Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1046

Cell-protein-material Interactionson Bioceramics and Model

Surfaces

BY

ÅSA ROSENGREN

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

Till de försummade

Nothing in life is to be feared. It is only to be understood.Marie Curie (1867-1934)

List of papers

This thesis is based on the following papers:

I Å. Rosengren, E. Pavlovic, S. Oscarsson, A. Krajewski, A. Ravaglioli and A. Pinacastelli, “Plasma protein adsorption pat-tern on characterized ceramic biomaterials”, Biomaterials, 2002; 23: 1237-1247

II Å. Rosengren, S. Oscarsson, M. Mazzocchi, A. Krajewski and A. Ravaglioli, “Protein adsorption onto two bioactive glass-ceramics”, Biomaterials, 2003; 24: 147-155

III Å. Rosengren, A. Vitina and S. Oscarsson, “Bone cell (MG63) adhesion on silicon wafers pre-coated with ceruloplasmin and prothrombin”, Submitted

IV Å. Rosengren, A. Vitina and S. Oscarsson, “Characterization of adsorbed 2-HS-glycoprotein and 1-antichymotrypsin and their influence on bone cell (MG63) adhesion”, Submitted

I have been responsible for all the planning, experimental work (except mi-crograin preparation, BET-analysis and Hg-porosimetry in paper I-II and cell culturing in paper III-IV), analysis of results and writing of papers I-IV.

Paper I and II are reprinted from [1, 2] copyright (2002, 2003) with permis-sion from Elsevier.

Papers not included in this thesis:

S.M. Barinov, F. Rustichelli, V.P. Orlovskii, A. Lodini, S. Oscarsson, S.A. Firstov, S.V. Tumanov, P. Millet and Å. Rosengren, “Influence of fluorapa-tite minor additions on behavior of hydroxyapatite ceramics”, Journal of Materials Science: Materials in Medicine, 2004; 15: 291-296

Å. Rosengren, S. Oscarsson, A. Krajewski, A. Ravaglioli and A. Piancastelli, "Serum protein adsorption pattern on ceramic biomaterials", In: A. Ravaglioli and A. Krajewski (Eds), Ceramics, cells and tissues: implants for spine, Ravenna: Tipografia Moderna, 1999; 5: 193-197

Å. Rosengren, S. Oscarsson, A. Krajewski, A. Piancastelli and A. Ravaglioli, "Protein adsorption onto biomterials after exposure to human serum", In: A. Ravaglioli and A. Krajewski (Eds), Ceramics, cells and tissues: ceramic-polymer composites, Ravenna: Tipografia Moderna, 1997; 4: 207-211

Contents

Introduction...................................................................................................15Biomaterials .............................................................................................15Biocompatibility.......................................................................................17Host response and wound healing at the biomaterial interface ................18Aim of the present investigation ..............................................................20

Proteins .........................................................................................................21Structure and properties ...........................................................................21Protein adsorption ....................................................................................22

Blood plasma .......................................................................................251-Antichymotrypsin ...........................................................................26

Ceruloplasmin......................................................................................262-HS-Glycoprotein.............................................................................27

Prothrombin .........................................................................................27

Surfaces.........................................................................................................29Bioceramics..............................................................................................29

Alumina and Zirconia ..........................................................................29Hydroxyapatite ....................................................................................30AP40 and RKKP..................................................................................31

Model surfaces .........................................................................................31Silicon and silicon oxide......................................................................31

Cells ..............................................................................................................33Bone cells .................................................................................................33

MG63...................................................................................................34Bone cell adhesion ...................................................................................34

Methods ........................................................................................................37Protein screening ......................................................................................37

Flow-based protein adsorption analysis...............................................37Bicinchoninic acid (BCA) protein assay .............................................39Two-dimensional (2-D) gel electrophoresis ........................................39Western immunoblotting .....................................................................40

Protein characterization............................................................................41Surface preparation..............................................................................41

Atomic force microscopy in tapping mode™......................................41Ellipsometry.........................................................................................43

Protein-cell interactions............................................................................45Cell adhesion test.................................................................................45Cycloheximide treatment.....................................................................46Fluorescence microscopy.....................................................................46

Results and discussion ..................................................................................48Papers I and II ..........................................................................................48Papers III and IV ......................................................................................52

Acknowledgements.......................................................................................57

Summary in Swedish ....................................................................................61Interaktioner mellan celler, proteiner och keramiska ytor........................61

References.....................................................................................................65

Abbreviations

ACT 1-Antichymotrypsin AFM Atomic force microscopy AHSG 2-HS-Glycoprotein AO Acridine orange AP40 Alloplast number 40 Apo D Apolipoprotein D Apo J Apolipoprotein J As Specific surface BCA Biocinchoninic acid BET Brunauer, Emmett and TellerC1 Complement factor 1 C3 Complement factor 3 CaP Calcium phosphate CE Capillary electrophoresis CP Ceruloplasmin Da Dalton DGEA Aspartic acid-Glycine-Glutamic acid-

AlanineDNA Deoxyribonucleic acid ECM Extra cellular matrix G Gibbs free energy H Enthalpy HA Hydroxyapatite Hg Mercury HPLC High-performance liquid chromato-

graphy IEF Isoelectric foccusing IgG Immunoglobulin G MG63 A specific type of osteosarcoma

cell lineMr Relative molecular weight PE Polyethylene PET Polyethylenterephthalate pI Isoelectric point PMMA Polymethylmetacrylate

PMN Polymorphonuclear granulocyte PT Prothrombin PTFE Polytetrafluoroethylene RGD Arginine-Glycine-Aspartic acid RKKP Ravaglioli, Krajewski andPiancastelliRNA Ribonucleic acid S Entropy SDS-PAGE Sodiumdodecylsulphate polyacryl-

amide gel electrophoresis SFM Scanning force microscopy T Absolute temperature TM Trade mark TZP Partially stabilized zirconia

2D-PAGE Two-dimensional polyacrylamide gel electrophoresis

Glossary

Adhesion (cellular) The close adherence (bonding) be-tween cells and (artificial) surfaces or between adjoining cells

Adhesion proteins Extra cellular proteins that enables cell adhesion

Affinity Attraction between matters (here: protein-material or protein-protein)

Antibody Protein produced by B lymphocytes that protects the organism against an antigen

Antigen Substance recognized by specific anti-bodies

Bioactivity Spontaneous interaction between a material and its biological environ-ment leading to a strong bond be-tween surrounding tissue and the material

Biodegradation Material breakdown mediated by the biological system

Biomaterial Substances other than food or drugs contained in therapeutic or diagnostic systems

Biocompatibility Ability of a material to perform with an appropriate host response in a specific application

Ceramics Non-metallic inorganic materials that usually are prepared by high tem-perature sintering

Chromatography Process whereby a (chemical or biol-ogical) mixture carried by a mobile phase is separated into its comp-onents as a result of differential distributions of the components between the mobile phase and a stationary phase.

Composites Solids which contain two or more distinct constituents or phases

Desorbent Solution or gas that disrupts the in-teraction between a surface and an adsorbed substance

Desorption Changing from an adsorbed state on a surface to a gaseous or liquid state

Electrophoresis Migration of charged species (e.g., proteins) in an electrical fields

Encapsulation When an implant is surrounded and walled off from normal tissue by a collagenous, relatively acellular tis-sue (resembles scar tissue)

Epitope Site on an antigen that is recognized by an antibody (typically a short amino acid sequence)

Extra cellular matrix Complex network of polysaccharides and proteins secreted by cells. Serves as a structural element in tissues and influences their development and physiology

Exudation Process by which fluid, proteins and blood cells escape from the vascular system into the injured tissue

Ex vivo Experiments or observations made outside the living body

Granulation tissue Highly vascularized connective tissue rich in collagen

Hemostatic system Biological system that serves to stop the flow of blood

Host response The reaction of a living system to the presence of a material

Hydration Binding of water molecules at an interface

In situ Experiments or observations made in an “original” setting

Integrins Cell adhesion receptors which bind to proteins in the extra cellular matrix

Intercalation Interaction between dyes and DNA/-RNA in which the dye molecule ins-erts itself between two neighboring base pairs of the DNA/RNA double helix

In vitro Experiments or observations made in an artificial setting such as reaction vessels, test tubes, culturing plates

In vivo Experiments or observations made in the living body

Isoelectric point The pH at which a molecule carries no net electrical charge

Leukocytes White blood cells Ligands Specific extracellular signal mole-

cules (typically proteins) Mobile phase (column) Liquid in which a sample is carried Molecular weight The sum of the relative atomic

masses of the constituent atoms of a molecule

Morphology The structural appearance of cells Parenchymal cells Cells from organ tissue Piezoelectric material Crystals that change their linear di-

mensions due to electrostatic stress Phagocytosis A three-step process in which an inj-

urious agent undergoes recognition, attachment of inflammatory cells, engulfment, killing or degradation.

Phenotype The physical and behavioral charact-eristics of an organism e.g., size, shape, metabolic activities and movement

Polymers Long-chain molecules that consist of a number of small repeating units (generally of organic nature)

Probe-broadening (AFM) Tip-shape-induced magnification of imaged features

Proliferation Cellular reproduction through divi-sion

Protease Enzyme that catalyzes the splitting of proteins into smaller fragments by a process known as proteolysis

Receptors Cell membrane bound proteins that bind biologically active compounds and initiate a response in the cell

Regeneration Replacement of injured tissue by parenchymal cells of the same type

Relaxation Time-dependent conformational changes of proteins

Stationary phase (column) Material that will provide retention by virtue of chemical interactions (not reactions) between the comp-onents of the (passing) sample and the material

Steady-state A non-equilibrium state of a system through which matter is flowing and in which all components remain at a constant concentration.

Tissue engineering Production of real tissue from cell culture ex vivo

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Introduction

BiomaterialsMany definitions have been proposed for the term biomaterial. Traditionally, it refers to a materials performance in vivo: …”any substance (other than drug) or combination of substances, synthetic or natural in origin, which can be used for any period of time, as a whole or as a part of a system which treats, augments, or replaces any tissue, organ or function of the body”… [3], but nowadays modern biomaterials science includes materials in biosen-soring, bioelectronics, diagnostics, array technologies as well as materials used for implantation and drug delivery. A broader definition like: …”substances other than food or drugs contained in therapeutic or diagnos-tic systems”… [4], therefore seems more appropriate. Biomaterials constitute a very profitable market. For example, the medical implant industry, on its own, has a total turnover approaching hundred bil-lion US dollars per year [5]. The field includes a wide variety of different materials and applications (table 1) but is by tradition divided into four main categories: metals, ceramics, polymers and composites [8-10].

Table 1. Medical implants used in the USA

Device Number/year Biomaterial (examples)

Intraocular lenses 2 700 000a PMMA Contact lenses 30 000 000a Silicone acrylate, Hydrogel Vascular grafts 250 000a PTFE, PET Hip and knee prostheses 1 285 000b Titanium, PE, Ceramics Catheters 200 000 000a Silicone, Teflon Heart valves 245 000b Treated pig valve Stents (cardiovascular) 1 750 000b Stainless steel Breast implants 250 000c SiliconeDental implants 300 000a Titanium, Ceramics Pacemakers 670 000b Polyurethane Renal dialyzers 16 000 000a CelluloseLeft ventricular assist devices >100 000a Polyurethane aCastner et al [6],bLysaght et al [7], cAmerican society of plastic surgeons, 2003 (www.plasticsurgery.org)

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Metals or rather metal alloys have preferably been used in hard tissue sur-gery (joint prostheses, bone plates/screws, dental implants) due to their ex-cellent mechanical properties but they have also found use in cardiovascular surgery (vascular stents) and other applications (cochlear implants, sutures). Their two major drawbacks are their at times too high mechanical strength, which can lead to a loosening of the implant, and their relatively poor corro-sion resistance which can harm the surrounding tissue and organs. Metallic implants are usually made of stainless steels, cobalt-chromium alloys or titanium alloys.

Ceramics are hard but brittle materials. This inherent brittleness has lim-ited their use in structural applications but they are commonly used in articu-lating devices and dental reconstructions, mainly because of their relative inertness to body fluids, high compressive strength and pleasing aesthetically appearance. Aluminum oxide with its high wear resistance is the most popu-lar ceramic and is used in hip joint replacements. Calcium phosphates and bioactive glasses do not have any good mechanical properties but they have the unique ability to bind to bone and are therefore used to generate bone either as particulates or coatings on metallic prostheses.

Polymers are by far the most widely used implant materials thanks to the great versatility of polymer engineering. They can be made hard, brittle, soft, flexible, inert and/or degradable. They are also relatively light, insulating, cheap and easy to provide. Additives such as plasticizers and anti-oxidants and other contaminants can, however, affect their biocompatibility. One of their most successful applications is in heart surgery where Teflon (PTFE) and Dacron (PET) have been used to replace the interventricular septum of the heart.

Composites are solids which contain two or more distinct constituents or phases (>atomic scale). Natural composites are bone, dentin, cartilage and skin. In designing a biomaterial one of the two constituents is added to en-hance e.g., strength and elasticity, as in fiber reinforced bone cement.

Other materials that are in use but don’t fall into the above mentioned strict groupings are the so-called engineered materials [11]. If metals, ce-ramics, polymers and composites were adopted from other areas of science and technology without any particular redesign, these materials represents a group of materials that are exclusively developed to fit the biological system. Within this group special efforts are made to create materials that have spe-cific, desirable biological interactions with its surroundings.

For example, surfaces that inhibit non-specific interactions (e.g., protein deposition) but carry natural or synthetic biomolecules for biorecognition may be generated through “self-assembly”- and immobilization-techniques; chemically and topographically patterned surfaces that control cell behavior (spatial distribution) and (eventually) cell growth are developed through e.g. micro-contact printing, micro-molding and photo-lithography; and systems

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that release growth factors and other hormones have been fabricated using many different techniques (i.e., microfabrication).

Other interesting new materials are the so-called “smart” materials e.g. shape-memory metals/polymers [12] and smart gels [13, 14]. Shape-memory materials have one shape at one temperature and another shape at a different temperature. This property can enable implantation of a bulky device in a more convenient way. Smart gels do also respond (and typically swell) to triggers such as temperature, but also to pH or specific molecules like anti-gens. The swelling mechanism can be linked to the release specific of drugs in response to for example excess antigen.

The true breakthrough in the biomedical materials field will, however, come with the advancement of tissue engineering [15, 16] (production of real tissue from cell culture ex vivo), but before any real progress can be made in this field we need to learn more about tissue organization, growth stimulus, biochemical signaling, biodegradable scaffolds etc.. Still, today’s biomaterials will continue to be relied upon to relieve human suffering for decades to come.

BiocompatibilityIf the term biomaterial describes the object and the research field, the term biocompatibility relates to the performance of the biomaterial. The term was coined at a conference on definitions in biomaterials in 1987 and was de-fined as follows: ...”the ability of a material to perform with an appropriate host response in a specific application”… [3]. The definition contains two key-expressions: 1) appropriate host response and 2) specific application. Obviously, depending on where the biomaterial is to be installed these two demands can mean very different things. However, there are some general issues that should be addressed. Toxicity: biomaterials should not be toxic unless specifically designed to be so (e.g., drug delivery), Blood compatibil-ity: thrombosis and embolization should be minimized, Inflammation: con-trolled, as opposed to uncontrolled (persistence/degree), inflammation is generally desired, Tissue integration: encapsulation always affects the per-formance of the biomaterial and may eventually lead to explantation, Physi-cal and mechanical properties: failure in any of the two unconditionally leads to unsatisfying clinical success, thus the material can appear as non-biocompatible, Site of implantation: depending on the circumstances, many biomaterials are in contact with more than one type of tissue or biological environment.

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Host response and wound healing at the biomaterial interfaceAs soon as a material is placed within the body it is covered in blood due to the incision made in the surrounding tissue. The blood-material interaction starts a series of biological reactions (host response [17-19]) (Figure 1) that in the best case lead to successful integration of the implant but in the worst case lead to a significant encapsulation.

The first event that takes place (nanoseconds) at the implant-tissue inter-face is that water molecules and salt ions reach the material surface. The distribution of these molecules and ions is highly surface dependent and is very important for proteins and other molecules that arrive later. For exam-ple, it may determine if proteins change their conformation, orientation, and coverage etc.

Shortly after the formation of the hydration layer (seconds), blood pro-teins and other macromolecules (lipids, sugars etc.) start to crowd the sur-face. Since blood contains several hundreds of different proteins, there will be a competition for the surface between the different species, resulting in unique protein mixtures at the interface. The adsorption of blood proteins is an important part of the body’s defense system as well as its hemostatic sys-tem since both rely on activation of normally inactive proteins following surface contact. This activation, which proceeds through a “cascade” of pro-tein reactions, provides signals to the surroundings that attract and activate cells such as platelets and leukocytes (white bloods cells).

Platelets and leukocytes are among the first arriving cells (hour) and serve to arrest bleeding and to “clean” and remove “dangerous” agents from the site of injury, respectively. Platelets emerge at the implant site mainly through the blood flow of injured blood vessels while leukocytes appear through exudation: referring to the migration of white blood cells (predomi-nately neutrophils or PMNs), proteins and other mediators of inflammation (e.g., serotonin, histamine) from intact blood vessels. Exudation is preceded by changes in vascular flow, caliber and wall permeability, and is stimulated by products released upon injury.

When the cells reach the implant site, they start to scan the protein cov-ered surface and its vicinity, looking for activation factors and places to at-tach to. For example, platelets preferentially attach to tissue elements such as collagen and von Willebrand factor and, after activation, to blood proteins such as fibrinogen, fibronectin and vitronectin. Neutrophils attach to i.a., immunoglobulin G (IgG) and complement-activated fragment C3b; both originally coming from blood.

Following attachment, a complex series of cell reactions is initiated (e.g., release of different active substances) which in the platelet case eventually results in formation of a clot or thrombus, in the neutrophil case leads to phagocytosis and in both cases encourage further cell recruitment and

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Figure 1. A simplified schematic view, showing possible events at the implant-tissue interface (Reprinted from [20] copyright (1991), with permission from Uni-versity of Toronto Press

activation. Phagocytosis is a process whereby inflammatory cells try to en-gulf, kill or degrade injurious agents.

Whilst neutrophils dominate in the first few hours of the inflammatory re-sponse, other phagocytic cells such as macrophages increase their presence within a day or so. Like the neutrophils, they will try to engulf the implant, but owing to the disparity in size between the biomaterial surface and the attached cell, success is limited and as a result frustrated phagocytosis may occur. This means that the cell opens up and releases reactive oxygen species and enzymes (mainly lysozyme); leaving behind cell and protein debris. Furthermore, the macrophages can fuse into multinuclear foreign body giant cells and thereby increase the inflammatory response further. Persistent in-flammatory stimuli caused by unfavorable chemical and physical biomaterial properties as well as motion at the implant site, lead to chronic inflamma-tion.

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The repair of the implant site starts in parallel with the inflammatory activi-ties. In most cases this means formation of a highly vascularized connective tissue (granulation tissue; 3-5 days) that matures into a collagen-rich fibrous tissue (>4 weeks). The collagen-rich fibrous tissue would in a normal wound (without an implant) be remodeled with time but the continuing presence of an implant prevents or perturbs this action.

The other, more unusual route of healing is replacement of the injured tis-sue by parenchymal cells of the same type (termed regeneration). This is seen when certain calcium phosphate materials, bioglasses and titanium are implanted into bone.

It is not clear what turns the balance towards encapsulation or regenera-tion but the direction taken is highly affected by (1) the in situ conditions, referring to the proliferative capacity of cells in the tissue or organ receiving the implant, the extent of injury and the persistence of the tissue framework of the implant site; (2) the biomaterial properties, i.e. the size, shape, topog-raphy, chemistry, physics and mechanics of the implant; (3) the surgical technique, including fitting of the implant (possible movement) and risk of infection; and (4) systemic factors, e.g. age, sex, pharmaceutical regime and general health status [17, 19].

Aim of the present investigation Why do some materials integrate in body tissue while others don’t? It is my personal belief that part of the solution to successful tissue integration lays in the layer of proteins that inevitably forms at the material-liquid interface. The body uses very specific signal pathways to heal wounds, but in an im-plant situation it can not handle the complex signals that derive from the non-specifically adsorbed protein layer and it therefore treats the implant as a foreign invader. The challenge is to design materials that attract specific (correct) proteins, in appropriate conformation and orientation, and thereby trigger normal wound healing and allow tissue integration. The protein re-cruitment can be either spontaneous or directed by surface modifications.

By studying the spontaneous adsorption of plasma proteins on bioceram-ics that experience different levels of tissue integration, I have tried to iden-tify such differences in the protein surface film that may herald long term clinical success or failure. To further examine the impact of specific proteins on tissue integration I selected some of the proteins that behaved differently and studied their interactions with a model surface and their influence on bone cell adhesion as a result of the adsorption process.

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Proteins

Proteins are basic constituents in all living organisms. Their central role in biological structures and functioning was recognized by chemists in the early 19th century when they coined the name for these substances from the Greek word proteios, meaning "of the first rank". Even though proteins are the major component of dry material of living organisms, their importance is related to their function rather than to their amount in tissue They play cru-cial roles in enzymatic catalysis, transport and storage, coordinated motion, mechanical support, immune protection, generation and transmission of nerve impulses, and control of growth and differentiation.

Structure and properties The basic structural units in proteins are called amino acids. They are linked together by peptide bonds forming polypeptide chains. One protein molecule consists of one or more polypeptide chains, each chain containing from fifty up to several thousand amino acids. There are 20 different types of amino acids in the body ranging from charged, polar to hydrophobic. It is the unique combination of amino acids that in the end determines the three di-mensional structure (conformation) of the protein molecule thereby its func-tion.

The three dimensional structure can be divided in to several levels of ar-chitecture [21, 22]. The order in which the amino acids are linked together by covalent bonds is called the primary structure. The secondary structurerefers to the folding of the polypeptide chain into periodic structures such as

helices and sheets. The folding relies on the hydrogen bonding between amide- and carbonyl groups in the main chain. The secondary structures arrange themselves into motifs and domains (tertiary structures), held to-gether by hydrogen bonding, hydrophobic-, electrostatic- and dipolar inter-actions, and sometimes disulfide bridges. A motif is a minor spatial ar-rangement formed by packing side chains from adjacent helices and sheets close to each other and a domain is a compact globular region com-posed of several motifs and secondary structures. The quaternary structureis the non-covalent association of independent tertiary structures.

To add further versatility and flexibility to the protein structure, most pro-teins carry sugars, nucleotides and/or lipids. These are covalently bonded but

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may vary with the biological cycle and state of health. That is: the same pro-tein can carry different sugars depending on the current circumstances.

The combinations seem endless and indeed more than 1 400 000 protein sequences (160 000 with a high level of annotation) have been determined up to date (www.expasy.org/sprot/). Nevertheless, proteins, as a group, have some general properties that is; they are amphiphilic and polyampholytes and in aqueous environments (biological fluids) they tend to locate their hydrophobic amino acid residues in the interior of the molecule leaving their polar residues on the surface.

Protein adsorption As soon as a solid surface comes in contact with a protein-containing (aque-ous) solution it is inevitably and spontaneously covered by proteins at the solid-liquid interface, thereby changing the properties of both the sorbent surface and in most cases the protein molecules.

The fundamental of a spontaneous adsorption process is that more energy is released (lowering the energy content in the system) than gained according to Gibbs law of free energy (at constant pressure and temperature):

adsG = adsH –T adsS < 0 (1)

where ads refers to the net change of the thermodynamical parameters: G(Gibbs free energy), H (enthalpy), S (entropy), and T (absolute temperature), as a result of the adsorption. Enthalpy (heat of reaction) refers to the making and breaking of chemical bonds; making bonds is energetically favorable ( H<0), and entropy refers to the free energy gain ( S>0) when going from order to disorder. According to the equation (1) adsG must be negative for the adsorption to be spontaneous.

The main contributions to the enthalpy and entropy of protein adsorption in aqueous environments are according to Norde [23] (Figure 2): (a) electro-static interactions between protein and sorbent surface and possible co-adsorption of ions; the optimal being charge neutralization without ion in-corporation (b) dispersion interactions: always attractive but dropping with decreasing molecular size and increasing distance between sorbent and pro-tein; (c) dehydration (or changes in the state of hydration) of the sorbent surface and parts of the protein molecule: even though proteins tend to bury their hydrophobic amino acid residues in the interior of the molecule up to 40-50% of the apolar structures can be available to water (entropically unfa-vorable), thus in the case of hydrophobic surfaces much is gained by making surface-protein contacts rather than protein-water contacts; (d) structuralrearrangements in the protein: can contribute positively to both entropy and enthalpy through decrease in ordered secondary structures and increase in

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Figure 2. Schematic view of a protein at a well-characterized surface

intra-molecular hydrogen bonding. The course of action is dependent on the polarity of the surface and the rigidity of the protein.

Apparently, no single factor can be used to predict the process of adsorp-tion. In fact they all contribute and are interdependent, being synergistic or antagonistic (Figure 3). However, dehydration and structural rearrangements are considered to be the dominating contributions.

The strength and nature of the above-mentioned interactions can affect the function of the protein by changing its native conformation and by orienting the protein in an unfavorable way on the surface (Figure 4). The conforma-tional changes often occur immediately upon adsorption, but time-dependent conformational changes (relaxation) are also evident. The relaxation nor-mally involves flattening (spreading) of the protein as the molecule strives to optimize its contact with the sorbent. The effect is more pronounced at low surface coverage than at high since then the lateral interactions between ad-sorbed molecules are negligible. This may lead to a structural heterogeneity in the adsorbed protein layer: the later-arriving molecules not finding enough space to relax on, hence keeping a more native-like structure up on adsorp-tion than the early-arriving molecules.

Another consequence is that relaxed proteins are less prone to detach from the surface thus affecting the reversibility of the adsorption process. In addition, if the proteins become detached they do not necessarily retrieve their native configurations thereby causing an adverse host reaction.

A third consequence concerns the amount of adsorbed proteins. At higher rates of attachment (due to high protein flux or high protein concentrations), larger amounts of proteins adsorb since there is less time available for re-laxation, in turn resulting in a more crowded surface. In general proteins form monolayers or sub-monolayers but di- and multilayers have also been reported [24].

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Figure 3. Interdependency of the major subprocesses that are involved in the overall protein adsorption process. Adsorption-promotion is denoted by a + sign and adsorp-tion-opposition by a – sign. (Reproduced with permission from Prof W Norde)

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Adsorption from protein mixtures is by far more complicated but obviously more relevant to the “real” biological situation. As a rule, proteins compete with each other (and other surface-active components) for available binding sites on the sorbent surface. An accepted theory is that the molecules with the highest arrival rate (e.i., small and/or abundant molecules) cover the sur-face first but are gradually displaced by molecules with higher affinity for the surface. A milestone was laid down by Vroman et al [25] who showed that the time course of adsorption is such that fibrinogen passes through an adsorption maximum eventually to be displaced by higher molar mass com-pounds. The phenomenon is referred to as the “Vroman effect” [26].

Figure 4. Schematic view, showing possible orientations and conformations of a protein at an interface

Blood plasma Plasma is the pale yellow liquid portion of blood that remains after the cells have been removed. Plasma constitutes 55% of the total blood volume and is composed of 91% water, 7% proteins, 0.95% minerals, 0.80% fats and 0.25% “extractives” (glucose, urea etc.) [27]. Plasma contains several hun-dreds of different proteins (and peptides) [28, 29] including, transport pro-teins (albumin, transferrin, and haptoglobin), coagulation factors (fibrino-gen), complement components (C1, C3), immunoglobulins (IgG), enzyme inhibitors, precursors of substances (angiotensin and bradykinin), and many others [30]. The relative protein abundances span at least 12 orders of mag-nitude, albumin (60%), globulins (36%) and fibrinogen (4%) being the major constituents.

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1-Antichymotrypsin1-Antichymotrypsin (ACT) is a negatively charged glycoprotein (Mr: 64-68

kDa) whose circulating level can increase 4-5 fold during inflammation [31-33]. ACT belongs to a large family of structural homologues, the serpins (serine protease inhibitor), whose three-dimensional globular structure is characterized by three -sheets (A, B and C) surrounded by nine -helices(A-I) (Figure 5) [34, 35]. It is an efficient inhibitor of chymotrypsin-like proteases [36, 37] and deficiency is associated with emphysema [38, 39] and chronic obstructive pulmonary disease [40]. The binding of ACT to the amy-loid- peptide and subsequent deposition of this complex in amyloid plaque deposits has been linked to progression of Alzheimer’s disease [41, 42].

Figure 5. Model of 1-antichymotrypsin (Reprinted from [43] copyright (1996), with permission from Elsevier)

Ceruloplasmin Human ceruloplasmin (CP or the sky-blue protein) is a copper-containing acute phase glycoprotein with a molecular weight of ~132 kDa (Figure 6). It consists of a single polypeptide chain arranged in six -barrel domains, which gives the protein a triangular symmetry [44]. The complete function of CP is not fully understood but it has at least four attributes: ferroxidase activity, antioxidant activity, copper transport and amine oxidase activity [45, 46]. Deficiency, as in Wilson’s disease, leads to deposition of copper in tissue, and eventually to death. CP in solution tends to bind leukocytes, erythrocytes, fibroblasts and endothelial cells [47], but after adsorption that property seems lost [48, 49].

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Figure 6. Model of ceruloplasmin (Reprinted from [44], copyright (1996), with permission from Elsevier)

2-HS-Glycoprotein 2-HS (Hermans-Schmid) glycoprotein (AHSG or human fetuin) is a nega-

tively charged sialylated disulfide-linked dimer with a molecular weight of 60 kDa (Figure 7) It belongs to a group of structurally well-preserved pro-

teins: the cystatin superfamily (PROSITE Database [50]: www.expasy.ch)but its full three-dimensional structure is so far not known. It contains bind-ing sites for Ca2+ and calcium apatite [51], it is selectively concentrated in bone and teeth [52], it is an effective inhibitor of calcification [53], and lack of AHSG results in severe systematic calcification in mice and humans [54]. AHSG has also been implicated in other biological process such as the im-mune response, insulin signaling, brain development, and transport of lipids [55-57]. Furthermore, it contains binding sites for TGF- like growth factors [58] and lectins [53].

Figure 7. Model of 2-HS-glycoprotein (Reprinted from [53] copyright (2001), with permission from Elsevier)

Prothrombin Prothrombin (PT or Factor II) is a vitamin-K dependent coagulation factor which in its active form, -thrombin, induces fibrin formation and platelet aggregation. The acidic single-chain glycoprotein (Mr: ~72 kDa) has a shape of a psuedoplanar “drop” [59, 60] and exists in at least eight apo-structures

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and cleavage products in the body [61] (Figure 8). Apart from its main func-tion to support the body with mature -thrombin, PT seems to play a role in bone mineralization [62, 63] and invasion/metastasis in bone [64, 65]. It supports cell adhesion of platelets, granulocytes, endothelial and smooth muscle cells, probably through RGD-mediated recognition [66-68].

Figure 8. Model of prothrombin (Reprinted from [59] copyright (1997), with per-mission from Elsevier)

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Surfaces

The surfaces used in the thesis are all ceramic in nature. The word ceramic comes from the Greek word keramos meaning “pottery” [69]. Ceramics are generally defined as inorganic materials having non-metallic properties. They are typically crystalline (but do include glassy structures) and are com-pounds formed between metallic and non-metallic elements such as alumi-num and oxygen (Al2O3-Alumina) and silicon and nitrogen (Si3N4-Siliconnitride). Classical properties are hardness, brittleness and chemical inertness. They are also thermal and electrically insulating, refractory, nonmagnetic, and heat and wear resistant; there are, of course, many exceptions to these generalizations.

In this thesis, traditional bioceramics were chosen to monitor protein ad-sorption behavior at “real” implant-like interfaces, and model surfaces were chosen to study the structural appearance and functionality of proteins as a result of the adsorption process.

BioceramicsCeramics that are used to augment or replace diseased, damaged or ageing tissues in the body are termed bioceramics. Most applications are in hard tissue surgery (replacements for hips, knees and teeth, maxillofacial recon-struction, otolaryngological implants etc.) but they are also finding use as heart valves, artificial tendons and ligaments, and more recently as scaffolds for tissue engineering and carriers in drug delivery systems.

According to Hench [70] and Billotte [71], bioceramics can be classified as (1) non-absorbable (relatively inert), (2) bioactive or surface reactive (semi-inert) and (3) biodegradable or resorbable (non-inert).

Alumina and Zirconia Alumina (Al2O3) and zirconia (ZrO2) are classic examples of relatively inert bioceramics, meaning that they undergo little or no chemical change during long-term exposure to the physiological environment. As such they do not cause any local or adverse systemic reactions but nor do they form any close bonds with surrounding tissues. The tissue response is (at least at early stages) characterized by the formation of a fibrous (but thin) capsule around

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the implant. The development of fibrous tissue is preceded by complex se-ries of protein reactions at the interface e.g. complement activation. Inert implants are attached to the physiological system through mechanical inter-locking (morphological fixation) or by tissue ingrowth into pores or equiva-lent (biological fixation) [70] .

The alumina used in this thesis is a high-purity alpha polycrystalline ma-terial and the zirconia is a partially yttria (Y2O3)-stabilized tetragonal poly-crystal (TZP) [72]. In aqueous environments, both surfaces become hydrated followed by dissociation of surface hydroxides [73]. These surface hydroxyl groups may ionize into Brønsted acids or bases and at physiological pH:s, alumina carries a weak positive net charge while zirconia carries a small negative net charge [73, 74]. As sintered bodies, zirconia has better me-chanical properties than alumina (fracture toughness, strength and elasticity) but alumina has superior tribological properties (friction and wear) [75, 76]. They are typically used as structural-supports and in applications where low friction is needed (e.g., joint prostheses).

HydroxyapatiteSynthetic (calcium)hydroxyapatite (Ca10(PO4)6(OH)2 or HA) is the manmade analogue of the mineral phase of bone. In contrast to “inert” materials, hy-droxyapatite develops a direct, adherent and strong bonding with (bone)tissue [70]. This property, termed bioactivity, refers to the ability to form a carbonatehydroxyapatite layer at the material surface. This material subsequently recruits bone producing cells (osteoblasts) which differentiate and produce bone matrix on the newly formed apatite [77]. The carbonate-hydroxyapatite synthesis is different for different bioactive materials. On CaP (calcium-phosphate)-surfaces it involves cell-mediated dissolution and precipitation processes [78]. It is not fully understood how blood proteins affect the carbonate-hydroxyapatite formation (and subsequent bone forma-tion) but they seem to slow down the solution induced-surface transforma-tion for some CaP-materials [79, 80].

Hydroxyapatite can be made porous/ dense, resorbable/solid and it can be produced in powders/blocks. The dissolution rate depends on the material’s crystal size, particle size, specific area, composition and substitution. Due to its poor mechanical properties, it is preferentially used as filler, coating, unloaded implant, composite or scaffold.

We used a polycrystalline hydroxyapatite substituted with 5% HPO42- and

2% CO32-, yielding a Ca/P molar ratio of 1.58. The granules were slightly

positively charged at pH: 7.4 (phosphate buffer) [74].

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AP40 and RKKP The two glass-ceramics AP40 (alloplast n:o 40) and RKKP (after Ravaglioli,Krajewski and Piancastelli) do also belong to the family of bioactive materi-als. However, being silicon oxide based systems they form carbonatehy-droxyapatite through a different mechanism than CaP-systems. The forma-tion includes surface dissolution, hydrogel formation (hydrated silica rich in Si-OH) and subsequent calcium phosphate deposition [77].

AP40 and RKKP are SiO2-CaO-Na2O-P2O5-systems which have been supplemented with MgO, K2O, and CaF2 to increase their bioactivity and resistance towards degradation [81]. RKKP was further supplemented with 0.5% La2O3 to improve its resistance towards alkali and 1% Ta2O5 to reduce the number of defects inside the glassy body. The lanthanum- and tantalum cations stabilize the molecular network of the glass-ceramics acting as am-photeres (both network former and modifier) and consequently induce the glassy system to be more refractory [81].

In contrast to completely amorphous glass, the glass-ceramics contain both amorphous and crystalline phases. The latter is an incorporation of <8% of hydroxyapatite crystals. The RKKP and AP40 materials are positively respectively negatively charged at physiological pH [82].

Model surfaces Since one part of this thesis deals with the functional behavior of protein as a consequence of the adsorption process we need to identify surfaces and analysis techniques that (1) permit characterization of proteins at the inter-face (in terms of e.g. conformation, orientation, layer formation) and (2) single out the protein-induced effects on for example cell activity stemming from a reduction of interfering surface properties (e.g. topography, variations in surface chemistry). These demands can not be satisfied for “real” implant surfaces since available techniques are unable to resolve such complex envi-ronments. By using more simple models with well-characterized surfaces (chemistry and topography) together with a limited number of proteins, de-tails in biological processes that normally are hidden can be monitored. In this work atomic force microscopy and ellipsometry were used for those protein characterization efforts which require surfaces of low surface rough-ness and optical reflectance.

Silicon and silicon oxide The silicon-silicon oxide (Si-SiOx) structure is one of the most intensively studied systems due to silicon’s critical role in the semiconductor industry [83]. In air, the silicon wafer consists of a bulk phase with silicon atoms

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arranged in a zinc-blende structure and a thin surface region made up of amorphous silicon oxide [84]. The native oxide attracts water and is there-fore fully hydroxylated (e.g., Si–OH) at room temperature [85]. Another consequence is that the outermost layer of the surface consists of structured water even at ambient conditions [85]. The thickness and morphology of the native oxide layer can be manipulated by different cleaning procedures [84] and by immersion in water. Water exposure enables further hydroxylation producing a “silica” gel (network of –Si(OH)2-O-Si(OH)2-OH) [86]. The silica surface is negatively charged under physiological conditions (pI: 2 -3.7) [87].

The silicon wafer surface used in this study was polished, n-doped and had a native oxide layer of 1.4 nm with a roughness of 2-3 Å.

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Cells

One way to test the function of proteins is to study their interactions with cells. Cells are programmed during development to respond to specific sets of signals (often in the form of proteins or peptides) that regulate the behav-ior of the cell and determine whether the cell lives or dies and whether it proliferates (multiplies by division) or stays quiescent. The cellular response is a function of the ability of surface receptors (proteins in the cell mem-brane) to recognize and bind the signaling molecules (ligands). The attach-ment involves recognition of specific epitopes (typically a minor amino acid sequence) on the protein or protein-bound sugars, lipids or nucleotides. These recognition sites might become exposed or hidden upon protein ad-sorption to surfaces thereby altering the function of the protein. For example proteins that normally don’t bind cells in solution can become activated and vice versa.

Bone cells Bone contains two distinct cell types, the osteoblasts, or bone-forming cells, and osteoclasts, or bone-resorbing cells. Together they are responsible for the continuous and essential remodeling of bone. They are equally important from a biomaterial and implantation point of view but since the thesis deals with interactions between proteins and osteoblast-like cells (MG63), only a short introduction of the osteoblastic cell type [88, 89] will be provided.

Osteoblasts (Figure 9) are derived from mesenchymal osteoprogenitor (or stem) cells found in bone marrow and periosteum [90]. They are located on the surface of bone where they are involved in bone deposition through se-cretion of organic components of bone matrix. The phenotypical characteris-tics include: synthesis and expression of collagen type I, bone sialoprotein, osteopontin, osteonectin, osteocalcin, and cell membrane enzymes and re-ceptors such as alkaline phosphatase (ALP) and vitamin D receptor [91]. After fulfilling their secretory activity, osteoblasts undergo either apoptosis or terminal differentiation to osteocytes surrounded by bone matrix. Os-teoblasts indirectly regulate bone mineralization and osteoclast differentia-tion/activity by providing regulating factors.

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Figure 9. SEM micrograph of osteoblasts residing on alumina (Reproduced with permission from Dr M Karlsson)

MG63 Since human-derived osteoblasts as a group are quite heterogeneous due to different stages of maturation, it can be advantageous to work with well-characterized osteoblast surrogates. A popular cloned osteoblast-like cell line of tumor origin is MG63 [92, 93]. According to Clover et al [92], the MG63 cell line is an appropriate model for studying cell adhesion and expression of cell adhesion receptors (integrins). In addition, MG63 cells are suitable for investigating regulation and production of osteocalcin but they are not repre-sentative for studying proliferation and alkaline phosphatase activity.

Bone cell adhesion To be able to deposit bone matrix in a controlled way at an implant interface, the osteoblasts need to attach and spread, or more commonly adhere, to the surface [94, 95]. Osteoblasts, as other cells, are surface sensitive creatures that don’t survive on naked materials. However in vivo, the materials are conditioned with proteins (and other biological molecules) from the sur-roundings (typically from blood) and with proteins secreted by cells (part of the extra-cellular matrix or ECM). Some of these proteins, called adhesionproteins, can then serve as ligands for specific adhesion receptors on the cell surface thereby providing points of anchorage as well as inducing internal signaling pathways leading to cell cycle progression and phenotype (physical and behavioral characteristics of the organism) expression.

The major cell adhesion receptors involved in adhesion to biomaterials (in contrast to cell-cell adhesion) are the integrins [96, 97]. Integrins are trans-

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membrane heterodimeric proteins consisting of non-covalently associated and sub-units. They exist in 24 varieties of which many have mutual and /or multiple ligands. The extracellular part of the integrin, the ligand-binding domain, contains multiple divalent cation binding sites which, when bound to cations, have a profound influence on the integrin function. The cations (Mn2+, Mg2+ and Ca2+) stabilize the structure of the integrin and coordinate the direct binding of ligands [98-100]. The coordination of ligand binding involves specific pairing of the and sub-units with aspartate(D)- or glu-tamate(E)- containing sequences in ligands via a carboxyl divalent cation coordination complex [101].

Mn2+ and Mg2+ generally promote firm adhesion whereas the influence of Ca2+ is concentration dependent. Low concentrations ( 0.1 mM) mediates weak adhesion (e.g., platelet rolling) whereas high (1-10 mM) inhibits adhe-sion [98, 102]. The physiological role of this metal dependent regulation is unclear especially during bone replacement when large amounts of Ca2+ are released.

The extracellular domain exists in an “open” and a “closed” conforma-tion; the open configuration commonly thought of as being available for ligand binding (Figure 10). The switch from closed to open (and vice versa) is normally triggered by stimuli received by other receptors resulting in in-tracellular modulation of the integrin’s cytoplasmic and tails thereby altering the conformation of the extracellular domain (inside-out signaling)[100, 103, 104]. The inside-out signaling is particularly important for circu-lating blood cells since their integrins usually needs to be activated before they can mediate adhesion whereas tissue cells, such as osteoblasts, normally maintain their integrins in an “open” state ready for adhesion. The exception is tissue cell migration which requires occasional de-activation of the ligand-binding ability.

Figure 10. Model for integrin activation (Reproduced with permission from Prof. S Johansson)

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Typical ligands for osteoblastic integrins are fibronectin, vitronectin, laminin and collagen [105] which provide specific aspartate(D) and glutamate(E) containing sequences such as RGD and DGEA. Ligand-binding does not necessarily involve interactions with just one recognition motif on the ligand; two synergistic sites are required for strong binding of the 5 1 in-tegrin to fibronectin [106].

Apart from being points of adhesion, ligands are signaling proteins that inform the cell about the state of its surroundings. Depending on the signal-ing strength and persistence (type, amount and combination of binding ligands) the cell responds via various downstream cellular activities resulting in focal contacts, spreading, migration, activation of growth factor receptors (crosstalk), and gene expression leading to differentiation and synthesis of matrix proteins, metalloproteases and receptors.

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Methods

Protein screening A conventional flow system together with bicinchoninic acid (BCA) protein assay, two-dimensional (2-D) gel electrophoresis and Western immunoblot-ting was used to probe preferential adsorption of proteins from biological solutions onto different biomaterials (Figure 11).

Flow-based protein adsorption analysis The principle of flow-based adsorption analysis is based on the dynamic interaction of proteins with adsorbing surfaces under continuous flow.

The experimental model relies on a traditional chromatographic set-up consisting of a mobile phase and a stationary phase [107]. The stationary phase, the biomaterial, is packed into a glass column which is connected to a pump system thereby allowing a flow of mobile phase over the material sur-face. The sample, human blood plasma, is injected onto the column and the proteins are captured by the biomaterial. Proteins that do not possess any affinity for the biomaterial will either pass directly through the column or be eluted by a low-stringency washing step. The bound proteins are then recov-ered by washing the column with a solution that disrupts the interaction be-tween the protein and the biomaterial (desorption). The proteins in the flow-through and the recovery liquids are readily collected in fractions for further analysis.

Total protein binding capacity The total protein binding capacity of the individual materials was calculated from equation (2):

Total binding capacity (mg/m2) = (mprotein in – mprotein out)/As = mads/As (2)

where the amount of adsorbed plasma proteins was obtained by measuring the protein content in the flow-through during the injection phase and the low-stringency washing step, and subtracting it from the applied protein amount. The protein content was measured by BCA protein assay and the specific surface area, As, was determined by the BET method and Hg-porosimetry [108].

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Figure 11. Schematic view showing the instrumental setup used for protein screen-ing

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Dynamic binding capacity The dynamic binding capacity, which is a relative measurement of available protein binding sites between different materials, was determined by com-paring the flow-through volumes at the 50% saturation point of the break-through curves between the different materials.

Relative binding capacity for individual proteins The relative binding capacity study is a purely qualitative comparison be-tween materials regarding the presence of individual proteins in the flow-through. It is evaluated by identifying the proteins in the flow-through at 25%, 50% and 75% of saturation i.e., when 25%, 50% and 75% of available protein binding sites have been occupied. The proteins were identified by 2-D gel electrophoresis.

Bicinchoninic acid (BCA) protein assay The BCA assay [109] is a coloring assay that is used for protein quantifica-tion. The method is based on the Biuret reaction during which Cu2+ is re-duced to Cu+ by peptide bonds in proteins under alkaline conditions. The Cu2+ is then detected by a chelating reaction with BCA yielding an intense purple color. The production of Cu+ is a function of protein concentration and incubation time allowing the protein content of unknown samples to be determined spectrophotometrically by comparison with known standards. The advantage of BCA assay over other assays is that it is relatively insensi-tive to detergents and denaturing agents. It is, however, more sensitive to sugars.

Two-dimensional (2-D) gel electrophoresis 2-D gel electrophoresis, first introduced by O’Farrell [110] and Klose [111], is a powerful and widely used method for the identification and analysis of proteins in complex protein mixtures (Figure 12). The technique, as all elec-trophoresis techniques, relies on the migration of charged species (e.g., pro-teins) in electrical fields. The proteins are sorted according to two independ-ent properties in two steps: the first dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second dimension step, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weight (Mr,relative molecular weight). Each spot on the resulting 2-D array (Figure 13) corresponds to a single protein species in the sample. Thousands of different proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein is obtained.

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A normal 2-D protocol involves thorough sample solubilization with highly concentrated urea, nonionic detergent, carrier ampholytes and reducing agent, IEF under denaturing conditions in a thin, nonsieving gel with an im-mobilized pH gradient, SDS-PAGE in homogenous or gradient slab gels and protein staining with silver or Coomassie Blue™ followed by evaluation with an appropriate image evaluation software [112, 113].

In addition to its ability to resolve thousands of proteins, the technique al-lows rapid comparison of protein composition in different samples, it does not require any protein labeling which could modify the protein structure, it enables detection of post- and co-translational protein modifications which can’t be predicted from the genome sequence and it is relatively economical compared to high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The practical limitations include poor solubility of hydrophobic and membrane proteins, narrow dynamic range, difficulty in focusing highly basic and acidic proteins, inadequate sensitivity, poor quan-titation and it is also quite labor-intense. Nevertheless it remains the tech-nique of choice for separation of proteins in complex biological samples. Recently 2-D gel electrophoresis has experienced an enormous upswing when combined with mass spectrometry for sensitive and thorough protein identification.

Western immunoblotting Immunoblotting [114] provides a simple and effective method for identify-ing antigens (proteins recognized by specific antibodies) in a complex mix-ture of proteins. Initially, the constituent proteins are separated by SDS-PAGE, or similar technique, and thereafter transferred electrophoretically or by diffusion onto a nitrocellulose filter. The immobilized proteins can then be identified using antibodies that bind the protein of interest and subsequent visualization of the resulting antibody-antigen complex. The visualization in this study relies on binding of an additional secondary antibody conjugated with a marker enzyme. The activity of the enzyme marker results in colored insoluble product when incubated with an appropriate chromogenic sub-strate. Immunoblotting is more sensitive than 2D-gel electrophoresis and is therefore used for identification of proteins in low concentrations.

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Protein characterization Atomic force microscopy (AFM) was used to study the orientation and con-formational changes of single proteins after adsorption on model surfaces. Ellipsometry was used to monitor the formation of protein layers during adsorption.

Surface preparation Surfaces are readily contaminated during regular storing. The contaminants occur in essentially three forms: (1) hydrocarbons, (2) metals, and (3) parti-cles (macrocolloids, dust, inorganic debris) [115].

In the case of silicon wafers, the contaminants are generally removed with wet chemical cleaning such as etching in “piranha” solution (2:1 H2SO4/H2O2) [116, 117]. The organic contaminants are removed by the oxi-dizing agent (H2O2) and the metals by the acid (H2SO4). The acidic and oxi-dative conditions increase the number of hydroxyl groups on the surface andenhance the growth of the natural oxide layer yielding increased surface roughness. There have been reports on possible sulfur contamination on silica after piranha treatment [118].

The last steps in cleaning are rinsing and drying which should be done in ultrapure (low in carbon) deionised water and by physical removal (e.g., flow of N2), respectively.

Atomic force microscopy in tapping mode™ Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a non-optical method that employs mechanical sensing to image the topogra-phy of solid surfaces [119]. A sharp tip mounted at the end of a flexible can-tilever is used to raster-scan the sample surface, registering the interaction force (typically in the order of 10-10 N for biomolecular interactions) between tip and sample at each position [120] (Figure 12). During tapping mode™, this means registering the amplitude damping of an oscillating cantilever as it touches the surface [121]. The damping is monitored by an optical detector that measures the variation of the point of incidence of a laser beam reflected from the backside of the cantilever. The optical signal alerts a feed-back system which controls a piezoelectric positioner (piezo) on which the sample rests. The piezo either raises or lowers the sample (relative to the tip) to maintain a constant amplitude of the oscillating cantilever and thereby can-cel any changes in the tip-sample interaction. The movement of the piezo, or more precisely the applied voltage, is proportional to the height of the sam-ple and is used to reconstruct a pseudo-three-dimensional profile of the sam-ple.

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General guidelines for protein imaging are to use atomically flat surfaces and sharp tips with high aspect ratios [120]. The reason for the former is quite obvious since it is a prerequisite for the tip to “see” the target molecules, and nothing but those molecules. The reason for the latter is that the tip imposes itself on the image making every image a convolution of the actual molecule topography and the shape of the tip.

Figure 12. Principle of the AFM (Reproduced with permission from Dr E Pavlovic)

At the worst this will generate a false appearance of the imaged object (Fig-ure 13) or at the best a slight magnification. The latter always occurs and is known as probe-broadening [120]. There are methods to remove the “tip effect” but these are usually quite time-consuming and require knowledge about the tip-shape [122-124].The displacement of the tip in the z direction is not affected by probe-broadening and, hence, the height of the (hard) ob-ject can be used for characterization. Nevertheless, the lateral dimensions work as guidelines to detect the orientation of objects especially if the differ-ences in dimensions between their axes are big enough [125, 126]

Protein imaging may be performed in air, liquid and vacuum [127, 128]. The advantage of liquid is that proteins can be imaged in their natural envi-ronment and that the solution conditions can be manipulated to minimize the force exerted by the tip on the sample [129]. Unfortunately, the AFM resolu-tion is worse in liquid than in air/vacuum since the molecules tend to be more mobile. There is also a risk that the proteins are moved or removed by the tip because proteins are less firmly attached in liquid than in air.

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Figure 13. Tip shape induced artifacts: the surfaces was scanned with a blunt “dou-ble” tip which magnifies and registers the same features twice

Imaging in air is easier but suffers from two main disadvantages. During drying of the samples, proteins loose most of their liquid which might cause structural changes or even denaturation of the proteins. The liquid is, how-ever, not completely removed leading to strong capillary forces between the tip and the sample and possible protein deformation. This effect is more pronounced in other modes such as contact mode but needs still to be con-sidered as a tapping mode™ artifact.

EllipsometryEllipsometry is one of the oldest [130] and most frequently used techniques for direct monitoring of the adsorption of proteins on solid surfaces [131-134]. It uses linear (circular) polarized light to determine the change of the polarization state by reflection at the sample surface. The polarization state is described by the two ellipsometric angles, (relative phase change) and (relative amplitude change) from which surface properties such as (effective) refractive index (N=n+ik) and (mean) film thickness (d) can be calculated.

An ordinary rotating-analyzer ellipsometer is equipped with a light source (laser; : 623.8 nm), a polarizer, a compensator (quarter-wave plate), an analyzer (rotating polarizer) and a detector (Figure 14). The laser sends out monochromatic light, which is linearly polarized by the polarizer and there-after transformed into circularly polarized light by the compensator. The latter improves the sensitivity of the instrument. Upon reflection, the light is elliptically polarized and then directed through a rotating element generating a sinusoidal intensity-signal at the detector plate. The amplitude and phase of the exiting light is compared with the corresponding parameters for the en-tering light beam.

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Ellipsometry is a good tool for protein adsorption analysis. It is sensitive (thickness resolution: ±1 Å), does not destroy the adsorbed proteins, does not require any protein labeling and can be performed in situ and in real-time. In addition it is robust and fairly simple to handle experimentally.

Figure 14. Principle of the rotating-analyzer ellipsometer

The technique requires atomically flat and optically reflective surfaces which limits the application. The main disadvantage is, however, its model depend-ency [131, 133]. To convert and into effective refractive index (N) and mean film thickness (d) some assumptions must be made. We have to as-sume that the electrolyte-protein-substrate system can be described by a layer model with homogenous optical parameters in different layers meaning constant density and refractive index within the film, and discrete boundaries towards each layer. This is sometimes not true, especially not within sub-monolayers of protein. The latter result in a pseudo response that is an aver-age of the optical properties of both surface and coating. However, keeping this in mind, ellipsometry can be used with confidence.

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Protein-cell interactions A cell adhesion test was developed to study the functional behavior of spe-cific proteins after adsorption. The test used model surfaces, cycloheximide treatment, fluorescence microscopy and divalent cations (Mg2+/Ca2+) for cell quantification, cell morphological studies and monitoring of specific/non-specific cell-substrate interactions.

Cell adhesion test Cell adhesion (attachment and spreading) can be used to evaluate the func-tion of adsorbed proteins in vitro (Figure 15). The number and morphology(structural appearance) of adhered cells gives an indication of the adsorbed proteins’ potential to regulate cell establishment at surfaces. A protein that promotes cell establishment is expected to give high numbers of attached and spread cells and vice versa. To further classify the proteinous effect, reference proteins such as fibronectin (cell adhesion protein) and albumin (cell repellant) are often included in the experiments.

Figure 15. Fluorescence microscopy pictures of different levels of cell adhesion to surfaces

The nature of the cell-substrate interaction can be monitored by comparing the number of attached cells with the number of spread cells and/or by in-cluding/excluding divalent cations (e.g., Mg2+/Ca2+) from the cell suspension(Figure 16). The first alternative relies on the assumption that cells need integrins for spreading but not for attachment; thus if the number of attached cells is higher than the number of spread cells then some of the attached cells use other interactions than ligand-integrin recognition for binding. The sec-ond alternative makes use of the basic condition that integrins require diva-lent cations to be able to bind ligands; thus if the number of attached cells dramatically decrease when Mg2+ and Ca2+ are excluded from the cell sus-pension then the cell attachment is integrin mediated. The test does not ex-

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clude the possibility that other receptors than integrins are involved, there-fore studies with “blocked” integrins are good complements.

A prerequisite for this type of experiments is that the system is kept free from other proteins and that the cells aren’t allowed to be influenced by the naked surfaces (topographical and physicochemical interactions) instead of the adsorbed proteins. The former is solved with cycloheximde treatment (see below) and careful washing of the cells after culturing and the latter is settled by using flat and fully (protein) covered surfaces.

Figure 16. Principles of cell attachment and spreading

Cycloheximide treatment In vivo (and in vitro), bone cells react on artificial surfaces by secreting pro-teins (part of the extra cellular matrix (ECM)) and thereby providing points of attachment. This secretion is undesirable when studying cell attachment to individual proteins.

Cycloheximide (actidione) [135] is a glutarimide antibiotic that effi-ciently inhibits protein synthesis in culture. It prevents amino acids to be transferred from aminoacyl transfer RNA into nascent peptides on ribosomes of the 80 S type [136].

Fluorescence microscopy Fluorescence microscopy provides a simple method to quantify cells and study their morphology on surfaces (Figure 17). The technique relies on the phenomena that, fluorescent molecules absorb light at one wavelength and emit it at another, longer wavelength [137]. If a cell, stained with such a dye, is illuminated at the dyes’ absorbing wavelength and then viewed through a

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filter that allows only light of the emitted wavelength to pass, it is seen to glow against a dark background.

The dye used in this thesis is acridine orange (AO) which is a selective cationic stain for nucleic acids. It interacts with DNA and RNA by intercala-tion and electrostatic attraction, respectively. The absorption band of AO lies between 440 and 480 nm (blue light) and the emission occurs between 520 nm (green fluorescence for DNA) and 650 nm (orange fluorescence for RNA). Fluorescence microscopy combined with a video camera, computer and image software, has developed into a powerful tool for imaging of cells.

Figure 17. Principle of the fluorescence microscope

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Results and discussion

Papers I and II

Å. Rosengren, E. Pavlovic, S. Oscarsson, A. Krajewski, A. Ravaglioli, A. Piancastelli (2002). Plasma protein adsorption pattern on characterized ceramic biomaterials. Biomaterials, 23;1237-1247

Å. Rosengren, S. Oscarsson, M. Mazzocchi, A. Krajewski, A. Ravaglioli (2003). Protein adsorption onto two bioactive glass-ceramics. Biomaterials, 24;147-155

The work in, papers I and II, was performed to investigate the dynamic and selective nature of protein adsorption from diluted human plasma onto five different bioceramics (Table 2). We used a conventional flow system, two-dimensional gel electrophoresis and Western immunoblotting to monitor possible quantitative and qualitative proteinous differences between the cho-sen materials.

Table 2. Chemical composition of the bioceramics in weight percent (wt%)

Alumina (Al2O3), zirconia (ZrO2) and hydroxyapatite ((Ca10(PO4)6(OH)2 or HA) were chosen since they are well-established materials for bone implants and because they represent different levels of tissue integration in vivo: the level of tissue integration being viewed by the authors as highly protein de-pendent. Alumina and zirconia induce a thin fibrous capsule while hy-droxyapatite forms a strong bond to surrounding tissue [70]. RKKP and AP40, two glass-ceramics, were chosen since they are being tested for bio-

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material applications, they represent an additional integration pathway and because they (despite their almost identical composition) have shown differ-ences when implanted into osteopenic rats [138, 139]. RKKP which contains 1% Ta5+ and 0.5% La3+ integrates into osteopenic bone whereas AP40 which lacks Ta5+ and La3+ does not.

Before performing the adsorption experiments we measured the pore vol-ume, the pore size distribution and the specific surface area of the materials by Hg-porosimetry and BET-method to investigate how much of the specific surface that was available for protein adsorption. Since these measurements are performed with non-protein related substances, it sometimes happens that the obtained surface availability doesn’t correspond to the practical availability. The practical availability can be evaluated with isotope labeled model proteins but is less useful for the complex protein mixture that we use since the properties of the model protein and the protein mixture seldom are the same.

Another possibility is to use a comparative method, such as the method referred to as the “dynamic binding capacity”-method in the “Methods” sec-tion. This method is based on the methodology of frontal analysis [140] which relies on the condition that materials with different numbers of bind-ing sites will give different elution fronts. That is; if the amount of binding sites is larger for one material than for another then the material with the largest amount of binding sites will have its elution front shifted to the right in the graph compared to the one with lower amount of binding sites. The method doesn’t give an absolute value for available binding sites but pro-vides a way to compare the protein availability for different materials.

When these two methods were used, we obtained two very different re-sults. According to the total area of the used particles, alumina, zirconia and hydroxyapatite should be able to adsorb 43 times more proteins than AP40 and RKKP but according to the dynamic binding capacity study it was evi-dent that the amount of binding sites only differed by a factor of 1.2. This strongly indicates that most of the specific surface for alumina, zirconia and hydroxyapatite was not available for proteins and the practically available surface areas were more or less the same for all materials.

Since this comparison hasn’t been made before, we drew the wrong con-clusions regarding the total protein binding capacities and the protein cover-age of the materials in paper I and II.

However, even though we are unable to calculate the exact total protein binding capacities we can obtain an indication of the protein coverage degree by comparing the total protein binding capacity values for AP40 and RKKP with the surface concentration for monolayer coverage and thereby draw a worst case scenario for the two materials. In papers I and II we used a monolayer concentration (2.5 mg/m2) determined by Fair and Jamieson [141] but more recent literature data [142] on the same model protein (albu-min) have shown that this value is too low and that 4 mg/m2 is more appro-

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priate. Using this concentration instead, the study indicates that RKKP and AP40 are fully covered and that the proteins on AP40 probably even form multilayers. If the “true” availability of the material surface is less than the specific surface values then the protein coverage degree will obviously in-crease. Furthermore, since the dynamic binding capacity study showed that all materials had approximately the same available surface we can conclude that it is highly probable that the surfaces of alumina, zirconia and hy-droxyapatite are fully covered with proteins as well.

Despite their physico-chemical differences, alumina, zirconia, hydroxya-patite, AP40 and RKKP showed similarities in depleting proteins from plasma (Table 3). In fact, AP40 and RKKP displayed identical adsorption patterns. Nevertheless, twelve of the twenty-six studied proteins preferred one material over another. Among these selective proteins, Apo D, C1s and C3 showed the highest relative affinity for zirconia. Hydroxyapatite had the highest relative binding capacity for 2-HS-glycoprotein and RKKP/AP40 had the highest relative binding capacity for fibrinogen and fibronectin. Alumina, zirconia and hydroxyapatite had higher relative binding capacities for 1-antichymotrypsin and prothrombin than did AP40/RKKP while zirco-nia, hydroxyapatite and AP40/RKKP had higher relative binding capacities for Apo J than did alumina. Zirconia and hydroxyapatite had higher relative binding capacities for ceruloplasmin than did alumina and AP40/RKKP, while alumina and zirconia had higher relative binding capacities for -1B-glycoprotein than did hydroxyapatite and RKKP/AP40. Alumina and hy-droxyapatite had higher relative binding capacities for SRBP than did zirco-nia. The latter was not studied for AP40 and RKKP.

The “selective” proteins have many diverse physiological functions. Pro-teins like complement factor C3 and C1s (inflammatory components), fi-bronectin (cell adhesion protein), fibrinogen and prothrombin (coagulation agents) are known to be important for the healing process whereas the influ-ence of the others is more diffuse [9]. The picture is further complicated by the fact that the proteins can be activated, passivated or remain indifferent upon adsorption. Other parameters that influence the host response are the quantity of the individual proteins and their respective distribution in inner layers versus outer layers. Furthermore, the protein layers rearrange them-selves with time due to displacement forces. Obviously, additional evalua-tions are needed.

In conclusion, the five bioceramics adsorbed quite similar amounts of proteins but the types of proteins were different for many of the materials which could explain their different levels of tissue integration in vivo. Fur-thermore, the presence of Ta5+ and La3+ in RKKP did not seem to affect the amounts or the types of proteins that were adsorbed.

Table 3. Identified non-adsorbed proteins in the flow-through as a function of saturation degree (25%, 50% and 75%) and identified adsorbed proteins in the desorption fraction (D). The presence of a protein in a fraction is marked with a + sign and the absence is marked with a - sign.

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Papers III and IV

Å. Rosengren, A. Vitina, S. Oscarsson (2004). Bone cell (MG63) adhesion on silicon wafers pre-coated with ceruloplasmin and prothrombin. Submitted

Å. Rosengren, A. Vitina, S. Oscarsson (2004). Characterization of adsorbed 2-HS-glycoprotein and 1-antichymotrypsin and their influence on bone

cell (MG63) adhesion. Submitted

Inspired by the previous work (papers I and II), we picked out four of the “selective” proteins for further characterization and evaluation. The goal was to monitor their orientation and (if possible) conformational changes after adsorption and to study their influence on bone cell adhesion as a result of the adsorption process. The choice fell on ceruloplasmin, prothrombin, 2-HS-glycoprotein and 1-antichymotrypsin (Figure 7-10).

The study was performed on ultraflat model surfaces (silicon wafers with a natural layer of silicon oxide/silica) to be able to characterize the adsorbed proteins with atomic force microscopy (AFM) and ellipsometry, and to sin-gle out the protein-induced effect on the cell activity by reducing interfering surface properties (e.g. topography, variations in surface chemistry). Fur-thermore, we sought to distinguish between integrin mediated cell adhesion and non-specific (i.e., divalent cation-independent) cell adhesion by regulat-ing the integrin’s ligand binding ability with divalent cations, Mg2+ and Ca2+.The cell results were compared with equivalent experiments on a plasma-treated polyolefin surface, Thermanox , which is frequently used as a posi-tive cell adhesion reference.

The dimensions of the adsorbed proteins on silicon wafers were measured with the AFM and the values were compared with available X-ray data (Ta-ble 4). Most of the AFM values differed from the crystal data. This was, however, expected since AFM imaging always suffers from probe broaden-ing and sometimes from probe compression (i.e., compression of the mole-cule by the probe) [120]. The former increases all lateral dimensions and the latter decreases the height. Nevertheless, by looking at the overall shapes of the molecules and comparing the AFM dimensions with the X-ray data, it was possible to speculate about the orientation of the adsorbed molecules. Accordingly, the molecules appeared to be oriented with their long axis par-allel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface (Figure 18).

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Table 4. Comparison of protein dimensions measured by AFM and X-ray crystallog-raphy

1 Length is the longest axis in the object. 2Width is the longest perpendicular bisector to the length. 3Ceruloplasmin is an equilateral triangle with side 75 Å and triangular height 65 Å. 4Without and with two extending loops. 5 Prothrombin is a pseudoplanar “drop-shaped” mole-cule.6 The crystal structure is not known. 7 Dimensions obtained from native uncleaved struc-tural homolog 1-antitrypsin [143], cleaved 1-antichymotrypsin [34] and 1-antichymotrypsin with partial loop insertion [40]

Figure 18. Schematic view, showing the orientations of a) ceruloplasmin, b) prothrombin and c) 1-antichymotryspin

We could also see that the molecules had globular shapes but the resolution was insufficient to reveal any further conformational details. We could, however, conclude that prothrombin (but none of the others) formed multi-layers at high proteins concentrations and that the ceruloplasmin sample contained two different size populations. The latter gave us the idea that the AFM could be used to study the stability of protein solutions over time and consequently we found that ceruloplasmin gradually rearranges itself proba-bly due to loss of copper [144].

Another important issue when studying cell-protein interactions on sur-faces is to minimize the influence of the underlying material. We therefore used ellipsometry to find out how well the surfaces could be covered with the individual proteins. The ellipsometric results together with the AFM data (Figure 19) showed that none of the protein coatings (except prothrombin) reached complete coverage. The prothrombin coating, however, consisted of

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Figure 19. Deposition of proteins on silicon wafers as a function of applied protein concentration

at least two levels of protein molecules. In conclusion, the cells could be influenced by the material in nearly all cases but it is unclear to what degree.

An osteosarcoma cell line (MG63) was used to study the functional be-havior (referring to cell binding and cell spreading capacity) of the proteins after adsorption. The cells were treated with cycloheximde and washed care-fully to keep the system free from other proteins and cell-related contami-nants. When applying the MG63 cells to the pre-coated silicon wafers and a pre-coated reference surface (Thermanox ), a clear difference in cell at-tachment (Figure 20) and cell spreading (Table 5) could be seen as assessed by fluorescence microscopy. Ceruloplasmin, 2-HS-glycoprotein and 1-antichymotrypsin stimulated cell attachment to silica, but suppressed at-tachment to Thermanox . The reason for this could be that the proteins adopted different conformations/orientations on the two materials or/and, as in the ceruloplasmin case, different protein moieties become adsorbed by the materials or/and that the materials exposed different amounts of naked sur-face. In the Thermanox case it is clear that most of the surface is covered with proteins since very few cells attached compared to the naked surface. However in the silica case we know from ellipsometry that some of the un-derlying surface might be available for the cells so the difference in coloni-zation could be explained by differences in the amount of exposed surface. Nevertheless, we could conclude from the spreading behavior and the Mg2+/Ca2+ dependency that at least some of the proteins must have

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Figure 20. Comparison of number of attaching cells per cm2 after 1.5 h on naked and protein coated silicon wafers and Thermanox as a function of salt addition. The following proteins were used as coatings: ceruloplasmin (CP), prothrombin (PT), 2-HS-glycoprotein (AHSG), 1-antichymotrypsin, fibronectin (FN), albumin (HSA) and human plasma (HP).

Table 5. Percentage of spread cells per cm2 as a function of protein-, surface- and salt type.

a different conformation or orientation on silica than on Thermanox since 15-44% of the cells adhered through integrin-mediated interactions to the pre-coated silica while the integrin-mediated interactions and the cell spread-ing were negligible on pre-coated Thermanox .

Prothrombin, on the other hand, stimulated cell attachment to both sur-faces. The cells slightly preferred pre-coated silica over pre-coated Ther-manox . The adhesion to silica and Thermanox was mediated both by integrins and by divalent cation-independent adhesion sites. In addition, the cells anchored to Thermanox through an unidentified divalent cation-dependent interaction. We believe that these different adhesion mechanisms are due to different protein conformations and orientations since the materi-als should be fully covered by proteins (as assessed by AFM, ellipsometry

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and Mg2+/Ca2+ dependency) and are thereby unable to experience any direct cell-material interactions. Further studies are, however, needed to confirm this.

These studies demonstrated how important the adsorption of proteins at material interfaces is for cell adhesion. By manipulating the conformation and orientation of individual proteins as well as the degree of protein cover-age, very different cell responses can be induced. Unfortunately, the studies also showed that the techniques today are insufficient to fully characterize a layer of adsorbed proteins. Even though we succeeded to monitor the overall orientation of single proteins, we failed to see any conformational details. In addition it is highly speculative if the orientation and conformation of single proteins are the same for protein in layers. Nevertheless, we believe that with the development of new as well as old imaging techniques we will soon be able to relate protein configurations to their functional behavior.

In summary, papers I-IV show how easily the composition of an adsorbed protein layer and the structure and functional behavior of individual proteins are manipulated by the properties of a material surface. By controlling these interactions it is possible to induce different cellular responses, a fact that might be put to use in the development of new biomaterials. These studies increase the fundamental knowledge regarding the cell-protein-material in-terface and are therefore of relevance for the scientific community.

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Acknowledgements

Many people have supported me and I would like to express my sincere gratitude to all of you: family, friends, colleagues, teachers, students and donors. Where would I have been without you…

The following people deserve special thanks:

Prof. Sven Oscarsson, my supervisor

You started off by saying “this isn’t going to be easy” – and boy you were right! Who knew what a ride we were up for?! Nevertheless, you always believed in me even though I sometimes doubted my own ability. You and I, together, created the independent critical researcher I am today.

Prof. Karin Caldwell and Dr. Per Kårsnäs, the heads of my two departments

Karin, thank you, for accepting me as a PhD-student at your department and always kindly sharing your vast and diverse knowledge in science. Per, thank you for providing the necessary financial means. I know it wasn’t easy…

Magnus Bergkvist, Maria Lundqvist, Helén Larsericsdotter, Elisabeth Pav-lovic, Greger Ledung, Jos Buijs, Arjan Quist and more recently, Simon Berner, the group

Having (very) different projects it wasn’t easy to help each other profes-sionally but the help I got personally; good laughs, small talk and solid friendship is invaluable.

Prof. Staffan Johansson, Prof. Carl Påhlsson, Prof. Hans Eklund, Prof. Pentti Tengvall, Prof. Jan Carlsson and Prof. Jan-Christer Jansson, the experts

Thank you for your time and expertise! You are all professionals in sum-marizing a lifetime of knowledge into effective 30 minutes.

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Dr Antonio Ravaglioli, Dr Adriano Krajewski, Dr Mauro Mazzocchi and Ing Andreana Piancastelli, the Italian crew

International collaborations are of crucial importance for a young scientist under development. Apart from the stimulating scientific environment, I am thankful for the true Italian moments…food and wine…

Anita Vitina, Dr Sven Karlsson, Ing. Jan-Åke Gustavsson, Lars Ling and DrDavid Eaker, the helping hands

Cell culturing, particle characterization, wafer cutting, purchase assistance and linguistic revision - I am sincerely grateful for all your practical help.

Peter Johansson, Ulrika Svensson and Ola Lundström, the illustrators

”En bild säger mer än tusen ord”, txs.

Tomas Sandberg, my roomie

Tomas, being undergraduate and graduate colleagues we have spent al-most ten years together - just like a better marriage. Somebody should have told us it was for better and for worse…

Karin Fromell, Hans Blom, Marjam Karlsson, Johan Lindholm, Maria Lönnberg, Bingze Xu, Erika Ledung, Helena Stärner, LarsErik Johansson, Lasse Nurkkula, Nedaa Hajem, the present and former PhD-students at CYB and IBK

The perfect PhD-student doesn’t eat, sleep or have a social life. I am glad that you weren’t perfect…..

All the people at Department for Surface Biotechnology (CYB) and Depart-ment for Biology and Chemical Engineering (IBK), the colleagues

None mentioned – none forgotten.

Södermanlands-Närkes Nationskör, the choir

True moments of joy! You have made me “lite snyggare och lite bättre”!

Uppsala Akademiska Fältrittklubb, the riding school

Wednesday, 4 pm – the most important appointment in my weekly life for the past five years. A special thanks to Birgitta Segerfeldt for your patience.

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People at Karlsrogatan 13V21-29 and 11B24-26, Kemitjejerna, Tunagänget, Grundskoletjejerna, Hällevikslägret, Dan Jensen, Joakim Strand, Anna Touminen, Emma Brännström, Cecilia Evansson, Neeme Nemvalts, Ulrika Svensson, Ulrika Åkerstedt and Gustav Olsson, the good people

It takes people to play - you were willing! Humble thanks!

Hans-Erik Carlsson, Anna Foberg and all the animals at Lugnet

Peace and harmony are rare phenomena when doing a PhD. Fortunately, I was lucky to meet you two who took care of me when times were rough. With your support and our wild horse rides, I was able to relax and be a hap-pier human being.

Gittan, Henrik, Kattis and Ingrid, the inner circle

You have listen, given advice and spoken the truth - I can’t express how grateful I am to have you as my friends.

Margit Rosengren, Stina Danielsson and Torsten Nenzen, family

You are important to me.

Torgil, Anita and Ylva, my father, my mother and my sister

One word – LOVE

Uppsala, November 2004

Åsa Rosengren

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Figure 21. A PhD at work – hard work…

Financial support:

KK-stiftelsen (the Knowledge Foundation) INCO Copernicus concerted action contract No IC15-CT98-0816 INTAS Project contract No 97-30443

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Summary in Swedish

Interaktioner mellan celler, proteiner och keramiska ytor En kroppsdel som går sönder eller nöts ut ersätts för det mesta med en ny konstgjord och förhoppningsvis likvärdig del. Svårigheten med att sätta in främmande föremål i kroppen är att den upplever föremålet som just främ-mande. En rad biologiska processer startas för att området där implantatet satts in ska återfå sin normala funktion och aktivitet. Om detta inkluderar den nya kroppsdelen eller ej, beror på interaktionen mellan föremålet och omgivande vävnad.

Under de första sekunderna efter implantering invaderas implantatytan av blodproteiner. Sammansättningen och organisationen i proteinlagret bestäms till största delen av materialets egenskaper, men även av proteinernas före-komst i omgivningen. Detta innebär att proteiner med stor affinitet (attrak-tion) för materialet kan återfinnas i lagret trots att de förekommer i liten mängd i omgivningen. Omvänt återfinns även proteiner med låg affinitet i lagret i och med att de förekommer i hög mängd i omgivningen. Samman-sättningen av lagret förändras snabbt i början men blir konstant med tiden. Detta ger varje material en unik blandning av proteiner på ytan.

De adsorberade proteinerna fungerar som signalmolekyler för kroppens celler. Cellerna ”läser av” proteinskiktet och agerar olika beroende på vad de ser. I de flesta fall innebär detta att området runt implantatet genomgår en lite längre inflammationsperiod med påföljande ärrbildning vilket i slutändan leder till en ogynnsam fibrös inkapsling av implantatet.

För att undvika detta försöker man idag ta fram implantat och då främst implantatytor som motverkar en kraftig inflammation och eventuell inkaps-ling, och som styr läkningsprocessen mot uppbyggnad av önskad vävnad. En viktig gren av biomaterialforskningen har därför inriktat sig på att förstå hur läkningsprocessen vid ett implantat fungerar och då mer specifikt hur celler och proteiner påverkar eller påverkas av olika material.

I denna avhandling som består av fyra enskilda studier har vi använt oss av in vitro försök dvs., försök utanför kroppen, för att studera interaktionen mellan blodproteiner och olika ytor. I de två första studierna undersöktes hur proteiner från human plasma adsorberades till fem olika biokeramer: alumi-

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niumoxid (Al2O3), zirkoniumoxid (ZrO2), hydroxyapatite (Ca10(PO4)6(OH)2)och två glaskeramer, AP40 och RKKP. AP40 och RKKP har samma kemis-ka grundsammansättning (SiO2-CaO-Na2O-P2O5 med tillsatser av MgO, K2Ooch CaF2) men innehåller olika halter av tantal- och lantan-joner. Materialen används främst i tand- och benapplikationer men nyttjas även inom andra anatomiska områden tex som hjärtklaffar och ligament. Materialen valdes för att de som implantat integreras på olika sätt i benvävnad: aluminiumoxid och zirkoniumoxid bildar en fibrös kapsel medan hydroxyapatit och glaske-ramer binder direkt till ben, om än via olika reaktionsvägar. Utöver detta har de mycket närbesläktade glas-keramerna, AP40 och RKKP, uppvisat intres-santa skillnader vid implantering i osteoporotiskt ben: RKKP men inte AP40 binder till osteoporotisk benvävnad. Vi var därför nyfikna på att undersöka om de olika biologiska fenomenen (delvis!) kan härröras till skillnader i proteinadsorption.

För att studera detta använde vi oss av ett konventionellt kromatografiskt flödessystem, ”protein assays”, tvådimensionell gelelektrofores och ”Wes-tern immunoblotting”.

Studierna visade att de olika keramiska materialen attraherade olika prote-iner och i olika mängder men att den totala mängden av adsorberade protei-ner var jämförbara. Vidare visade studien att inslag av tantal- och lantanjo-ner i glassystem inte påverkade vilka typer av proteiner som adsorberades.

Nästa fråga att besvara var då: Vilka proteiner ser cellerna? Eftersom blod innehåller en mängd olika proteiner med en mängd olika funktioner (tex transport av syre, stimulering av koagulation och försvar mot infektioner), är det av stor vikt att ”rätt” proteiner fastnar på implantatet. Vad som avses med ”rätt” i detta sammanhang är inte helt klart men vanligtvis brukar man hänvisa till en lagom mängd inflammations- och koagulationsproteiner samt en stor mängd cell adhesion (cell ”bindande”) proteiner. De senare stimule-rar celletablering vid ytor och anses därför viktiga för att omgivande vävnad ska kunna binda till implantatet.

Biokeramerna adsorberade en mängd olika typer av blodproteiner men endast ett tjugotal av dessa kunde med säkerhet identifieras. Nära hälften av de identifierade proteinerna uppvisade någon form av preferens för något utav de studerade materialen tex., inflammationsproteinet C3 föredrog zir-koniumoxid medan koagulationsfaktorn, fibrinogen, och cell adhesions pro-teinet, fibronektin, föredrog RKKP och AP40.

Sammantaget visar de två första studierna att biokeramerna attraherar un-gefär lika mycket blodproteiner men att de olika typerna av attraherade blodproteiner skiljer sig mellan materialen. Detta skulle kunna påverka läk-ningsprocessen i en viss riktning.

En ytterligare betänklighet då det gäller proteinadsorptionsstudier på im-plantatmaterial är att vissa proteiners funktioner kan komma att ändras när proteinerna fastnar på en yta till exempel kan nya funktioner framkallas och

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gamla regleras ned. Funktionsändringen är en konsekvens av att proteinerna ändrar sin konformation (form/struktur) eller orienterar sig på ett specifikt sätt på implantatytan och därigenom exponerar eller gömmer interaktionssä-ten på molekylen.

I de två sista studierna valde vi därför ut några av de proteiner som skiljt sig mellan biokeramerna för att närmare studera hur de orienterar sig på en yta, om de förändrar sin konformation och om deras funktion påverkas av adsorptionsprocessen. Valet föll på ceruloplasmin, protrombin, 2-antichymotrypsin och 2-HS-glykoprotein. Ceruloplasmin transporterar koppar i kroppen och protrombin är ett blodkoagulationsprotein. 2-Antichymotrypsin hämmar proteaser (proteiner som klyver andra proteiner) och 2-HS-glykoprotein deltar i metabolismen av ben.

Proteinernas konformation och orientering studerades med atomkrafts-mikroskopi (AFM) och deras etablering i lager undersöktes med ellipsome-tri. Dessa tekniker fodrar att ytan som proteinerna adsorberats till har en mycket liten ytråhet ( 1-2 Å) och därför valdes en ultraflat modellyta (kisel med ett naturligt lager av kiseloxid) istället för de biomaterial vi tidigare studerat.

AFM-studien visade att samtliga proteiner antog en globulär form och att de låg med sin längsta sida parallell mot kiselytan. Tyvärr tillät inte instru-mentets upplösning att ytterligare strukturella detaljer urskiljdes och vi kun-de därför inte klargöra om proteinerna förändrat sin konformation efter ad-sorption. Det vi däremot kunde visa var att ceruloplasmin-provet innehöll ceruloplasmin-molekyler av två olika storlekar och att protrombin-molekylerna bildar lager vid högre koncentrationer. Det senare kunde även verifieras med ellipsometri.

I övrigt visade ellipsometri-studien (med stöd av resultat från AFM) att inget av de andra proteinerna bildade multilager eller ens monolager dvs endast delar av ytan kunde täckas med proteiner. Detta beror troligtvis på att proteinerna repellerar varandra elektrostatiskt och/eller steriskt.

Slutligen undersöktes hur proteinernas förmåga att binda benceller (MG63-celler) påverkades efter adsorption och huruvida vidhäftningen till cellerna (adhesionen) var specifik eller ospecifik dvs om förankringen be-drevs via speciella vidhäftningsproteiner (integriner) i cellens membran eller inte. Det senare kunde studeras genom att reglera integrinernas funktion med magnesium- och kalciumjoner.

Innan MG63-cellerna såddes ut på de proteinbelagda kiselytorna förbe-handlades cellerna med cykloheximid som stänger av cellernas egen protein-produktion och med buffert för att minska risken för att andra proteiner för-orenar ytorna. Cellernas antal och morfologi studerades med fluorescens-mikroskopi och jämfördes med resultat från ytterligare en yta, Thermanox ,som behandlats på samma sätt som kiselytan. Naken Thermanox stimule-

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rar celltillväxt och användes i den här studien som en positiv cell adhesions-kontroll.

Resultaten visade att samtliga proteiner, utom protrombin, stimulerade cellulär adhesion till kisel men inte till Thermanox . Protrombin däremot, stimulerade cell vidhäftning till bägge materialen men i något högre grad till kisel. Vidare visade studien att en viss procent (15-57%) av de vidhäftade cellerna bredde ut sig (”spred” sig) på de proteinbelagda kiselunderlagen. Flest antal utsträckta celler hade protrombin på kisel och lägst antal hade 2-antichymotrypsin på kisel. När magnesium och kalcium uteslöts från cell-suspension minskade vidhäftningen av celler kraftigt till alla material.

Vidhäftningen till kiselytor belagda med ceruloplasmin, 2-antichymotrypsin och 2-HS-glykoprotein berodde delvis på interaktioner mellan de adsorberade proteinerna och cellens integriner och delvis på inter-aktioner mellan det nakna materialet och cellen. Däremot, berodde vidhäft-ningen till protrombin-beklädd kisel troligtvis bara på den första interaktio-nen eftersom kiselytan, enligt AFM- och ellipsometristudierna, bör vara helt täckt med proteiner. Vidhäftningsmekanismen till Thermanox var inte lika tydlig och verkade, förutom de redan beskrivna interaktionerna, bero på yt-terligare en obestämd interaktion.

Sammanlagt indikerar cellstudien att proteinernas funktion förändras som funktion av adsorptionsprocessen och att olika material inducerar olika funk-tioner hos proteinerna. Orsaken är troligtvis att proteinerna antar olika kon-formation och orientering, men det återstår att bevisa i det här fallet eftersom AFM:et inte lyckades urskilja strukturella detaljer på proteinerna och efter-som Thermanox i sin kommersiella form inte lämpar sig för AFM- och ellipsometristudier. I ceruloplasmin-fallet finns ytterligare en lösning dvs att de olika materialen attraherar olika populationer av samma protein, därav skillnaden i funktion.

Avhandlingen i sin helhet visar tydligt hur svårt det är att förutse vad pro-teiner på en implantatyta kommer att signalera till kroppens celler men för-hoppningsvis kan den här typen av studier bidra till en mer grundläggande förståelse för protein-material interaktioner och är därför relevanta för ut-vecklingen av biomaterial.

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References

[1] Rosengren Å, Pavlovic E, Oscarsson S, Krajewski A, Ravaglioli A, Pian-castelli A. Plasma protein adsorption pattern on characterized ceramic bio-materials. Biomaterials 2002;4:1237-1247

[2] Rosengren Å, Oscarsson S, Mazzocchi M, Krajewski A, Ravaglioli A. Pro-tein adsorption onto two bioactive glass-ceramics. Biomaterials 2003;1:147-155

[3] Williams DF. Definitions in biomaterials. Amsterdam: Elsevier, 1987 [4] Peppas NA, Langer R. New challenges in biomaterials. Science

1994;5154:1715-1720 [5] Kasemo B. Biological surface science. Surf Sci 2002;1-3:656-677 [6] Castner DG, Ratner BD. Biomedical surface science: Foundations to fron-

tiers. Surf Sci 2002;1-3:28-60 [7] Lysaght MJ, O'Loughlin JA. Demographic scope and economic magnitude

of contemporary organ replacement therapies. ASAIO J 2000;515-521 [8] Park JB, Bronzino JD. Biomaterials: principles and applications. Boca

Raton: CRC Press LLC, 2003 [9] Ratner BD, Hoffman AS, Schoen FJ, Lemons JE, editors. Biomaterials

science: an introduction to materials in medicine. 1st ed. San Diego: Aca-demic Press; 1996.

[10] Williams DF. The science and applications of biomaterials. Int J Mater Prod Technol 1995;3-6:360-377

[11] Tirrell M, Kokkoli E, Biesalski M. The role of surface science in bioengi-neered materials. Surf Sci 2002;1-3:61-83

[12] Lendlein A, Kelch S. Shape-memory polymers. Angew Chem2002;12:2034-2057

[13] Miyata T, Asami N, Uragami T. A reversibly antigen-responsive hydrogel. Nature 1999;6738:766-769

[14] Peppas NA. Hydrogels and drug delivery. Curr Opin Colloid Interface Sci1997;5:531-537

[15] Berthiaume F, Yarmush M. Tissue engineering. In: Bronzino JD. The bio-medical engineering handbook. Boca Raton: CRC Press LLC, 2000.

[16] Bianco P, Robey PG. Stem cells in tissue engineering. Nature2001;6859:118-121

[17] Anderson JM, Gristina AG, Hanson SR, Harker LA, Johnson RJ, Merritt K, Naylor PT, Schoen FJ. Host reactions to biomaterials and their evaluation. In: Ratner BD, Hoffman AS, Schoen FJ, Lemons JE. Biomaterials science: an introduction to materials in medicine. San Diego: Academic Press, 1996. 165-214

[18] Black J. Biological performance of materials: fundamentals of biocompati-bility. New York: Marcel Dekker, 1999

66

[19] Williams DF. Biofunctionality and biocompatibility. In: Cahn RW, Haasen P, EJ K. Materials science and technology: medical and dental materials.Weinheim: VCH, 1991. 1-27

[20] Thomsen P, Ericson LE. Inflammatory cell response to bone implant sur-faces. In: Davies JE. The bone-biomaterial interface. Toronto: University of Toronto Press, 1991. 153-164

[21] Branden C-I, Tooze J. Introduction to protein structure. New York: Garland publishing, 1999

[22] Stryer l. Biochemistry. New York: W.H. Freeman and company, 1988 [23] Norde W. Colloids and interfaces in life sciences. New York: Marcel Dek-

ker, 2003 [24] Wahlgren M, Arnebrant T. Protein adsorption to solid surfaces. Trends

Biotechnol 1991;6:201-208 [25] Vroman L, Adams AL. Findings with the recording ellipsometer suggesting

rapid exchange of specific plasma proteins at liquid/solid interfaces. Surf Sci 1969;438-446

[26] Slack SM, Horbett TA. The Vroman effect - a critical review. ACS Symp Ser 1995;112-128

[27] Schneck DJ. An outline of cardiovascular structure and function. In: Bronz-ino JD. The biomedical engineering handbook. Boca Raton: CRC Press LLC, 2000.

[28] Anderson NL, Anderson NG. The human plasma proteome. History, char-acter, and diagnostic prospects. Mol Cell Proteomics 2002;11:845-867

[29] Rose K, Bougueleret L, Baussant T, Boehm G, Botti P, Colinge J, Cusin I, Gaertner H, Gleizes A, Heller M, Jimenez S, Johnson A, Kussmann M, Menin L, Menzel C, Ranno F, Rodriguez-Tome P, Rogers J, Saudrais C, Villain M, Wetmore D, Bairoch A, Hochstrasser D. Industrial-scale pro-teomics: From liters of plasma to chemically synthesized proteins. Pro-teomics 2004;7:2125-2150

[30] Putnam FW, editor. The plasma proteins: structure, function and genetic control. Second ed. Volume 4. Orlando: Academic press; 1984. 1-420 p.

[31] Aronsen KF, Ekelund G, Kindmark CO, Laurell CB. Sequential changes of plasma proteins after surgical trauma. Scand J Clin Lab Invest 1972;S127-136:

[32] Berninger RW. Alpha1-antichymotrypsin. J Med 1985;1, 2, 3:101-127 [33] Schwick HG, Haupt H. Purified human plasma proteins of unknown func-

tion. Jpn J Med Sci Biol 1981;5:299-327 [34] Baumann U, Huber R, Bode W, Grosse D, Lesjak M, Laurell CB. Crystal

structure of cleaved human a1-antichymotrypsin at 2.7 .ANG. resolution and its comparison with other serpins. J Mol Biol 1991;3:595-606

[35] Wei A, Rubin H, Cooperman BS, Christianson DW. Crystal structure of an uncleaved serpin reveals the conformation of an inhibitory reactive loop. Nat Struct Biol 1994;4:251-258

[36] Beatty K, Bieth J, Travis J. Kinetics of association of serine proteinases with native and oxidized a-1-proteinase inhibitor and a-1-antichymotrypsin. J Biol Chem 1980;9:3931-3934

[37] Schechter NM, Jordan LM, James AM, Cooperman BS, Wang ZM, Rubin H. Reaction of human chymase with reactive site variants of a1-antichymotrypsin. Modulation of inhibitor versus substrate properties. JBiol Chem 1993;31:23626-23633

67

[38] Faber JP, Poller W, Olek K, Baumann U, Carlson J, Lindmark B, Eriksson S. The molecular basis of alpha 1-antichymotrypsin deficiency in a het-erozygote with liver and lung disease. J Hepatol 1993;3:313-321

[39] Poller W, Faber JP, Weidinger S, Tief K, Scholz S, Fischer M, Olek K, Kirchgesser M, Heidtmann HH. A leucine-to-proline substitution causes a defective a1-antichymotrypsin allele associated with familial obstructive lung disease. Genomics 1993;3:740-743

[40] Gooptu B, Hazes B, Chang W-SW, Dafforn TR, Carrell RW, Read RJ, Lomas DA. Inactive conformation of the serpin a1-antichymotrypsin indi-cates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease. Proc Natl Acad Sci 2000;1:67-72

[41] Abraham CR. Reactive astrocytes and a1-antichymotrypsin in Alzheimer's disease. Neurobiol Aging 2001;6:931-936

[42] Zhang S, Janciauskiene S. Multi-functional capability of proteins: a1-antichymotyrpsin and the correlation with Alzheimer's disease. J. Alz-heimer's Dis 2002;2:115-122

[43] Pearce MC, Rubin H, Bottomley SP. Conformational change and intermedi-ates in the unfolding of a1-antichymotrypsin. J Biol Chem 2000;37:28513-28518

[44] Zaitseva I, Zaitsev V, Card G, Moshkov K, Box B, Ralph A, Lindley P. The x-ray structure of human serum ceruloplasmin at 3.1 .ANG.: nature of the copper centers. J Biol Inorg Chem 1996;1:15-23

[45] Lindley P, Card G, Zaitseva I, Zaitsev V. Ceruloplasmin: the beginning of the end of an enigma. Perspect Bioinorg Chem 1999;51-89

[46] Saenko EL, Yaropolov AI, Harris ED. Biological functions of ceruloplas-min expressed through copper-binding sites and a cellular receptor. J Trace Elem Exp Med 1994;2:69-88

[47] Puchkova LV, Sasina LK, Aleinikova TD, Zakharova ET, Gaitskhoki VS. Reconstitution of the intercellular transfer pathway of the peptide moiety of ceruloplasmin in mammals. Biochemistry (Moscow) 1997;7:697-703

[48] Broadley C, Hoover RL. Ceruloplasmin reduces the adhesion and scavenges superoxide during the interaction of activated polymorphonuclear leuko-cytes with endothelial cells. Am J Pathol 1989;4:647-655

[49] Curtis ASG, Forrester JV. The competitive effects of serum proteins on cell adhesion. J Cell Sci 1984;17-35

[50] Sigrist CJA, Cerutti L, Hulo N, Gattiker A, Falquet L, Pagni M, Bairoch A, Bucher P. PROSITE: A documented database using patterns and profiles as motif descriptors. Briefings in Bioinformatics 2002;3:265-274

[51] Schinke T, Amendt C, Trindl A, Poeschke O, Mueller-Esterl W, Jahnen-Dechent W. The serum protein a2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in min-eralization and calcium homeostasis. J Biol Chem 1996;34:20789-20796

[52] Triffitt JT, Gebauer U, Ashton BA, Owen ME, Reynolds JJ. Origin of plasma .alpha.2HS-glycoprotein and its accumulation in bone. Nature1976;5565:226-227

[53] Jahnen-Dechent W, Schafer C, Heiss A, Grotzinger J. Systemic inhibition of spontaneous calcification by the serum protein a2-HS glycopro-tein/fetuin. Z Kardiol 2001;Suppl. 3:iii/47-iii/56

[54] Ketteler M, Bongartz P, Westenfeld R, Ernst Wildberger J, Horst Mahnken A, Bohm R, Metzger T, Wanner C, Jahnen-Dechent W, Floege J. Associa-tion of low fetuin-A (AHSG) concentrations in serum with cardiovascular

68

mortality in patients on dialysis: a cross-sectional study. Lancet2003;9360:827-833

[55] Dziegielewska KM, Brown WM. Fetuin. New York: Springer-Verlag, 1995 [56] Kalabay L, Cseh K, Pajor A, Baranyi E, Csakany GM, Melczer Z, Speer G,

Kovacs M, Siller G, Karadi I, Winkler G. Correlation of maternal serum fe-tuin/a2-HS-glycoprotein concentration with maternal insulin resistance and anthropometric parameters of neonates in normal pregnancy and gestational diabetes. European Journal of Endocrinology 2002;2:243-248

[57] Jersmann HPA, Dransfield I, Hart SP. Fetuin/a2-HS glycoprotein enhances phagocytosis of apoptotic cells and macropinocytosis by human macro-phages. Clin Sci 2003;3:273-278

[58] Demetriou M, Binkert C, Sukhu B, Tenenbaum HC, Dennis JW. Fetuin/a2-HS glycoprotein is a transforming growth factor-b type II receptor mimic and cytokine antagonist. J Biol Chem 1996;22:12755-12761

[59] Martin PD, Malkowski MG, Box J, Esmon CT, Edwards BFP. New insights into the regulation of the blood clotting cascade derived from the x-ray crys-tal structure of bovine meizothrombin des F1 in complex with PPACK. Structure 1997;12:1681-1693

[60] Soriano-Garcia M, Padmanabhan K, De Vos AM, Tulinsky A. The calcium ion and membrane binding structure of the Gla domain of calcium-prothrombin fragment 1. Biochemistry 1992;9:2554-2566

[61] Stubbs MT, Bode W. A player of many parts: the spotlight falls on throm-bin's structure. Thromb Res 1993;1:1-58

[62] Romberg RW, Werness PG, Riggs BL, Mann KG. Inhibition of hydroxya-patite-crystal growth by bone-specific and other calcium-binding proteins. Biochemistry 1986;5:1176-1180

[63] Sakiyama H, Nonaka T, Masuda R, Inoue N, Kuboki Y, Iijima M, Hiraba-yasi Y, Takahagi M, Yoshida K, Kuriiwa K, Yoshida M, Imajoh-Ohmi S. Characterization of mineral deposits formed in cultures of a hamster tar-trate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP). Cell Tissue Res 2002;2:269-279

[64] Lecrone V, Li W, Devoll RE, Logothetis C, Farach-Carson MC. Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins. Cell Calcium 2000;1:35-42

[65] Sano T, Gabazza EC, Zhou H, Takeya H, Hayashi T, Ido M, Adachi Y, Uchida A, Suzuki K. The zymogen prothrombin stimulates cell locomotion and calcium influx in murine osteosarcoma cells by different mechanism from thrombin. Int J Oncol 1999;6:1197-1203

[66] Byzova TV, Plow EF. Networking in the hemostatic system. Integrin al-phaiibbeta3 binds prothrombin and influences its activation. Journal of Bio-logical Chemistry 1997;43:27183-27188

[67] Byzova TV, Plow EF. Activation of .alpha.v.beta.3 on vascular cells con-trols recognition of prothrombin. Journal of Cell Biology 1998;7:2081-2092

[68] Chuang HYK, Mitra SP. Adsorption and reactivity of human prothrombin to artificial surfaces. J Biomed Mater Res 1984;6:695-705

[69] Report of the committee on definition of the term ceramics. J Am Ceram Soc 1920;7:526-536

[70] Hench LL. Bioceramics: from concept to clinic. J Am Ceram Soc1991;7:1487-1510

[71] Billotte WG. Ceramic biomaterials. In: Bronzino JD. The biomedical engi-neering handbook. Boca Raton: CRC Press, 2003.

[72] Stevens R. Introduction to zirconia. Magnesium elektron Ltd, 1986

69

[73] Kasprzyk-Hordern B. Chemistry of alumina, reactions in aqueous solution and its application in water treatment. Adv Colloid Interface Sci2004;1,2:19-48

[74] Krajewski A, Piancastelli A, Malavolti R. Albumin adhesion on ceramics and correlation with their Z-potential. Biomaterials 1998;7-9:637-641

[75] Hulbert SF. The use of alumina and zirconia in surgical implants. In: Hench LL, Wilson J. An introduction to bioceramics. Singapore: World Scientific, 1993. 25-40

[76] Piconi C, Maccauro G. Zirconia as a ceramic biomaterial. Biomaterials1999;1:1-25

[77] Kim HM. Ceramic bioactivity and related biomimetic strategy. Curr Opin Solid State Mater Sci 2003;4-5:289-299

[78] LeGeros RZ. Properties of osteoconductive biomaterials: calcium phos-phates. Clin Orthop 2002;395:81-98

[79] Radin S, Ducheyne P. Effect of serum proteins on solution-induced surface transformations of bioactive ceramics. J Biomed Mater Res 1996;3:273-279

[80] Radin S, Ducheyne P, Berthold P, Decker S. Effect of serum proteins and osteoblasts on the surface transformation of a calcium phosphate coating: a physicochemical and ultrastructural study. J Biomed Mater Res 1998;2:234-243

[81] Ravagioli A, Krajewski A. Bioceramics: materials, properties and applica-tions. London: Chapman and Hall, 1992

[82] Krajewski A, Malavolti R, Piancastelli A. Albumin adhesion on some bio-logical and non-biological glasses and connection with their zeta -potentials. Biomaterials 1996;1:53-60

[83] Huff HR. An electronics division retrospective (1952-2002) and future opportunities in the twenty-first century. J Electrochem Soc 2002;5:S35-S58

[84] Carim AH, Dovek MM, Quate CF, Sinclair R, Vorst C. High-resolution electron microscopy and scanning tunneling microscopy of native oxides on silicon. Science 1987;4815:630-633

[85] Zhuravlev LT. The surface chemistry of amorphous silica. Zhuravlev model. Colloids Surf A: Phys Eng Asp 2000;1-3:1-38

[86] Iler RK. The chemistry of silica: solubility, polymerization, colloid and surface properties and biochemistry. 1979

[87] Bousse L, Mostarshed S, Van der Shoot B, De Rooij NF, Gimmel P, Goepel W. Zeta potential measurements of tantalum pentoxide and silicon dioxide thin films. J Colloid Interface Sci 1991;1:22-32

[88] Ducy P, Schinke T, Karsenty G. The osteoblast: A sophisticated fibroblast under central surveillance. Science 2000;5484:1501-1504

[89] Mackie EJ. Osteoblasts: novel roles in orchestration of skeletal architecture. Int J Biochem Cell Biol 2003;9:1301-1305

[90] Aubin JE, Triffitt JT. Mesenchymal stem cells and osteoblast differentia-tion. Principles of Bone Biology 2002;59-81

[91] Triffitt JT. Osteogenic stem cells and orthopedic engineering: summary and update. J Biomed Mater Res 2002;4:384-389

[92] Clover J, Gowen M. Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype? Bone1994;6:585-591

[93] Heremans H, Billiau A, Cassiman JJ, Mulier JC, de Somer P. In vitro culti-vation of human tumor tissues. II. Morphological and virological charac-terization of three cell lines. Oncology 1978;6:246-252

70

[94] Anselme K. Osteoblast adhesion on biomaterials. Biomaterials 2000;7:667-681

[95] Garcia AJ, Keselowsky BG. Biomimetic surfaces for control of cell adhe-sion to facilitate bone formation. Crit Rev Eukaryot Gene Expr 2002;2:151-162

[96] Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;1:11-25

[97] Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell2002;6:673-687

[98] Chen J, Salas A, Springer TA. Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster. Nat Struct Biol 2003;12:995-1001

[99] Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to in-tegrins. J Biol Chem 2000;29:21785-21788

[100] Xiong JP, Stehle T, Goodman SL, Arnaout MA. Integrins, cations and ligands: making the connection. J Thromb Haemostasis 2003;7:1642-1654

[101] Xiong J-P, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, Ar-naout MA. Crystal structure of the extracellular segment of integrin avb3 in complex with an Arg-Gly-Asp ligand. Science 2002;5565:151-155

[102] Leitinger B, McDowall A, Stanley P, Hogg N. The regulation of integrin function by Ca2+. Biochim Biophys Acta 2000;2-3:91-98

[103] Carman CV, Springer TA. Integrin avidity regulation: are changes in affin-ity and conformation underemphasized? Curr Opin Cell Biol 2003;5:547-556

[104] Humphries MJ, McEwan PA, Barton SJ, Buckley PA, Bella J, Mould AP. Integrin structure: heady advances in ligand binding, but activation still makes the knees wobble. Trends Biochem Sci 2003;6:313-320

[105] Siebers MC, ter Brugge PJ, Walboomers XF, Jansen JA. Integrins as linker proteins between osteoblasts and bone replacing materials. A critical re-view. Biomaterials 2004;2:137-146

[106] Garcia AJ, Schwarzbauer JE, Boettiger D. Distinct Activation States of a5b1 Integrin Show Differential Binding to RGD and Synergy Domains of Fibronectin. Biochemistry 2002;29:9063-9069

[107] Janson J-C, Ryden L, Editors. Protein purification: principles, high resolu-tion methods and applications. 1998

[108] Allen T. Surface area and pore size determination. London: Chapman and Hall, 1997

[109] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of pro-tein using bicinchoninic acid. Anal Biochem 1985;1:76-85

[110] O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. JBiol Chem 1975;10:4007-4021

[111] Klose J. Protein mapping by combined isoelectric focusing and electropho-resis of mouse tissues. A novel approach to testing for induced point muta-tions in mammals. Humangenetik 1975;3:231-243

[112] Berkelman T, Stenstedt T. 2-D Electrophoresis: principles and methods.Amersham Biosciences, Edition AB,

[113] Issaq HJ, Conrads TP, Janini GM, Veenstra TD. Methods for fractionation, separation and profiling of proteins and peptides. Electrophoresis2002;17:3048-3061

[114] Otto JJ. Immunoblotting. Methods Cell Biol 1993;Antibodies in Cell Biol-ogy:105-117

71

[115] Mouche L, Tardif F, Derrien J. Particle deposition on silicon wafers during the wet cleaning processes. J Electrochem Soc 1994;6:1684-1691

[116] Pintchovski F, Price JB, Tobin PJ, Peavey J, Kobold K. Thermal character-istics of the sulfuric acid-hydrogen peroxide silicon wafer cleaning solution. J Electrochem Soc 1979;8:1428-1430

[117] Tamminen A, Anttila O. Cleaning processes for silicon wafers. VTT Symp1999;30th R3-Nordic Contamination Control Symposium, 1999:431-438

[118] Amick JA. Cleanliness and the cleaning of silicon wafers. Solid State Tech-nology 1976;11:47-52

[119] Binnig G, Quate C, Gerber C. Atomic force microscope. Phys Rev Lett1986;9:930-933

[120] Morris VJ, Kirby AR, Gunning AP. Atomic force microscopy for biologist.London: Imperial College Press, 1999

[121] Burnham N, Behrend O, Oulevey F, Gremaud G, Gallo P-J, Gourdon D, Dupas E, Kulik A, Pollock H, Briggs G. How does a tip tap? Nanotechnol-ogy 1997;67-75

[122] Nagase M, Namatsu H, Kurihara K, Makino T. Critical dimension meas-urement in nanometer scale by using scanning probe microscopy. Japanese Journal of Applied Physics, Part 1: Regular Papers, Short Notes & Review Papers 1996;7:4166-4174

[123] Ramirez-Aguilar KA, Rowlen KL. Tip Characterization from AFM Images of Nanometric Spherical Particles. Langmuir 1998;9:2562-2566

[124] Markiewicz P, Goh MC. Atomic force microscopy probe tip visualization and improvement of images using a simple deconvolution procedure. Langmuir 1994;1:5-7

[125] Bergkvist M, Carlsson J, Karlsson T, Oscarsson S. TM-AFM threshold analysis of macromolecular orientation: a study of the orientation of IgG and IgE on mica surfaces. J Colloid Interface Sci 1998;2:475-481

[126] Bergkvist M. Methods for studying orientation of surface adsorbed proteins. In: AT H. Encyclopedia of surface and colloid science. New York: Marcel Dekker, 2002. 3328-3341

[127] Haggerty L, Lenhoff AM. STM and AFM in biotechnology. Biotechnol Prog 1993;1:1-11

[128] Keller D. Scanning force microscopy in biology. In: Baszkin A, Norde W. Physical chemistry of biological interfaces. New York: Marcel Dekker, 2000.

[129] Muller DJ, Fotiadis D, Scheuring S, Muller SA, Engel A. Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. Biophys J 1999;2:1101-1111

[130] Rothen A. Measurements of the thickness of thin films by optical means, from Rayleigh and Drude to Langmuir, and the development of the present ellipsometer. In: Passaglia E, Stromberg RR, Kruger J, editors. Symposium proceedings on ellipsometry in the measurements of surfaces and thin films.Volume 256: National Bureau of Standards; 1964. p 7-21.

[131] Arwin H. Ellipsometry. Phys Chem Biol Interface 2000;577-607 [132] Elwing H. Protein adsorption and ellipsometry in biomaterial research.

Biomaterials 1998;4-5:397-406 [133] Ivarsson B, Lundstroem I. Physical characterization of protein adsorption

on metal and metal oxide surfaces. Crit Rev Biocomp. 1986;1:1-96 [134] Langmuir I, Schaefer VJ. Optical measurements of the thickness of a film

adsorbed from a solution. J Am Chem Soc 1937;1406

72

[135] Jost JL, Kominek LA, Hyatt GS, Wang HY. Cycloheximide: properties, biosynthesis, and fermentation. Drugs Pharm Sci 1984;Biotechnol. Ind. An-tibiot.:531-550

[136] Obrig TG, Culp WJ, McKeehan WL, Hardesty B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J Biol Chem 1971;1:174-181

[137] Rost FWD. Fluorescence microscopy. Cambridge University Press, 1995 [138] Belmonte MM, De Benedittis A, Muzzarelli RAA, Mengucci P, Biagini G,

Gandolfi MG, Zucchini C, Krajewski A, Ravaglioli A, Roncari E, Fini M, Giardino R. Bioactivity modulation of bioactive materials in view of their application in osteoporotic patients. J Mater Sci: Mater Med 1998;9:485-492

[139] Fini M, Giavaresi G, Aldini NN, Torricelli P, Morrone G, Guzzardella GA, Giardino R, Krajewski A, Ravaglioli A, Belmonte MM, De Benedittis A, Biagini G. The effect of osteopenia on the osteointegration of different bio-materials: histomorphometric study in rats. J Mater Sci: Mater Med2000;9:579-585

[140] Kasai KI, Oda Y, Nishikata M, Ishii SI. Frontal affinity chromatography: theory for its application to studies on specific interactions of biomolecules. J Chromatogr 1986;376:33-47

[141] Fair BD, Jamieson AM. Studies of protein adsorption on polystyrene latex surfaces. J Colloid Interface Sci 1980;2:525-534

[142] Norde W, Giacomelli CE. BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states. J Biotechnol2000;3:259-268

[143] Kim S-J, Woo J-R, Seo EJ, Yu M-H, Ryu S-E. A 2.1 .ANG. resolution structure of an uncleaved a1-antitrypsin shows variability of the reactive center and other loops. J Mol Biol 2001;1:109-119

[144] Rydén L. Ceruloplasmin. In: Lontie R. Copper proteins and copper en-zymes. Boca Raton: CRC Press, 1984. 37-100

Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

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A doctoral dissertation from the Faculty of Science and Technology, UppsalaUniversity, is usually a summary of a number of papers. A few copies of thecomplete dissertation are kept at major Swedish research libraries, while thesummary alone is distributed internationally through the series ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science and Technology.(Prior to October, 1993, the series was published under the title “ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science”.)