Research interests of the Interface Chemistry and Biomaterials Group
Institute of Physical and Theoretical Chemistry, University of Regensburg

Influence of surface properties on interactions with proteins and cells

Influences of protein films on antibacterial or bacteria-repellent surface coatings in a model system using silicon wafers

Rainer Müller and Verena Katzur, Institute of Physical and Theoretical Chemistry, University of Regensburg
Andreas Eidt, Karl-Anton Hiller, Gottfried Schmalz and Helmut Schweikl, Department of Operative Dentistry and Periodontology, University Hospital Regensburg
Stefan Ruhl, Department of Oral Biology, State University of New York at Buffalo
Satoshi Imazato, Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry

The project was supported by Kuraray Medical Inc., Dental Material Division (Tokyo, Japan).

Published in: Biomaterials 2009, 30, 4921-4929.

Abstract
Immobilisation of defined chemical functionalities to biomaterial surfaces is employed to optimize them not only for tissue compatibility but also for prevention of bacterial infection. Grafting surfaces with chains of poly(ethylene glycol) (PEG) results in bacterial repellence whereas modification with cationic groups conveys them with bactericidal properties. Since biomaterials in situ will become exposed to a protein-rich environment, it is necessary to investigate the influence of prior protein adsorption on the antibacterial activity of this type of chemical surface modification. In the present study, we immobilised short-chain PEG and two pyridinium group-containing methacrylate monomers, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and 6-methacryloyloxyhexylpyridinium chloride (MHPC), to silicon wafer model surfaces to investigate the influence of prior protein adsorption on the bactericidal activity of the surface coating towards subsequently attached bacteria. Adsorbed amounts of human serum albumin and salivary proteins were found to be two times higher on cationic compared to PEG-modified surfaces. An analogous tendency was found for attachment of Streptococcus gordonii and S. mutans to the same surfaces without prior protein exposure. However, most bacteria attached to cationic surfaces were found to be dead. Prior exposure of cationic surfaces to protein solutions drastically altered bacterial attachment dependent on the type of protein solution and bacterial species employed. Significantly, the original bactericidal activity of pyridinium-coated surfaces was found greatly reduced upon adsorption of a protein film. As a conclusion we propose that future approaches should combine the protein- and bacteria-repellent properties of PEG-coatings with the bactericidal function of charged cationic groups.

Figure 1: Viability testing of surface-attached bacteria. (A) Fluorescence signals obtained after life-dead staining of S. gordonii attached to differently modified wafer surfaces, green cells are alive, red cells are dead. (B) Amounts of living bacteria derived from the life-dead analysis which suggests that surface-bound pyridinium groups exert bactericidal properties but only in the case that the surface was not covered by a protein layer.

Adhesion of eukaryotic cells and Staphylococcus aureus to silicon model surfaces

Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg
Stefan Ruhl, Karl-Anton Hiller, Gottfried Schmalz and Helmut Schweikl, Department of Operative Dentistry and Periodontology, University Hospital Regensburg

The project was funded by the University Hospital Regensburg within the ReForM-C-program.

Published in: Journal of Biomedical Materials Research Part A 2008, 84, 817-827.

Abstract
Silicon wafers modified by silanisation with different functional groups are used to study the bioactivity of surfaces with varying physicochemical properties. Oxidation of the wafers created very hydrophilic surfaces, and moderately wettable surfaces were produced by coating with poly(ethylene glycol) (PEG). Immobilization of hydrocarbon chains to the wafers produced hydrophobic surfaces, and hydrophobicity was further increased by fluorocarbon coatings. The oxidized and the hydrocarbon -modified surfaces supported the adhesion of human MG-63 osteoblasts and 3T3 mouse fibroblasts as well as Staphylococcus aureus 8325-4. Adhesion of osteoblasts and fibroblasts, however, was inhibited on highly hydrophobic fluorocarbon surfaces, whereas adhesion of S. aureus was supported. Coating of the fluorocarbon surface with fibronectin increased the number of attached eukaryotic cells, but the accumulation of bacteria remained unchanged. In contrast, surface coatings with PEG-groups inhibited the binding of S. aureus, however, the adhesion of the eukaryotic cells was high. The number of S. aureus on PEG-modified surfaces covered with fibronectin increased about 2-fold, yet it was still decreased to 25-30% related to the number of bacteria on other surfaces. These findings provide evidence that the PEG-modified surfaces showed selective bioactivity, preventing the attachment of a microbial pathogen but supporting the adhesion of eukaryotic cells.

Figure 2: Cell adhesion to differently modified silicon wafer surfaces as determined by the crystal violet assay (A) and low-vacuum SEM (B). Hydrophilic OX and moderately hydrophilic PEG surfaces supported the adhesion of MG-63 osteoblasts, while adhesion was slightly decreased on hydrophobic OTS (CH3) and nearly suppressed on highly hydrophobic fluorocarbon HFS (CF3) modified surfaces. On HFS (CF3) modified surfaces, the few remaining cells appeared rounded and very small with almost no visible contact to the surface. In the presence of serum the cell density increased and cells developed some contact to the surface. After coating the surfaces with fibronectin, osteoblasts on HFS now appeared extremely flattened indicating similar close contact as observed on coated oxidized surfaces.

Proliferation of osteoblasts and fibroblasts on model surfaces of varying roughness and surface chemistry

Helmut Schweikl, Karl-Anton Hiller and Gottfried Schmalz, Department of Operative Dentistry and Periodontology, University Hospital Regensburg
Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg
Carsten Englert, Richard Kujat and Michael Nerlich, Department of Trauma Surgery, University Hospital Regensburg

The project was funded by the University Hospital Regensburg within the ReForM-C-program.

Published in: Journal of Material Science Materials in Medicine 2007, 18, 1895-1905.

Abstract
Physical and chemical properties of the surfaces of implants are of considerable interest for dental and orthopedic applications. We used self-assembled monolayers (SAMs) terminated by various functional chemical groups to study the effect of surface chemistry on cell behavior. Cell morphology and proliferation on silicon wafers of various roughnesses and topographies created by chemical etching in caustic solution and by corundum sandblasting were analyzed as well. Water contact angle data indicated that oxidized wafer surfaces displayed high hydrophilicity, modification with poly(ethylene glycol) (PEG) created a hydrophilic surface, and an amino group (NH₂) led to a moderately wettable surface. A hydrophobic surface was formed by hydrocarbon chains terminated by CH3, but this hydrophobicity was even further increased by a fluorocarbon (CF₃) group. Cell proliferation on these surfaces was different depending primarily on the chemistry of the terminating groups rather than on wettability. Cell proliferation on CH₃ was as high as on NH₂ and hydrophilic oxidized surfaces, but significantly lower on CF₃. Precoating of silicon wafers with cell culture serum had no significant influence on cell proliferation. Scanning electron microscopy indicated a very weak initial cell-surface contact on CF₃. The cell number of osteoblasts was significantly lower on sandblasted surfaces compared with other rough surfaces but no differences were detected with 3T3 mouse fibroblasts. The different surface roughnesses and topographies were recognized by MG-63 osteoblasts. The cells spread well on smooth surfaces but appeared smaller on a rough and unique pyramid-shaped surface and on a rough sandblasted surface.

Figure 3: Scanning electron micrographs of human MG-63 osteoblasts on smooth and rough silicon wafers. The cell morphology was analyzed on an oxidized smooth wafer surface (A), a surface etched in 10% aqueous potassium hydroxide solution for 5 min (B), a surface etched in 10% aqueous potassium hydroxide solution for 60 min (C), and a surface sandblasted with corundum particles (D). The cells appear extremely flattened on a smooth wafer surface (A) and on a surface etched in KOH for 5 min (B). The morphology of the cells changed on surfaces etched in KOH for 60 min (C), and a sandblasted surface (D). There is no continuous contact between the cells and the wafer surface for KOH 60 and SB, and the cells span across the ridges. We thank Brigitte Bay and Helga Ebensberger for technical support

Fluorescence-based bacterial overlay method for simultaneous in situ quantification of attached bacteria

Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg
Gerhard Gröger, Department of Prosthodontics, University Hospital Regensburg
Karl-Anton Hiller, Gottfried Schmalz and Stefan Ruhl, Department of Operative Dentistry and Periodontology, University Hospital Regensburg

The project was funded by the University Hospital Regensburg within the ReForM-C-program.

Published in: Applied Environmental Microbiology 2007, 73, 2653-2660.

Abstract
For the quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small-size material specimens. A new method was established for quantification of surface-attached biotinylated bacteria by in situ-detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, S. mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers, were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. Only in certain instances, mostly with saliva or serum, masking of the surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.

Figure 4: Detection of surface attached Streptococcus gordonii DL1 by the fluorescence-based bacterial overlay method. Silicon wafers were used as model surfaces of varying roughness and surface chemistry. Hydrophilic surfaces were generated after oxidation or coating with poly(ethylene glycol) (PEG) whereas hydrophobic surfaces were obtained by modification with hydrocarbon (OTS) or fluorocarbon moieties (HFS). HSA=human serum albumin.

Chemiluminescence-based detection and comparison of protein amounts adsorbed on differently modified silica surfaces

Rainer Müller, Institute of Physical and Theoretical Chemistry, University of Regensburg
Karl-Anton Hiller, Gottfried Schmalz and Stefan Ruhl, Department of Operative Dentistry and Periodontology, University Hospital Regensburg

The project was funded by the University Hospital Regensburg within the ReForM-C-program.

Published in: Analytical Biochemistry 2006, 359, 194-202.

Abstract
The biological consequences of protein adsorption on biomaterial surfaces are considered to be of outmost importance for their biocompatibility. A new method based on amino group-labelling coupled to a chemiluminescence reaction for direct determination of proteins adsorbed on material surfaces was employed. This method was used to explore the effects of surface chemistry and surface roughness on protein adsorption in a silicon oxide model system. Corundum sandblasting was applied to silicon wafers to create roughened surfaces while immobilisation of fluorocarbon-, hydrocarbon-, and poly(ethylene glycol)-containing silanes produced surfaces of varying wettability. The adsorption behaviour of two complex body fluids, human serum and saliva, as well as of two purified components, human serum albumin and fibronectin, was strongly influenced by the surface parameters. A general tendency to higher amounts of adsorbed protein was found on roughened surfaces, modification with poly(ethylene glycol) or with fluorocarbon moieties reduced protein adsorption. The values obtained with the new method could be confirmed by a colorimetric determination of protein amounts adsorbed on identically modified silica beads and were in accordance with to those previously reported utilizing established methods for protein quantification. The presented method, that was methodically simple to perform and allowed the simultaneous measurement of a large number of samples, may be of future value for high throughput surveying of biomaterials in terms of their protein adsorption characteristics.

Figure 5: Detection of surface-adsorbed proteins by a newly developed chemiluminescence-based assay. Silicon wafers were used as model surfaces of varying roughness and surface chemistry. Hydrophilic surfaces were generated after oxidation or coating with poly(ethylene glycol) (PEG) whereas hydrophobic surfaces were obtained by modification with hydrocarbon (OTS) or fluorocarbon moieties (HFS). HSA=human serum albumin and FN=fibronectin.

Last update on 17.11.2009 - For more informations please contact Dr. Rainer Müller