ToF-SIMS Investigations on Dental Implant Materials and Adsorbed Protein Films

Diplomarbeit von Falk Bernsmann

Introduction:

Biofilms play an important role in the health sector, in bioanalytics, in the food industry and in engineering science because the adsorption of organic molecules can alter the physical, biological and chemical properties of a surface. This work deals with the formation of biofilms on dental implant materials. When a dental implant is placed in the oral cavity, within seconds its surface is covered by a biofilm called pellicle consisting mainly of proteins and other macromolecules. Since the adsorption of proteins is a highly selective process, the proportions of proteins found in the pellicle differ significantly from the ones found in saliva. The pellicle is of great physiological importance because it serves as lubricant, as diffusion barrier to demineralising agents and as reservoir for remineralising electrolytes. Furthermore, proteins in the pellicle play an important role in the colonisation of the surface by bacteria and thus in the formation of dental plaque. On the one hand there are proteins, like amylase, that exhibit specific binding sites for bacterial adsorption. On the other hand enzymes, like lysozyme, immobilized in the pellicle have anti-bacterial properties. Since the adsorption process of proteins is a subject not yet fully understood, this work shall further investigate the adsorption of the proteins amylase, lysozyme and serum albumin on two experimental dental implant materials. The chosen method is Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) because it offers the following advantages: The mass distribution of molecules adsorbed on a sample’s surface can be measured with a high mass resolution and a high surface sensitivity. It is possible to create depth profiles with a depth resolution of below one nanometre. And the analysis of non-conducting samples is possible without further preparation steps. ToF-SIMS has already been used to analyse adsorbed protein films on different substrates but up to now there are no ToF-SIMS studies of protein films on dental implant materials. The difficulty in interpreting mass spectra of proteins is their complexity. Every protein consists of a combination of the same twenty amino acids which dissociate within the ToF-SIMS analysis to numerous fragments. Hence one has to take into account the intensities of many different masses for analysis. To reduce the number of variables (i.e. masses), Principal Component Analysis (PCA) is used. This multivariate technique concentrates the variance of the spectra onto only a few variables, called Principal Components (PC).

This work is subdivided into the following sections: In this first section an introduction to the subject is given. The second section deals with previous work on the analysis of adsorbed protein films by ToF-SIMS and multivariate data analysis. Theoretical aspects of the examined systems and the applied techniques are detailed in the third section. The experiments are described in the fourth section. The fifth section contains the following results: First, mass spectra of dental implant materials are examined to determine their elemental surface composition. Then mass spectra of proteins adsorbed to silane coated silicon substrates are analysed to develop the methods and programmes necessary for distinguishing different proteins by their mass spectra. Since this does not work very well, the examined system is simplified to proteins adsorbed directly to silicon substrates. Here the different proteins can be recognized by their mass spectra and the developed statistical models perform well in evaluation tests. The adsorption conditions are varied to obtain the best results. Additionally the mutual influences of two proteins adsorbed at the same time or consecutively to the same silicon substrate are studied. The results obtained are confirmed by enzymatic activity measurements. Finally the mass spectra of proteins adsorbed to dental implant materials are examined. With only little modifications in data pre-treatment, the programmes developed to analyse the spectra of proteins on silicon can be used to distinguish between different proteins adsorbed to dental implant materials. Again, information on the mutual influence of the proteins upon adsorption is obtained. The sixth section gives a summary and an outlook on possible future investigations.

Table of Contents:

Text Sample:

Chapter 4, Experimental aspects:

4.1, Chemicals:

Bovine serum albumin fraction V, approximately 99%, Sigma-Aldrich, Germany.

Lysozyme from chicken egg white, approximately 95%, Sigma-Aldrich, Germany.

Amylase from human saliva, Fluka BioChemika, USA.

Bovine serum albumin fluorescein conjugate, Invitrogen, Germany.

Glutardialdehyde solution, 50% in water, Merck-Schuchardt, Germany.

3-Aminopropyl-tri(ethoxy)silane, minimum 98%, Sigma-Aldrich, Germany.

Sodium di(hydrogen)phosphate monohydrate, minimum 99.5%, Fluka BioChemika, Switzerland.

Disodium hydrogenphosphate, minimum 99%, Riedel-de Ha¨en, Germany.

Bidistilled water with a resistivity of 182 kΩm from a ‘Milli-Q A10’ water purification system, Millipore, USA.

Dehydrated, denatured ethanol, Department of Chemistry, Technische Universität Kaiserslautern, Germany.

Hydrogen peroxide, 35%, Department of Chemistry, Technische Universität Kaiserslautern, Germany.

Sulphuric acid, 95 - 97%, Department of Chemistry, Technische Universität Kaiserslautern, Germany.

Sodium hypochlorite solution, approximately 13% of active chlorine, Department of Chemistry, Technische Universit¨at Kaiserslautern, Germany.

Dehydrated toluene, work group of Professor Thiel, Department of Chemistry, Technische Universität Kaiserslautern, Germany.

4.2, Sample preparation:

4.2.1, Dental material:

The dental implant materials and the samples of bovine enamel are provided by the work group of Professor Matthias Hannig, Universit¨atsklinikum Homburg, in pieces of roughly five millimetres by five millimetres size. The dental implant materials are polymer matrices containing apatite particles. The two examined types are called FAT and FAW for fluoroapatite with a more transparent or more whitish appearance. They are made of the following substances:

silanized (for FAW) or unsilanized (for FAT) fluoroapatite (Ca5(PO4)3F) particles, bis-phenol-A-glycidyl-di(methacrylate), tri(ethylene glycol)-di(methacrylate), poly(methacryl)oligo(maleic acid), camphor quinone, strontium.

The surfaces are polished. Before being used, the substrates are ultrasonically cleaned for five minutes in a solution of one percent of sodium hypochlorite. To remove a possible deposit of sodium hypochlorite, they are ultrasonically cleaned for another five minutes in bidistilled water and rinsed three times in bidistilled water.

4.2.2, Protein films on silanised substrates:

Approximately ten millimetres by five millimetres large silicon wafer pieces serve as substrate in this case. First they are cleaned for fifteen minutes in a solution consisting to two thirds of concentrated sulphuric acid and to one third of a thirty-five percent solution of hydrogen peroxide. This cleaning solution is called piranha solution for its ability to remove most organic matter. Afterwards the substrates are rinsed with bidistilled water and dried with nitrogen. Then they are placed for one hour under protective gas in a mixture of 20 ml of dry toluene and 0.5 ml of 3-aminopropyl-tri(ethoxy)silane (APTES). First the APTES hydrolises with the residual water in the solution to form a silanol. The latter can covalently bind to the oxide atoms of the silicon oxide to form a monolayer as shown in figure 14. At the same time the silane can also polymerise. To remove polymerised silane from the surface, the samples are given into an ultrasonic bath for fifteen minutes in ethanol.Then they are rinsed with bidistilled water. Silanised substrates are imaged with a scanning electron microscope (SEM) by Dr. Stefan Trellenkamp, Nano+BioCenter Kaiserslautern. When the samples are not sonicated in ethanol, many aggregates of polymerised silane can be found on the surface. Their number is strongly reduced after sonication. Glutardialdehyde serves as link between the silane and the protein. It shall bind to the silane as shown in figure 16. Therefore the samples are placed for one hour in a five percent (volume / volume) solution of glutardialdehyde in pH 7 buffer solution. The pH-value of the buffer solution is adjusted by mixing a 0.1 molar solution of sodium di(hydrogen)phosphate (NaH2PO4) and a 0.1 molar solution of disodium hydrogenphosphate (Na2HPO4). It is controlled with a pH electrode ‘Testo 252’ by ‘Testo’, Germany. After the adsorption of glutardialdehyde the samples are rinsed with buffer solution. The actual protein adsorption also takes place in pH 7 buffer solution for one hour with protein concentrations of two grammes per litre. All adsorption steps are effectuated at room temperature. To remove loosely bound protein, the samples are rinsed with fresh buffer solution. In a last step they are rinsed another three times with bidistilled water to remove buffer salts and dried with argon.