Reduced bacterial adhesion on titanium surfaces micro-structured by ultra-short pulsed laser ablation

Despite enormous advance in modern implant medicine, the prevalence of patients suffering from implant-associated infections is still in the order of 20% [1]. These infections are caused by bacterial biofilms, a sessile multi-microbial community surrounded by self-produced extracellular polymers. Intrinsic resistance to antibiotic agents and host immune defense mechanisms make the treatment of biofilms difficult [2]. Implant research aims at avoiding biofilm formation from the early beginning by developing novel implant materials, which inhibit bacterial adhesion and growth. A multitude of chemical implant functionalizations, ranging from metallic nanoparticles to antimicrobial polymers, were already reported to inhibit microbial colonization in vitro [3–6]. However, chemical modifications may be an obstacle for medical device approval. This problem could be avoided by creating bacteria-repellent properties solely from surface structuring. A micro-structure design with potential anti-adhesive effects is the Sharklet™ surface. It is inspired by the skin of fast-moving sharks and consists of diamond-shaped, geometrically ordered micro-structures [7]. It was shown that Sharklet™ surfaces produced from poly-(dimethyl siloxane) elastomers (PDMS) reduce bacterial adhesion of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Mycobacterium abscessus [8–12]. Moreover, the structure also decreases attachment of algal spores, blood platelets, endothelial and epithelial cells [11, 13, 14].

The aim of the present study was to generate a Sharklet™-like micro-structure on the common implant material titanium and to assess bacterial biofilm formation on this surface. The exact mechanism of the Sharklet™ effect has not yet been completely understood. It is hypothesized that particular arrangement of topographical features with appropriate dimensions create a stress gradient within the cell membrane during initial contact resulting in lower attachment rates [15]. Therefore, in addition to Sharklet™, groove and grid structures, which have similar dimensions, were examined. All structures were generated by ultra-short pulsed laser ablation. SEM images of micro-structured titanium are shown in Figure 1. The samples were colonized with S. aureus, a major implant-associated pathogen. Biofilm was grown up to 24 h and bacterial surface coverage was quantified at the structured/smooth titanium interface.

Figure 1:

SEM images of analyzed micro-structures.

All micro-structures were generated on disk shaped titanium specimen (grade 4, Ø 12 mm) as 1×1 mm squares by ultra-short pulsed laser ablation. For surface machining of titanium samples, a commercially available amplified Ti-Sapphire femtosecond laser system (Femtopower Compact Pro, Femtolasers Produktions GmbH, Vienna, Austria) was used. This system delivers sub-30-fs pulses at 800 nm wavelength with energy of up to 1 mJ, and a repetition rate of 1 kHz. Three different structural designs were implemented: (A) the Sharklet™ like design consisted of 2 μm wide grooves of varying lengths (from 4 μm to 16 μm). These grooves were arranged into a periodic, diamond-like pattern at a fixed spacing of 2 μm between adjacent features. The grooves depth was ≥2 μm; (B) linear grooves of 2 μm width, >=2 μm depth and a fixed spacing of 2 μm between adjacent grooves; and (C) grid structures generated by orthogonal overlapping of two groove structure with dimensions the same as B. A mask projection technique was used for structuring. In doing so, a 100 μm wide square mask (for Sharklet™ like) and 100 μm wide line mask (for groove and grid structure) was imaged onto the sample surface with a 50-times magnification using a 50× microscope lens (Leica HCX PL APO L 50×/0.55 UVI, Leica Microsystems, Mannheim, Germany) integrated in an autofocus system (INH200, Vistec, Milpitas, CA, USA). This technique enables high quality, reproducible structuring and a finer depth control of the surface features.

As shown in Figure 2A, with beginning of biofilm growth, a reduced surface coverage on Sharklet™ like micro-structures could be observed. This trend was maintained up to 24 h of cultivation (Figure 2A, B). Statistical analysis revealed a total p-value of 0.074 for differences between smooth and Sharklet™ like structured titanium. After 24 h, the differences reached statistical significance (Table 1). The detailed p-values are listed in Table 1. The reduced biofilm surface coverage is in line with findings from Chung et al. [8]. They detected a similar effect for S. aureus on Sharklet™-structured PDMS surfaces. The rate of reduction was higher than 40%, as also found by Reddy et al. [12] and May et al. [10]. The reduction rate determined in the present study was about 15%. The most plausible explanation is that this is related to the different materials used. Whereas S. aureus biofilm formation on PDMS typically takes 14 days [8], biofilm formation on titanium needs only several hours. This may influence the effectiveness of the Sharklet™-like micro-structured titanium.

Figure 2:

Bacterial colonization on micro-structured titanium

Prior to each experiment, Staphylococcus aureus (DSM 2569, German collection of microorganisms and tissue culture cells, Braunschweig, Germany) was pre-cultivated in tryptic soy broth supplemented with 10% yeast extract (TSB) for 96 h to reach late stationary phase. Pre-cultures were diluted in 50% TSB supplemented with 25 mM glucose and adjusted to an optical density at 600 nm of 0.001 (approx. 1×10⁶ cfu/mL). Cells were allowed to adhere to titanium specimens for 1 h at 37 °C under gentle rotation at 50 rpm. Then, medium was exchanged and biofilm was cultivated up to 24 h at 37 °C without agitation. After 1, 4.5, 6, 7.5, 9, 10.5, 12, 13.5, 15 and 24 h, test specimens were removed, fluorescently stained using LIVE/DEAD^®BacLight™ Bacterial Viability Kit (Life Technologies, Darmstadt, Germany) and fixed using 2.5% glutardialdehyde in PBS. Bacterial colonization was analyzed by CLSM (Leica TCS SP2, Leica Microsystems, Mannheim, Germany). For each structure, 4 pictures were taken at the structure/smooth interface. Percent surface coverage on structured and smooth titanium was calculated using ImageJ 1.48v (Wayne Rasband, National Institutes of Health, USA, http://imagej.nih.gov/ij/). Surface coverage over time and standard deviation are depicted in A, C and E for Sharklet™-like, groove and grid micro-structures, respectively. B, D and F show representative microscopic images of bacterial colonization after 24 h at Sharklet™-like, groove and grid interphase, respectively. Smooth titanium is shown on the left side of the dotted line and micro-structured titanium on the right side.

Interestingly, the groove and the grid micro-structures showed the same effect as found for the Sharklet™-like structure (Figure 2C, E). Bacterial surface coverage was decreased on structured surfaces compared to smooth ones from the beginning of the biofilm growth up to 24 h (Figure 2D, F). Statistical analysis revealed a total p-value of 0.013 (for groove structures) and 0.017 (for grid structures) and therefore statistically significant differences for structured/smooth titanium. The detailed p-values are listed in Table 1. As for the Sharklet™-like structure, the rate of reduction was approximately 15% for both structures. Equivalence testing also supported that biofilm surface coverage on all micro-structures was largely comparable (Table 2).

The patented Sharklet™ structure was originally developed for inhibition of settlement of swimming algal spores [13]. Afterwards, their effectiveness was also proven for bacterial attachment [8–12]. The mechanism proposed to explain inhibition of algal spore attachment – the increasing of intracellular stress gradients due to cell contact with two geometrically different features [15] – cannot be used for explanation of reduced bacterial attachment. The dimension of a single bacterial cell (e.g. S. aureus has a diameter of 1 μm) is smaller than the 2 μm spacing between neighboring feature of the Sharklet™ structure. A single cell cannot contact two neighboring features and experience an impact of stress gradient. Other mechanisms should be responsible for the anti-adhesive effect of these micro-structured surfaces. According to the results of this study, micro-structured surfaces become unfavorable for initial cell attachment even if the size is larger than the bacterial cell. Possibly, further reduction of structural dimensions can increase the anti-adhesive effect, because bacterial cells will experience intracellular stress gradients proposed by Schumacher et al. [15]. This question will be clarified in further studies. Apart from the stress gradient, it can be assumed that physical interactions between attached bacterial cells and surrounding surface features negatively influence biofilm development [8, 9]. Even though the effect mechanism for reduced S. aureus surface coverage is not known yet, the current study together with previously published investigations demonstrate an attachment inhibitory effect of micro-structures per se. The essential question whether micro-structured surfaces mainly reduce cell attachment or if they also alter bacterial biofilm development should be addressed in future studies.

In conclusion, the present study demonstrates that (1) solely due to micro-structuring of titanium samples reduced surface coverage of S. aureus could be achieved; and (2) different pattern designs with similar structure feature dimensions show similar effectiveness. Therefore, it can be presumed that the effect of Sharklet™ is more a result of structure feature dimensions than structure design.

Concerning clinical applications, the effectiveness of the micro-structures should be further improved (e.g. by optimization of structure dimensions) and evaluated for their interaction with host tissue. The latter may be important for the structure design choice. This study serves as prove-of-principle, showing that micro-structures carry potential for anti-bacterial functionalization of titanium surfaces, and is therefore a promising basis for further investigations.