Probing the nanoadhesion of Streptococcus sanguinis to titanium implant surfaces by atomic force microscopy

Abstract

As titanium (Ti) continues to be utilized in great extent for the fabrication of artificial implants, it is important to understand the crucial bacterium-Ti interaction occurring during the initial phases of biofilm formation. By employing a single-cell force spectroscopy technique, the nanoadhesive interactions between the early-colonizing Streptococcus sanguinis and a clinically analogous smooth Ti substrate were explored. Mean adhesion forces between S. sanguinis and Ti were found to be 0.32±0.00, 1.07±0.06, and 4.85±0.56 nN for 0, 1, and 60 seconds contact times, respectively; while adhesion work values were reported at 19.28±2.38, 104.60±7.02, and 1,317.26±197.69 aJ for 0, 1, and 60 seconds, respectively. At 60 seconds surface delays, minor-rupture events were modeled with the worm-like chain model yielding an average contour length of 668±12 nm. The mean force for S. sanguinis minor-detachment events was 1.84±0.64 nN, and Poisson analysis decoupled this value into a short-range force component of -1.60±0.34 nN and a long-range force component of -0.55±0.47 nN. Furthermore, a solution of 2 mg/mL chlorhexidine was found to increase adhesion between the bacterial probe and substrate. Overall, single-cell force spectroscopy of living S. sanguinis cells proved to be a reliable way to characterize early-bacterial adhesion onto machined Ti implant surfaces at the nanoscale.

Keywords: atomic force microscopy; bacterial adhesion; biophysics; dental implants; titanium.

Figure 1

Ti substrate characterization. Notes: (A) Representative SEM image of the employed Straumann machined Ti discs. (B) Consistent with previous reports, contact angle measurements were found to be 67.0°±5.0°, demonstrating a slightly hydrophilic nature. (C) AFM surface profiles for two independent machined Ti discs, obtained from 10×10 μm scans. (D) AFM 3D reconstruction image showing the topography of machined Ti surfaces with high-resolution (Z=700 nm). Abbreviations: AFM, atomic force microscopy; SEM, scanning electron microscopy; Ti, titanium; 3D, three-dimensional.

Figure 2

Streptococcus sanguinis–Ti adhesive interactions probed by atomic force microscopy. Notes: Representative force-curves for the unbinding of S. sanguinis bacterial probes after 0, 1, and 60 seconds surface contact times. Insets show the comparison between bacterial probes and the poly-DOPA coated probes (controls) at each time point for both studied parameters (average of three independent probes) (*P<0.05, Kruskal–Wallis). Abbreviation: Ti, titanium.

Figure 3

Worm-like chain modeling and Poisson analysis of Streptococcus sanguinis–Ti unbinding events. Notes: (A) Histogram for the predicted contour length obtained for minor-detachment events across three independent S. sanguinis probes (n=661 events). Inset represents the fitting process carried out on the analysis software. (B) Force histogram for minor-detachment events observed at increased contact times. (C) By plotting average unbinding force against variance for each cell probe, it is possible to predict values for F_SR and F_LR. The analysis for four independent S. sanguinis probes is shown (R²=0.92). Abbreviations: F_LR, long-range force; F_SR, short-range force; Ti, titanium.

Figure 4

Effect of 2 mg/mL CHX on Streptococcus sanguinis–Ti interaction. Notes: Adhesion force and adhesion work values following the addition of 2 mg/mL CHX in TRIS buffer. Increased values for both parameters was observed (*P<0.05, Kruskal–Wallis). Insets are representative force-curves demonstrating the changes in unbinding behavior after CHX exposure. Box plot represents medians and bars represent minimum and maximum values. Abbreviations: CHX, chlorhexidine; Ti, titanium.