Bacterial Adhesion on Polyelectrolyte Modified Microstructured Titanium Surfaces
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Bacterial Adhesion on Polyelectrolyte Modified Microstructured Titanium Surfaces Argelia Almaguer-Flores1, Yolloxóchilt R. Sánchez-Cruz1, Jung Hwa Park2, René OlivaresNavarrete2, Michel Dard3, Rinna Tannenbaum2, Zvi Schwartz2 and Barbara D. Boyan2 1
Laboratorio de Genética Molecular, Facultad de Odontología, Universidad Nacional Autónoma de México, Circuito exterior s/n, Ciudad Universitaria, 04510 México D. F. México 2 Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA 3 Institut Straumann AG, Peter Merian-Weg 12, 4052 Basel, Switzerland Email: argelia.almaguermac.com
ABSTRACT Micron-scale and submicron-scale surface roughness enhance osteoblast differentiation on titanium (Ti) substrates and increases bone-to-implant contact in vivo. However, bacterial adhesion is also strongly influenced by roughness and surface chemistry. The aim of this study was to investigate if chemical surface modifications alter initial bacterial attachment. To achieve this, two polyelectrolyte layers [chitosan (Ch) and poly(L-lysine) (PLL)] were used to coat Ti surfaces with different roughness (PT [Ra 3.0 µm) with 1 mm thickness and 15 mm diameter. The disks were cleaned with a sequential acetone-isopropanol-ethanol-distilled water protocol and then sterilized with oxygen plasma followed by autoclave. Chitosan (Ch, Mw = 125,000 - 350,000 g/mol, deacetylation degree 80 - 89%, medical grade) was obtained from NovaMatrix (Drammen, Norway). Poly(L-lysine) (PLL, Mw = 150,000 - 300,000 g/mol, medical grade) was purchased from Sigma-Aldrich (St. Louis, MO). Glacial acetic acid, sodium chloride, acetone, isopropanol, and ethanol were obtained from Sigma-Aldrich (St. Louis, MO). PLL solutions were 0.1 mg/ml in 0.15 M aqueous NaCl. The Ch solution was 1.5 mg/ml dissolved in 0.1 M acetic acid containing 0.14 M aqueous NaCl. The Ch15 solution was prepared at 60oC for 15 minutes. Polyelectrolyte solutions were filtered through a sterilized PTFE filter (pore size 0.2 μm) and 300 µl were added to the cleaned and sterilized PT and SLA surfaces at room temperature for 2 hours. Each coating was followed by a 5 minute rinse in either 0.15 M NaCl for PLL or 0.14 M NaCl for Ch and dried. Anti-microbial test Nine reference strains (Table 1) were tested on the experimental surfaces. Lyophilized bacterial stocks (American Type Culture Collection, Rockville, MD) were rehydrated in Mycoplasma broth base (BBL, Becton-Dickinson and Co., Sparks, MD). All strains were grown on Mycoplasma agar base (BBL, Becton-Dickinson and Co., Sparks, MD) supplemented with
5% defibrinated sheep blood, 5 μg/ml hemin (Sigma-Aldrich) and 0.3 μg/ml menadione (SigmaAldrich) under anaerobic conditions (80% N2, 10% CO2 and 10% H2). Table I. Reference strains employed for the adhesion assays Species Aggregatibacter actinomycetemcomitans serotipe b Actinomyces israelii Campylobacter rectus Eikenella corrodens Fusobacterium nucleatum subsp. nucleatum Parvimonas micra Porphyromonas gingivalis Prevotella intermedia Streptococcus sanguinis
Strain*
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