Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1046 Cell-protein-material Interactions on Bioceramics and Model Surfaces BY ÅSA ROSENGREN ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2004 ! "" #$ % & ' & & ( ) * + ' ) , ' -) "") .!EE3 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. Pinacastelli, “Plasma protein adsorption pat- tern on characterized ceramic biomaterials”, Biomaterials, 2002; 23: 1237-1247 II Å.
Ravaglioli, “Protein adsorption onto two bioactive glass- ceramics”, Biomaterials, 2003; 24: 147-155 III Å. Oscarsson, “Bone cell (MG63) adhesion on silicon wafers pre-coated with ceruloplasmin and prothrombin”, Submitted IV Å. Oscarsson, “Characterization of adsorbed D2-HS-glycoprotein and D1-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. Rosengren, “Influence of fluorapa- tite minor additions on behavior of hydroxyapatite ceramics”, Journal of Materials Science: Materials in Medicine, 2004; 15: 291-296 Å. Piancastelli, "Serum protein adsorption pattern on ceramic biomaterials", In: A. Krajewski (Eds), Ceramics, cells and tissues: implants for spine, Ravenna: Tipografia Moderna, 1999; 5: 193-197 Å.
Ravaglioli, "Protein adsorption onto biomterials after exposure to human serum", In: A. Krajewski (Eds), Ceramics, cells and tissues: ceramic- polymer composites, Ravenna: Tipografia Moderna, 1997; 4: 207-211 Contents Introduction.17 Host response and wound healing at the biomaterial interface .18 Aim of the present investigation .21 Structure and properties .29 Alumina and Zirconia .30 AP40 and RKKP.31 Silicon and silicon oxide.34 Bone cell adhesion .37 Flow-based protein adsorption analysis.37 Bicinchoninic acid (BCA) protein assay .39 Two-dimensional (2-D) gel electrophoresis .41 Atomic force microscopy in tapping mode™.43 Protein-cell interactions.45 Cell adhesion test.46 Results and discussion .48 Papers I and II .48 Papers III and IV .57 Summary in Swedish .61 Interaktioner mellan celler, proteiner och keramiska ytor.65 Abbreviations ACT D1-Antichymotrypsin AFM Atomic force microscopy AHSG D2-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 Teller C1 Complement factor 1 C3 Complement factor 3 CaP Calcium phosphate CE Capillary electrophoresis CP Ceruloplasmin Da Dalton DGEA Aspartic acid-Glycine-Glutamic acid- Alanine DNA 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 line Mr 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 andPiancastelli RNA 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., 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., 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 Introduction Biomaterials Many 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]. 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 Silicone Dental implants 300 000a Titanium, Ceramics Pacemakers 670 000b Polyurethane Renal dialyzers 16 000 000a Cellulose Left ventricular assist devices >100 000a Polyurethane a Castner et al [6],bLysaght et al [7], cAmerican society of plastic surgeons, 2003 (www.org) 15 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., 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., 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. micro-contact printing, micro-molding and photo-lithography; and systems 16 that release growth factors and other hormones have been fabricated using many different techniques (i. Other interesting new materials are the so-called “smart” materials e.
shape-memory metals/polymers [12] and smart gels [13, 14].