted HA precursor was prepared using the following procedure. Di- ammonium hydrogen phosphate was prepared with a concentration of 0.2 M.

For the formation of 0.05% strontium substituted calcium hydroxyapatite, (Ca_9.95Sr_0.05(PO₄)₆(OH)₂). written as 005SrHA in the following), the calculat- ed concentrations of calcium nitrate and strontium nitrate were 0.317 M and 0.013 M, respectively, and both chemicals were mixed in deionised water with stirring. To avoid the rapid growth of crystalline grains, solutions of pure calcium nitrate and mixed calcium nitrate containing strontium nitrate were added drop-wise to a diammonium hydrogen phosphate solution. The entire procedure was performed under vigorous stirring overnight. The precipitate was then kept stationary in the mother liquor for another 24 h.

025SrHA (Ca_7.5Sr_2.5(PO₄)₆(OH)₂) and 050SrHA (Ca₅Sr₅(PO₄)₆(OH)₂) were synthesized using the same method.

Filtration of translucent HA and SrHA nano-ceramic precursors

For the second and key step of forming the HA and SrHA translucent ceramic precursors, a 20 mL suspension was removed from each mother liquor and ultrasonicated to ensure a homogeneous suspension. The filtrated precipitate was made into a precursor through a simple glassware-based filtration system that used one laboratory pump. The samples were dried in air at room temperature after filtration. The vacuum pump system was connected to a filter unit. A polycarbonate filter paper (Whatman® Nuclepore™ Track- Etched Membranes) with a diameter of 47 mm and a pore size of 0.2–0.4 μm was used throughout the entire procedure.

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Sintering of as-prepared HA and SrHA translucent nano- ceramics

Four types of HA and strontium-doped HA precursors (HA, 005SrHA, 025SrHA, 050SrHA) were dried and then placed in an ordinary non-vacuum furnace for sintering. The samples were heated to 1000°C at a rate of 5°C/min and kept at 1000°C for 2 hours. Then, the samples were to room temperature cooled at the same rate.

Biomimetic HA deposition

Bioactive bone-replacement implants have exhibited better bonding to host bone than inert implants. The mode-of-action has been described as the material’s ability to form a HA coating on its surface in body fluids ³². For this process to occur, a negative surface charge is needed. This technique can also be used as a coating method to provide a HA-coated implant, which is called biomimetic HA coating.

HA coatings on implants have shown good fixation to the host bone with the coating acting as a bonding layer between the bone and the implant ⁴⁴. Such an interface is important for orthopaedic applications, as it will improve the implant’s stability and integration with the surrounding bone. There is a keen interest in developing biomaterials that can control the process of biomineralisation at the implant surface, thus aiding in the formation of a strong bone implant interface ^45–48.

Bone formation is actually a complicated process that combines ion adsorption, the formation of apatite crystals, and ion exchange. Kokubo et al. pro- posed a method that could mimic certain steps involved in bone formation ⁴⁹, which has been widely used to test the bioactivity of a defined biomaterial surface, as described above. The soaking solution has an ion concentration close to that of human blood plasma and is a simulated body fluid (SBF).

SBF can be applied to assess the bioactivity of new functionalized materials, particularly when testing surface modifications of metallic implants.

Characterization techniques

Different analysis techniques were used in this thesis. (1) Scanning electron microscopy (SEM), (2) X-ray photoelectron spectroscopy (XPS), (3) Atomic force microscope (AFM) and (4) X-ray diffraction (XRD) are the principle techniques used in the papers included in this thesis. Other techniques, such as confocal laser scanning microscopy, nano-indentation, Fourier transform infrared spectroscopy, and thermal gravity analysis, were also used.

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SEM uses a focused beam of high-energy electrons to generate different signals at the surface of a solid specimen, revealing for example, the morphology, chemical composition, and crystalline structure of material. This technique has been used for the analysis of the micro- and nano-structures of material surfaces (Fig. 4). In this thesis, a Zeiss SEM (LEO 1550 and Mer- lin) was used (Fig. 5). The materials studied in this thesis were non- conducting and to avoid charging in the microscope chamber the samples must be covered with a thin layer of a conductive metal (Pt/Au) or carbon.

XPS, which is known as electron spectroscopy for chemical analysis (ES- CA), is obtained by irradiating a material using X-rays, and the kinetic energy and number of electrons that escape from the top of the material are measured simultaneously. XPS is used to analyse the surface chemistry of the studied material (e.g. the surface of a single crystalline rutile before and after treatment). The technique is surface-sensitive and quantitative, and can measure the elemental composition. A physical electronics quantum 2000 (Al Kα X-ray source) was used.

AFM is one type of scanning probe microscopy, and measures the force between a probe and the sample. AFM was used to determine the surface topography and adhesion force in this thesis. Peak force quantitative nanomechanical mapping (PF-QNM) was used to determine the surface adhesion of single crystalline rutile before and after FN adsorption. A Bruker multi- mode 8 AFM was used.

X-ray diffraction is a tool that can be used to determine the crystalline phas- es in a material. A beam of incident X-rays diffracts at crystalline atoms, and by detecting the angles and intensities of the diffracted beams the crystal structure of a material can be determined. XRD (Siemens diffractometer D5000 (Cu Kα radiation)) was used to determine the crystallinity of materials in this thesis, such as hydroxyapatite and strontium substituted hydroxyapatite.

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Figure 4. Example of an SEM micrograph, which shows the bulk structure of a translucent hydroxyapatite nano-ceramic plate.

Figure 5. Images of the SEM setup used throughout this thesis (Merlin, Zeiss, Ger- many)

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Results and Discussion

Micro-patterned nanocrystalline diamond surfaces

Figure 6. SEM micrographs of micrometer-sized nanocrystalline diamond before (left) and after (right) culturing with mouse ganglion cells. Confocal laser microscopy showing neonatal mouse spiral ganglion neuron growth on the NCD pillars; most

sprouting neurites remained on the structured NCD surface (middle).

The results show that human and murine inner-ear ganglion neurites and, potentially, neural progenitor cells can attach to patterned NCD surfaces (Fig. 6) without an extracellular matrix coating. SEM and confocal laser scanning microscopy revealed adhesion and neural growth, specifically in an ordered manner along the nail-head-shaped NCD pillars, rather than in non- textured areas. This pattern was established when the inter-NCD pillar distance varied between 4 and 10 µm. At a distance of 14 µm, the neurons initi- ated random growth.

These findings demonstrate that regenerating auditory neurons show a strong affinity for the NCD pillars, and the technique could be used for neural guidance and the creation of new neural networks. Together with the NCD’s unique anti-bacterial and electrical properties (NCD can be made electrically conductive using boron doping), structured NCD surfaces could provide designed neural/electrode interfaces to create independent electrical stimulation signals in CI electrode arrays for the neural population.

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Translucent HA nano-ceramics

An analysis of HA and 005SrHA before and after sintering showed that good translucency was obtained after filtration for both materials. Figure 7 shows the dense microstructure of HA and 005SrHA nano-ceramics. The translucency decreased after sintering. 025SrHA and 050SrHA appeared not to be translucent. The HA and 005SrHA ceramics showed a highly compact structure and inter-granular pores were difficult to detect. A significant morphology transformation was observed with increasing strontium concentrations in the case of 025SrHA and 050SrHA, which were rod-like particles (Fig. 8 a–

b). Moreover, the grain morphology of sintered 025SrHA and 050SrHA ceramics was primarily between quadrangular and hexagonal, with a size in the range of 100–300 nm, and the ceramics were porous (Fig. 8 c–d).

Figure 7. SEM images of the final nano-ceramic products, as revealed by SEM (A) Low magnification HA, (B) high magnification HA, (C) low magnification

005SrHA, and (D) high magnification 005SrHA.

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Figure 8. The morphologies of (a) 025SrHA precursors, (b) 050 SrHA precursors, (c) 025SrHA ceramics, and (d) 050SrHA ceramics.

The pores, grain size and grain boundaries of a ceramic are the main factors that affect its translucency and mechanical strength. Porosity is the most important factor for the translucency of the material, and a porosity lower than 0.1% can obtain an initial transparency ⁵⁰. The elimination of pores happens mainly during sintering after grain growth. Because the green body was composed of spherical nanoparticles, the mass transformation and growth of grain boundaries is more homogeneous than observed for green bodies with rods and irregular-shaped grains. Sintering under a vacuum may help to achieve a higher translucency. Based on the results of this study, by controlling the morphology of ceramic nanoparticles, other functional translucent ceramics could be fabricated via this simple technique.

MC3T3 cells are a mouse calvarial model of early stage pre-osteoblasts ⁵¹. MC3T3 cells undergo different stages of differentiation and proliferates rapidly, making them a popular choice for osseous toxicity and developmental testing ⁵². MC3T3 cells were used to evaluate whether translucent HA ceramics can be used as a window material. Bright field and CFDA staining showed that the MC3T3 cells were viable over the 7 days of culture (Fig. 9).

The results show that the material is not toxic and cells can be easily seen through the tHA nano-ceramics, indicating that tHA has the potential to be used as a window material.

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Figure 9. Fluorescence images of MC3T3 cells seeded on tissue culture polystyrene dishes (TCP) covered by translucent hydroxyapatite nano-ceramics after 7 days (a)

40x, (b) 100x.

Biomineralized single-crystal rutile surfaces

Two processes were used to evaluate biomineralization on single-crystal rutile surfaces: (1) samples were soaked in SBF containing FN and (2) FN was adsorbed onto samples surfaces followed by soaking in SBF without FN. By soaking each substrate in the two different processes, different HA growth was observed after 1 week of incubation. Fig. 10 shows the SEM results obtained after biomineralization on each face for 1 week. In process I, a few crystals could be found at the centre of each substrate, suggesting that no significant differences in the growth rate occur during the first week on each face. However, in comparison to the (001) and (100) faces, fewer spherical HA crystals with plate-like structures could be found on the (110) face. Significant differences in HA growth were found in process II, during which the (001), (100) and (110) faces exhibited a faster biomineralization than obtained with process I. In addition, HA growth on the (110) face was the slowest. The morphologies of (001) and (100) were sphere-like particles, and the size of particles on the (001) face was 2.05 ± 0.28 µm, which was larger than that of the particles on the (100) faces (1.70 ± 0.24 µm).

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Figure 2. SEM images of biomineralisation on the (001), (100) and (110) faces after being soaked in processes I and II for 1 week. The shapes of some individual

calcospherites are also presented to illustrate the nucleation period.

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31

Conclusion

In paper I, the findings demonstrate that auditory neurons have a strong affinity for the NCD pillars, and the technique could be used for neural guidance and the creation of new neural networks. Together with the NCD’s unique anti-bacterial and electrical properties, patterned NCD surfaces could provide designed neural/electrode interfaces to create independent electrical stimulation signals in CI electrode arrays for the neural population. The results obtained from our current study suggest that NCD surfaces fabricated from a topographical perspective are a new strategy for developing potential interface biomaterials that can support neuron adhesion and migration.

In paper II, two types of translucent ceramics, HA and 0.05% strontium- substituted calcium HA, were fabricated successfully using a simple filtration method. Optical transmittance tests confirmed that the translucency of each body was mostly retained after calcination at 1000 °C, and both ceramics achieved good mechanical properties compared to commercial HA products. The presented theoretical model confirms the importance of a proper filtration pressure and time for producing a dense precursor and finally a sintered translucent ceramic.

In paper III, biomimetic tHA nano-ceramics were used as a window ma- terial to directly observe cells. The attachment and spreading of MC3T3 cells can be observed through tHA nano-ceramics. The findings made it possible to observe cell behaviour through bioceramics via light microscopy, revealing a function of such HA materials for live detection in biomedical applications.

In paper IV, the obtained findings indicated that the interfacial energies could be altered by pre-adsorbed FN, which led to different HA growth ki- netics on the (001), (100) and (110) surfaces.

The above studies demonstrated that synthesis/surface structuring (paper I and II), characterization (paper II and IV) and functionalization (paper IV), and cell studies (paper I and III) can be used to evaluate biomaterials with defined surfaces. The use of these advanced tools in material characteriza- tions will provide further evidence to confirm that a material will be well accepted in biomedical applications.

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32

Sammanfattning (Summary in Swedish)

Hos en vanlig människa, finns det cirka 3400 hårceller i örat och balanse- rande organ. Dock kommer alla dessa hårceller aldrig regenerera efter att ha skadats. Detta innebär att varje patient som har drabbats av hörselproblem kommer kunna vara i behov av att transplantera in ett cochleaimplantat. När det kommer till cochleaimplantat är en svår utmaning att skapa en yta som kan styra tillväxten av neuroner med utmärkt affinitet och biokompatibilitet.

Genom att studera befintliga cochleaimplantat visar det sig att dessa produk- ter endast har 12-20 elektroder för att ersätta funktionen av 3400 hårceller, vilket är långt ifrån en idealisk situation, då varje individuell neuronfiber mottager olika våglängder när stimulering tas emot från utsidan. Dessa måste sedan separeras för att realisera hörseln. Emellertid är det mycket svårt att göra tusentals elektroder på en mycket liten ytarea. Detta ledde till att vi valde ett spännande material med utmärkt biologisk affinitet och biokompatibilitet - nanokristallin diamant (NCD). Mikrostrukturerade NCD-pelare tillverkades med hjälp av tillverkningstekniker lånade från mikroelektronik- industrin såsom kemisk ångdeponering, litografi och plasmaetsning. Slutlig- en studerades en sådan plattform med mänskliga hörselneuroner som är tagna från patienter, genom att samarbete med läkare från Uppsalas Akade- miska sjukhus. Resultaten är väldigt lovande, de visade att neuroner kan styras in i systematisk tillväxt till följt av denna mikropelarstruktur.

Hydroxyapatit (HA), betraktas som ett polykristallint keramiskt biomaterial med bioaktiva och biokompatibla egenskaper, det har använts i stor utsträckning inom tvärvetenskapliga områden såsom fysik, kemi, biologi och medicin. HA uppvisar osteo-ledande, icke-giftiga och icke-immunogena egenskaper, särskilt vid reparation av ben och tänder. Det är dock svårt att direkt observera HA-celler eller HA-vävnadsinteraktioner med konvention- ella ljus mikroskopimetoder eftersom HA normalt är opakt. Att kunna göra HA-keramer transparenta kommer därför att utvidga dess biomedicinska tillämpningar. Transparenta polykristallina keramer uppvisar förbättrad vär- mebeständighet och hårdhet jämfört med enkristalla, även om de har lägre optisk transmittans. Det finns ett växande intresse av att tillämpa sådana keramikbaserade material inom biomedicin och biomaterial, i synnerhet vid direkt observation av benmatriser in vitro samt perkutana enheter. En transparent keram behöver en kompakt och porfri mikrostruktur, eftersom porer- na ofta bidrar till opaciteten i en keram. Emellertid är det en utmaning att uppnå full förtätning av polykristallin keramik, eftersom det krävs ett lågt

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33 vakuum samt en hög temperatur och tryck under tillverkningen. Nyligen framtagna metoder som ljusbågssintring (SPS), het isostatisk pressning (HIP), och trycklös sintring (PLS) är exempel på metoder som har använts.

Vi beskriver tillverkningen av halvgenomskinliga HA, nanokeramisk HA med substituerat strontium (SrHA) och nanokeramisk Al2O3 genom ett enkelt filtreringssystem och användningen av dem som ett nytt bio-fönstermaterial för detektering av cellbeteende samt celltillväxt.

Titandioxid är känd som ett bioaktivt material. Dock sker aldrig den cell- substrala interaktionen vid implantering direkt med den kala ytan hos materialet utan istället genom den extracellulära matrisen (ECM), där bio- makromolekyler (t ex proteiner, polysackarider, proteoglykaner) adsorberas på implantatytan vid exponering av biologiska vätskor. Typerna och mäng- derna av adsorberade proteiner bestämmer tillgängligheten av de bioaktiva platser som kan vara avgörande för cell-substrat-interaktioner. Egenskaper- na, inklusive orientering, konformation, packningstäthet av de adsorberade proteinerna avgör om de bioaktiva platserna känns igen av membranbundna receptorer, genom vilka cellerna förhör sin omgivning. Enkristallint rutil (001), (100) och (110) plan valdes som modeller för att undersöka det kom- plexa samspelet mellan ytbioaktivitet och proteinadsorption. Vidhäftning på ytan för varje plan före och efter adsorption av fibronektin (FN) bestämdes genom analys med atomkraftsmikroskopi (AFM) med peak force quantitative nanomechanical mapping (PF-QNM). Resultaten tyder på att ytenergin hos FN pre-adsorberat (001), (100) och (110) ytor har förbättrats, vilket leder till efterföljande acceleration av HA-formation.

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34

Acknowledgement

First, I would like to thank those who have encouraged and supported me to accomplish such a challenging 4-year adventure in epic Uppsala and vintage Uppsala University. The present thesis is a team effort that would not have been completed without your help.

I would like to dedicate the prologue to my supervisors as atokenof my affectionandgratitude: Assoc. Prof. Wei Xia, Assoc. Prof. Mikael Karlsson and Prof. Håkan Engqvist, I would like to thank you for consistently training me to be a diversified researcher and ensuring the usefulness and advantages in each of my projects. You are ideal supervisors who have never directly regulated my research but inspiringly illuminated the path for me in an effec- tive way. More profoundly, the methodology I have learned from you, prob- ing the dialectical relation between idealised and scientific states as well as how to practically manage my projects, will motivate me in my later career to courageously embark on a future full of adventure.

I am sincerely grateful to Prof. Zhong Li (ECUST) and Prof. Johan Liu (CHALMERS) for your earlier enlightenment of my research training and tolerating my clumsy start in the lab. I also would like to give my thanks to Prof. Xuhong Qian for offering splendid dialogue and kind encouragement, Prof. Pengkang Jin for always being around and collaborative in complete brotherhood and Prof. Bin Zhu for continuously boosting my perseverance and inspiring me to trust my struggle. You raise me up, to walk on stormy seas, and to be the best that I can be.

Special thanks to Mr. Stephen Guan, Mr. Zhenyu Zhao, Mr. Yongjun Zhou, Mrs. Xiaoming Ling and Mr. Yi Zhao for illuminating my business ideas, trials and entrepreneurship.

I would like to thank Assoc. Prof. Joakim Bergström, Prof. Lars Öster- lund and Prof. Klaus Leifer for always sharing your creative ideas and ac- cepting me in those challenging and innovative projects. I also would like to thank Prof. Olle Björneholm and Dr. Johan Söderström for the first year of XPS training in Department of Physics and Astronomy. Furthermore, I would like to thank Dr. Hao Li from Uppsala University Hospital, for bridg- ing wonderful research collaboration and for the inter-disciplinary sparks.

I would like to express my appreciation for all the staff and members within our MIM group: Caroline, Cecilia, Fredrik, Gemma, Marjam, Sara, Henry, Charlotte, Ingrid, Michael, Shiuli, Song, Tao, Torbjörn, Celine, Le,

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35 Jun, Luimar and Satwik, for taking good care of our lab resources and estab- lishing a great atmosphere for my experiments.

I would like to express my deepest gratitude to Ernesto Vargas Catalan for your kind help with the Swedish summary and for your presence, Fredrik Edin for contributing strong credits in our ‘Diamond All Ears’ project, Hu Li, for contributing nice discussions to our single rutile project, Dr. Kai Hua and Peng Zhang, for sharing your expertise and Dr. Chaofan Zhang for es- tablishing our earliest memories in Sweden.

I would also like to thank Sara and Per-Richard for your kind help with daily administrative affairs during my PhD studies and Victoria for your strong experimental support when I was working with the cleanroom facili- ties.

I would like to thank Mr. Gerald Pettersson for creating wonderful links between research and industry and Dr. Moa Fransson for launching business strategic discussions and offering your critical help in the patent applications.

Worth noting are Dr. Baoyuan Wang and Chen Xia, my tough comrades- in-arms; I appreciate those exciting moments when we were supporting each other and always sharing positive wishes for our scientific results. I believe we will get there and our joint efforts will be rewarded, sooner or later. Fur- thermore, Dr. Wenjing Dong, Dr. Xunying Wang, Yanyan Liu and Wei Zhang, I appreciate you hosting me in Wuhan and always offering as much kind help as possible.

Finally, I would like to thank my family and friends, particularly my par- ents, for their persistent care during my stay in Sweden. It would not be possible to accomplish this journey, full of twists and turns, without your strong support. Thank you for offering me a better sense of visualisation of the di- versities of the world, and your love.

Finally, I would like to express appreciation for the financial support from the funding agencies or institutions below: the patent grant from Uppsala Innovation Centre, the travel grant from Uppsala Berzelii Technology Centre for Neurodiagnostics, the travel grant from the Graduate School on Ad- vanced Materials for the 21st Century, the travel grant from the Liljewalchs Scholarship Council, the travel grant from Nanjing Yunna Nanotech Lth., the travel grant from The Committee of 2015 China (Nantong) Jianghai Tal- ents Entrepreneurship Week and the travel grant from The Committee of China (Nanjing) 321 Plan of Attracting Leading Entrepreneurial High-Tech Talents.

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References

1. Z. Pan, H. Sun, Y. Zhang, and C. Chen, Phys. Rev. Lett., 2009, 102, 055503, 1–

2. R. F. Davis, Z. Sitar, B. E. Williams, H. S. Kong, H. J. Kim, J. W. Palmour, J. 4.

A. Edmond, J. Ryu, J. T. Glass, and C. H. Carter, Mater. Sci. Eng. B, 1988, 1, 77–104.

3. A. R. Hind, S. K. Bhargava, and A. McKinnon, Adv. Colloid Interface Sci., 2001, 93, 91–114.

4. I. Dion, C. Baquey, and J. R. Monties, Int. J. Artif. Organs, 1993, 16, 623–627.

5. O. A. Williams, Diam. Relat. Mater. , 2011, 20, 621–640.

6. P. Metzler, C. von Wilmowsky, B. Stadlinger, W. Zemann, K. A. Schlegel, S.

Rosiwal, and S. Rupprecht, J. Craniomaxillofac. Surg., 2013, 41, 532–538.

7. L. Yang, T. J. Webster, and B. W. Sheldon, MRS Proc., 2011, 1138, 12–16.

8. L. Yang, L. Zhang, and T. J. Webster, Nanomedicine, 2011, 6, 1231–1244.

9. N. L. Opie, L. N. Ayton, N. V. Apollo, K. Ganesan, R. H. Guymer, and C. D.

Luu, Artif. Organs, 2014, 38, E82–94.

10. M. Amaral, P. S. Gomes, M. A. Lopes, J. D. Santos, R. F. Silva, and M. H.

Fernandes, Acta Biomater., 2009, 5, 755–763.

11. F. R. Kloss, R. Gassner, J. Preiner, A. Ebner, K. Larsson, O. Hächl, T. Tuli, M.

Rasse, D. Moser, K. Laimer, E. A. Nickel, G. Laschober, R. Brunauer, G.

Klima, P. Hinterdorfer, D. Steinmüller-Nethl, and G. Lepperdinger, Biomaterials, 2008, 29, 2433–2442.

12. W. Jakubowski, G. Bartosz, P. Niedzielski, W. Szymanski, and B. Walkowiak, Diam. Relat. Mater., 2004, 13, 1761–1763.

13. L. C. Palmer, C. J. Newcomb, S. R. Kaltz, E. D. Spoerke, and S. I. Stupp, Chem. Rev., 2008, 108, 4754–4783.

14. A. V. Belyakov and A. N. Sukhozhak, Glas. Ceram., 1995, 52, 14–19.

15. R. Chaim, R. Marder-Jaeckel, and J. Z. Shen, Mater. Sci. Eng. A, 2006, 429, 74–78.

16. V. K. Balla, S. Banerjee, S. Bose, and A. Bandyopadhyay, Acta Biomater., 2010, 6, 2329–34.

17. M. Long and H. . Rack, Biomaterials, 1998, 19, 1621–1639.

18. J. J. Klawitter and S. F. Hulbert, J. Biomed. Mater. Res., 1971, 5, 161–229.

19. R. Van Noort, J. Mater. Sci., 1987, 22, 3801–3811.

20. B. Kasemo, J. Prosthet. Dent., 1983, 49, 832–837.

21. T. Kokubo, F. Miyaji, H.-M. Kim, and T. Nakamura, J. Am. Ceram. Soc., 1996, 79, 1127–1129.

22. K. Hayashi, K. Uenoyama, N. Matsuguchi, and Y. Sugioka, J. Biomed. Mater.

Res., 1991, 25, 515–523.

23. M. . Casaletto, G. . Ingo, S. Kaciulis, G. Mattogno, L. Pandolfi, and G. Scavia, Appl. Surf. Sci., 2001, 172, 167–177.

24. F. Zhang, Z. Zheng, Y. Chen, X. Liu, A. Chen, and Z. Jiang, J. Biomed. Mater.

Res., 1998, 42, 128–133.

(37)

37 25. K. Lee, M. Kim, and H. Kim, J. Mater. Chem., 2010, 20, 3791–3798.

26. S. Liu, J. Yu, and M. Jaroniec, Chem. Mater., 2011, 23, 4085–4093.

27. T. Ohno, K. Sarukawa, and M. Matsumura, New J. Chem., 2002, 26, 1167–

1170.

28. J. B. Lowekamp, G. S. Rohrer, P. a M. Hotsenpiller, J. D. Bolt, and W. E.

Farneth, J. Phys. Chem. B, 1998, 102, 7323–7327.

29. F. Lindberg, J. Heinrichs, F. Ericson, P. Thomsen, and H. Engqvist, Biomaterials, 2008, 29, 3317–3323.

30. C. Lindahl, P. Borchardt, J. Lausmaa, W. Xia, and H. Engqvist, J. Mater. Sci.

Mater. Med., 2010, 21, 2743–2749.

31. R. Xin, Y. Leng, J. Chen, and Q. Zhang, Biomaterials, 2005, 26, 6477–6486.

32. T. Kokubo and H. Takadama, Biomaterials, 2006, 27, 2907–2915.

33. Q. Shi, J. Wang, J. Zhang, J. Fan, and G. D. Stucky, Adv. Mater., 2006, 18, 1038–1042.

34. Y. W. Gu, K. A. Khor, and P. Cheang, Biomaterials, 2003, 24, 1603–1611.

35. X. Lu and Y. Leng, Biomaterials, 2005, 26, 1097–1108.

36. P. Zhang, Z. Hong, T. Yu, X. Chen, and X. Jing, Biomaterials, 2009, 30, 58–

37. L. Y. Santiago, R. W. Nowak, J. P. Rubin, and K. G. Marra, Biomaterials, 70.

2006, 27, 2962–2969.

38. H. Q. Nguyen, D. A. Deporter, R. M. Pilliar, N. Valiquette, and R.

Yakubovich, Biomaterials, 2004, 25, 865–876.

39. X. Liu, P. K. Chu, and C. Ding, Mater. Sci. Eng. R Reports, 2004, 47, 49–121.

40. A. Oyane, M. Uchida, C. Choong, J. Triffitt, J. Jones, and A. Ito, Biomaterials, 2005, 26, 2407–2413.

41. L. Gan and R. Pilliar, Biomaterials, 2004, 25, 5303–5312.

42. V. M. Donnelly and A. Kornblit, J. Vac. Sci. Technol. A, 2013, 31, 050825.

43. F. Laermer, Microelectron. Eng., 2003, 67-68, 349–355.

44. R. J. Furlong and J. F. Osborn, J Bone Jt. Surg Br, 1991, 73, 741–745.

45. S. Mann, D. D. Archibald, J. M. Didymus, T. Douglas, B. R. Heywood, F. C.

Meldrum, and N. J. Reeves, Science, 1993, 261, 1286–1292.

46. K. Lilliendahl and J. Solmundsson, Mar. Biol., 2006, 149, 979–990.

47. H. A. Lowenstam, Science, 1981, 211, 1126–1131.

48. R. S. Greco, F. B. Prinz, and R. L. Smith, Nanoscale technology in biological systems, CRC Press, 2005.

49. T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, and T. Yamamuro, J. Biomed.

Mater. Res., 1990, 24, 721–734.

50. R. Apetz and M. P. B. Van Bruggen, J. Am. Ceram. Soc., 2003, 86, 480–486.

51. H. Sudo, H. A. Kodama, Y. Amagai, S. Yamamoto, and S. Kasai, J. Cell Biol., 1983, 96, 191–198.

52. E. M. Czekanska, M. J. Stoddart, J. R. Ralphs, R. G. Richards, and J. S. Hayes, J. Biomed. Mater. Res. - Part A, 2014, 102, 2636–2643.

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Acta Universitatis Upsaliensis

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1345

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology.

(Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)

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urn:nbn:se:uu:diva-277121