Development of Laser-Structured Liquid-Infused Titanium with Strong

Research Article www.acsami.org

Development of Laser-Structured Liquid-Infused Titanium with Strong Biofilm-Repellent Properties Katharina Doll,*,† Elena Fadeeva,‡,∥ Joern Schaeske,† Tobias Ehmke,§ Andreas Winkel,† Alexander Heisterkamp,§,∥ Boris N. Chichkov,‡,∥ Meike Stiesch,† and Nico S. Stumpp*,† †

Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany ‡ Department of Nanotechnology and §Department of Biomedical Optics, Laser Zentrum Hannover e.V., Hollerithalle 8, 30419 Hannover, Germany ∥ Institute of Quantum Optics, Leibniz University of Hannover, Welfengarten 1, 30167 Hannover, Germany S Supporting Information *

ABSTRACT: Medical implants are commonly used in modern medicine but still harbor the risk of microbial infections caused by bacterial biofilms. As their retrospective treatment is difficult, there is a need for biomedical materials that inhibit bacterial colonization from the start without using antibacterial agents, as these can promote resistance development. The promising concept of slippery liquid-infused porous surfaces (SLIPS) possesses enormous potential for this purpose. In the present study, this principle was applied to titanium, a common material in implantology, and its biofilmrepellent properties were demonstrated. To simplify prospective approval of the medical device and to avoid chemical contamination, surface structuring was performed by ultrashort pulsed laser ablation. Four different structures (hierarchical micro- and nanosized spikes, microsized grooves, nanosized ripples, and unstructured surfaces) and five infusing perfluoropolyethers of different viscosities were screened; the best results were obtained with the biomimetic, hierarchical spike structure combined with lubricants of medium viscosities (20−60 cSt at 37 °C, 143 AZ, and GPL 104). The surfaces exhibited extremely low contact angle hysteresis, as is typical for liquid-infused materials and a reliable 100-fold reduction of human oral pathogen Streptococcus oralis biofilms. This characteristic was maintained after exposure to shear forces and gravity. The titanium SLIPS also inhibited adherence of human fibroblasts and osteoblasts. Toxicity tests supported the explanation that solely the surface’s repellent properties are responsible for the vigorous prevention of the adhesion of bacteria and cells. This use of physically structured and liquid-infused titanium to avoid bioadhesion should support the prevention of bacterial implantassociated infections without the use of antibacterial agents. KEYWORDS: slippery liquid-infused porous surface, biofilm, implant infection, Streptococcus oralis, titanium, laser structuring

■

catheter patients.7,8 These infections are caused by bacterial biofilms, which are attached bacterial cells of multiple species embedded into a self-produced polysaccharide matrix and altered in their phenotype.9 Implants are prone to bacterial colonization, as the biomaterial itself influences the host’s immune response. Frustrated phagocytosis, a possible result of insufficient implant degradation and endocytosis by macrophages, leads to selective reduction in antibacterial activity at the implant site.10 Once developed, biofilms exhibit several resistance mechanisms.11 The extracellular polysaccharide matrix reduces antibiotic penetration, leading to lower local concentrations of antibiotic. Bacteria inside the biofilm exhibit reduced metabolic activity and a unique physiology. This causes

INTRODUCTION Implantssuch as joint prostheses, pacemakers, stents, mechanical heart valves, and dental implantshave become a standard treatment procedure in modern medicine. Their total revenue in Germany has continuously increased over the last few years, and the largest proportion by far are dental prostheses with ∼50%.1,2 The improvements in implants include implementation in drug delivery systems, application of micro- and nanotechnology, and combination with tissue engineering procedures.2 This is one of the fastest growing areas of medical technology,1,2 and the number of implanted medical devices is steadily increasing.3 In parallel, the prevalence of implant-associated infections is also increasing.4−6 This leads to follow-up treatments that are a burden to the patient and to rising health care costs. The rate of implant-associated infection is between 1 and 5% in orthopedics but is as high as 20% for dental implants and © 2017 American Chemical Society

Received: December 16, 2016 Accepted: February 23, 2017 Published: February 23, 2017 9359

DOI: 10.1021/acsami.6b16159 ACS Appl. Mater. Interfaces 2017, 9, 9359−9368

Research Article

ACS Applied Materials & Interfaces

used to obtain uniform isotropic structures. A mask projection technique was used to fabricate grooves. A 250 μm wide slit mask was projected onto the sample surface with 50-fold demagnification and using a 50× objective (Leica HCX PL APO L 50×/0.55 UVI, Leica Microsystems, Mannheim, Germany) integrated into an autofocus system (INH200, Vistec, Milpitas, USA). This provided structures with 5 μm groove width and periodicities of 10 μm. The depth of the structure was ∼2 μm. The use of the mask projection technique assures high quality, reproducible structuring, and finer depth control of the surface features. Ripples were also fabricated by using an achromatic lens with a focal distance of 200 mm. The scanning speed in the x-direction was 1000 μm/s, and the translation steps along the y-direction were 14 μm. Ripples were fabricated at a laser fluence of 0.5 J/cm2. All structures were initially visualized by scanning electron microscopy. The average structure roughness (Ra) and the average maximum height of the profile (Rz) were characterized using a MahrTalk Perthometer (Mahr GmbH, Göttingen, Germany) with MarSurf XR 20 surface texture analysis software. At least four measurements were performed within the central area of each structure type. The evaluation length was 2.00 mm, and the BFW A 4-45-2/90 20496 standard stylus with a 2 μm tip radius and a 90° tip angle was used. For SLIPS generation, specimens were dip coated with a fluorinated polymer (Antispread E 2/30 FE 60, Dr. Tillwich GmbH Werner Stehr, Horb-Ahldorf, Germany). The chemical functionalization avoids oil creeping on the surface. Five perfluoropolyether lubricants (DuPont de Nemours, Neu-Isenburg, Germany) with different viscosities were spin coated onto the surfaces, thus ensuring a thin and homogeneous liquid film: Krytox GPL 100 (4 cSt at 40 °C), Krytox 143 AZ (18 cSt at 38 °C), Krytox GPL 104 (60 cSt at 40 °C), Krytox GPL 105 (160 cSt at 40 °C), and Krytox GPL 106 (240 cSt at 40 °C). Prior to bacterial experiments, SLIPS and uncoated control samples were sterilized by irradiation with UV light (253.7 nm) for 20 min. Contact Angle Measurements. Contact angle measurements were performed using a contact angle goniometer (OCA 40, Software SCA 202 V.3.61.4, DataPhysics Instruments GmbH, Filderstadt, Germany) at 21 °C and air humidity of 25%. One sample with a diameter of 12 mm was prepared for each condition, and the measurement was repeated four times with water droplets of 10 μL placed at different positions. For static contact angle, the means were calculated of the horizontally positioned specimen’s right and left water contact angles. Contact angle hysteresis was measured by tilting the surface. The difference between the advancing and receding contact angles just before the droplet starts sliding was calculated as contact angle hysteresis. The samples were tilted to a maximum of 50°. If water droplets did not start sliding at this angle, they were defined as pinned. Optical Coherence Tomography. For visualizing lubricants on the structured titanium surfaces, swept source optical coherence tomography (OCS1300SS, Thorlabs, Newton, USA) was used. In this system, a rapidly tuned narrowband laser source is broken into two arms: one reference arm and one sample arm, which scans the item of interest.26,27 Light propagating in the sample arm is backscattered, reflected from various structures inside the sample and superimposed with light from the reference arm.26,27 An axial scan can be constructed from the depth-resolved spectral interference signal. Scanning the laser beam over the sample allows adjacent interferograms to be stitched, which results in two or even three-dimensional imaging. The axial resolution of the used system is specified as 9 μm in water. For data evaluation, B-scans (2D images) were loaded as an image sequence into ImageJ 1.48v (Wayne Rasband, National Institutes of Health, USA, http://imagej.nih.gov/ij/). Because interfaces inside the sample lead to strong scattering and reflection, due to changes in refractive index, they give a very strong signal in the B-scan and can be clearly identified. Threshold fading of all unimportant information was applied from the center of each sample. The resulting image showed the full titanium interface and a part of the lubricant interface, which was extrapolated to fit the titanium surface. The lubricant thickness (geometric path length) was calculated by dividing the lubricanttitanium distance present in the optical coherence tomography images

increased resistance to antibacterial substances that affects molecules and cellular pathways that are mainly activated in free-living cells. The combination of lowered immune response and increased bacterial resistance makes it difficult to treat implant-associated infections. For medical applications, the ideal implant would inhibit bacterial attachment from the very beginning. As antimicrobial treatment can be omitted, the risk of developing bacterial resistance is minimized. Slippery, liquid-infused porous surfaces (SLIPS) were introduced by Aizenberg’s group12,13 and possess great potential for this purpose. These were inspired by Nepenthes pitcher plants,13 which capture a liquid film on their peristomes. This liquid film inhibits the adherence of the insect’s feet and makes them slide within the pitcher.14 The SLIPS functionality has been transferred to various surfaces by combining surface structuring, chemical modification, and infusion with lubricants. Three main criteria have to be fulfilled in their design: (1) the lubricant has to adhere stably to the surface, (2) the surface must be preferentially wetted by the lubricant rather than by the repellent liquid, and (3) the lubricant and the repellent liquid must be immiscible.13 Liquidinfused surfaces made of polymers or silicone have already been shown to withstand bacterial colonization of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus.12,15−18 It was also possible to create these surfaces from titanium, a common biomaterial for dental implants and joint replacement.19,20 So far, the generation of titanium SLIPS has been based on surface structuring like etching20 or chemical functionalization19 that involve (toxic) chemical compounds, which may be an obstacle for medical device approval. Furthermore, titanium SLIPS antibiofilm properties have not yet been demonstrated.21 The aim of this study was to create liquid-infused titanium using ultrashort pulse laser ablation, a chemical agent-free manufacturing process. The laser ablation technique enables structuring without influencing the material’s biocompatibility and can be selectively applied to specific implant areas. After structuring and hydrophobization, lubricants build up a liquid layer on the surface. Furthermore, the ability of titanium SLIPS to reduce microbial colonization should be demonstrated. As dental implants are the most frequently used implant type, the oral commensal Streptococcus oralis was chosen as the model organism. The Gram-positive bacterium has been shown to be an important initial colonizer that influences biofilm formation in the human mouth.22−24 Once attached, it serves as a binding site for late colonizing, pathogenic bacteria that promote the development of peri-implantitis.24,25

■

EXPERIMENTAL SECTION

Titanium SLIPS Fabrication. Titanium SLIPS were generated from disk-shaped titanium specimens (grade 4) and were 3 or 12 mm in diameter. Control samples were finished with 45 μm diamond abrasive polishing wheels. For surface structuring, a commercially available amplified Ti-Sapphire femtosecond laser system (Femtopower Compact Pro, Femtolasers Produktions GmbH/Spectra Physics, Vienna, Austria) was used. It 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 structures were implemented: spikes, grooves, and ripples. An achromatic lens with a focal distance of 200 mm was used to focus for the fabrication of spikes. The laser beam was scanned along its linear polarization direction (x-direction). The scanning speed in the x-direction was 800 μm/s, and the translation step in the y-direction was 15 μm. Spike structures were generated by laser ablation at a fluence of 8 J/cm2. Circularly polarized laser light was 9360


Research Article

ACS Applied Materials & Interfaces Table 1. Characteristics of Surface Structures Used for SLIPS Generation surface spikes grooves ripples unstructured a

category hierarchic microstructure nanostructure flat

main feature dimension

sub feature dimension

average roughness Ra

[μm]

[μm]

[μm]

10−20 5/10 0.7 n.a.a

≤0.2 n.a.a ≤0.2 n.a.a

2.15 0.46 0.07 0.16

± ± ± ±

0.10 0.01 0.00 0.01

average maximum profile height Rz [μm] 11.53 1.81 0.69 1.02

± ± ± ±

0.77 0.06 0.15 0.08

N.a., not applicable.

(optical path length) by the lubricants refractive index, which was 1.3 for all lubricants tested (as specified by the distributer, H. Costenoble GmbH Co. KG., Eschborn, Germany). Bacterial Strains and Culture Conditions. Cultures of Streptococcus oralis (ATCC 9811, American Type Culture Collection, Manassas, USA) were stored at −80 °C as glycerol stocks. Prior to experiments, bacteria were grown in trypton soya broth (Oxoid Limited, Hampshire, UK) supplemented with 10% yeast extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for 24 h. Experiments were performed using nine specimens for each condition. For biofilm formation, precultures were diluted in the same medium supplemented with 50 mM glucose (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and adjusted to an optical density at 600 nm of 0.1. According to standard plate counting, this corresponds to 4 × 1010 CFU/ml for S. oralis. Specimens were incubated in the bacterial suspension, and the biofilm was allowed to grow for 18 h at 37 °C under static conditions. Before evaluation, samples were gently rinsed twice with phosphate-buffered saline to remove nonbound cells. For analysis of initial bacterial adhesion, precultures were washed twice with 50 mM TRIS HCl buffer, pH 7.5, to remove any growth medium and adjusted to an optical density at 600 nm of 0.7 in the same buffer. According to standard plate counting, this corresponds to 3 × 1011 CFU/ml for S. oralis. Specimens were incubated with the bacterial suspension, and adhesion was allowed for 5 h at 37 °C under constant agitation at 150 rpm. Nonadherent cells were removed by rinsing six times with sterile distilled water. Confocal Laser Scanning Microscopy Analysis. Bacteria were fluorescently stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies, Darmstadt, Germany). The two fluorescent dyes were applied simultaneously as a 1:2000 dilution in phosphate-buffered saline and incubated for 15 min at ambient temperature. Cells were fixed with 2.5% glutardialdehyde in phosphate-buffered saline at 4 °C for 15 min. Imaging was performed by confocal laser-scanning microscopy (CLSM, Leica TCS SP2, Leica Microsystems, Mannheim, Germany) with image areas of 1200 × 1200 μm2 or 190 × 190 μm2. The acquired biofilm stacks were loaded into the Imaris x64 6.2.1 software package (Bitplane AG, Zurich, Switzerland) and processed to give 3D images. The biofilm volume per image was calculated using the surface mode. Quantification of initial bacterial adhesion was performed with ImageJ 1.48v. By dividing the total surface area covered with bacteria by the mean surface area of a single bacterium, the number of adhering bacteria per image was calculated. Toxicity Screening. S. oralis precultures were diluted in trypton soya broth and the optical density at 600 nm was adjusted to 0.1. Bacterial suspension was mixed with 5, 10, or 20% lubricant, Antispread, or sterile distilled water as control and incubated in a 96-well plate for 24 h at 37 °C under agitation at 500 rpm. Absorption at 600 nm was measured using a multimode reader (Synergy 2, BioTek, Bad Friedrichshall, Germany). Bacterial viability was quantified with the BacTiter-Glo Microbial Cell Viability Assay (Promega, Mannheim, Germany). The BacTiter-Glo reagent was first mixed with bacterial suspension. After 5 min of incubation at ambient temperature, luminescence was measured using a multimode reader (Synergy 2, BioTek). Absorption and luminescence values were normalized against the water control samples. Cytocompatibility and Cell Adhesion Testing. For cytocompatibility testing, human gingival fibroblasts (HGF, Provito, Berlin, Germany) were precultured for 24 h in Dulbecco’s modified Eagle

medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (PAN-BIOTECH GmbH, Aidenbach, Germany), 100 U/ml penicillin, and 100 μg/mL streptomycin (Biochrom AG, Berlin, Germany) at 37 °C in a 5% CO2, 95% humidified air atmosphere in a 96-well plate. Culture medium was replaced by fresh medium, followed by the addition of 5, 10, or 20% lubricant, Antispread, or sterile distilled water as control. Cells were incubated for 24 h under constant agitation at 500 rpm. For cellular viability to be quantified, CellTiter-Blue reagent (CellTiter-Blue Cell Viability Assay, Promega, Mannheim, Germany) was added followed by 4 h incubation at 500 rpm and 37 °C. The fluorescence was recorded using a multimode reader (λex = 530 nm, λem = 590 nm, Synergy 2, BioTek), and the values were normalized against the control samples. Cell adhesion testing was performed using human gingival fibroblasts and normal human osteoblasts (NHOst, Lonza, Walkersville, USA). Cells were seeded onto nine specimens of each condition at a concentration of 75,000 or 50,000 cells/well for fibroblasts or osteoblasts, respectively, and incubated for 24 h at 37 °C in a 5% CO2, 95% humidified air atmosphere. For live/dead staining, Calcein-AM (Invitrogen, Thermo Fisher Scientific, Waltham, USA) and propidium iodide (Sigma-Aldrich, St. Louis, USA) were added to a final concentration of 1 μg/mL each. After 15 min of incubation at 37 °C, fluorescence was recorded with a Zeiss Axio Scope.A1 fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) using a 20× immersion objective. Statistical Analysis. Data were documented and evaluated with GraphPad Prism 5.02 software (GraphPad Software, Inc., La Jolla, USA). The d’Agostino and Pearson omnibus normality test was used to test for Gaussian distribution. Contact angle measurement, biofilm formation screening, and tests of the toxicity of lubricants to bacteria and fibroblasts were analyzed using ordinary two-way ANOVA with Bonferroni’s test for multiple comparison. Detailed analysis of spike SLIPS and their long-term effects were evaluated using the Kruskal− Wallis test with Dunn’s test for multiple comparison. Family wise significance level was set to α = 0.05 for all statistical analyses.

■

RESULTS AND DISCUSSION Surface Structure Characterization. Previously, it had been shown that aluminum SLIPS based on hierarchical, microand nanostructures show different characteristics regarding lubricant immobilization stability.28 To address these findings, hierarchical micro- and nanosized spikes, microsized grooves, and nanosized ripples were generated by ultrashort pulsed laser ablation for titanium SLIPS formation. All surfaces were initially characterized by scanning electron microscopy and roughness measurements. The main surface characteristics are summarized in Table 1. The hierarchical biomimetic spike structure (Figure 1A) was inspired by leaves of the lotus Nelumbo nucifera. Its components are much smaller than the laser beam focus and exhibit certain regularity in pattern, which is why it is called a “quasi-periodic surface structure”. The structure selforganizes spontaneously under femtosecond laser irradiation29 and consists of a grain-like, convex microstructure, which is additionally covered by nanosized irregular undulations.30 The groove structure (Figure 1B) is an operator-defined geometric microstructure, which was generated by the mask projection 9361


Research Article


Figure 1. Differently structured titanium samples used for SLIPS generation. Scanning electron microscopic images of laser-generated spikes (A), grooves (B), and ripples (C) and unstructured titanium (D) used for SLIPS fabrication.

technique. Its feature dimensions were 5 μm in width with a periodicity of 10 μm. The ripple nanostructure (Figure 1C) is a quasi-periodic surface structure, which also self-organizes under femtosecond laser irradiation. It has multiple nanosized parallel features, which are additionally covered with nanosized subfeatures.30 Unstructured, polished titanium served as a control surface (Figure 1D). To our knowledge, groove-like structures have not yet been used for SLIPS generation. In contrast, nanostructured aluminum SLIPS, generated by sol− gel chemistry or electrodeposition, have already been shown to withstand ice accretion and are stable under flow conditions,31,32 and the comparison of nanostructured and hierarchically structured aluminum SLIPS (comparable to spikes) showed that the former was more stable under flow conditions.28 Contact Angle Measurement. SLIPS generated from differently structured titanium, infused with Krytox lubricants of varying viscosities, were initially characterized by measurements of water contact angles. The uncoated, structured titanium specimens showed distinctly different static contact angles (Figure 2A). Spikes and unstructured titanium exhibited hydrophobic contact angles of ∼130°. Ripples showed superhydrophobic properties with a static contact angle of 150°. In contrast, grooves had a slightly hydrophilic contact angle of ∼70° parallel to the grooves’ direction. When these structures were converted into SLIPS, the static water contact angles showed no statistical differences. They were slightly hydrophobic (∼100°) regardless of structure and lubricant (Figure 2A, C). This indicates that wettability was similar for all lubricants evaluated. Besides static contact angles, contact angle hysteresis was measured. A characteristic property of SLIPS is their strong liquid repellency, signified by their very low contact angle hysteresis of 2−5°.13,32,33 The easy sliding of the droplet is due to the high apparent contact angle, small contact areas, low interfacial surface tension, and an increasing effective droplet mass.34 For spike SLIPS, the measured water contact angle hysteresis was below 5° for all lubricants tested (Figure 2B, D). For groove, ripple, and unstructured SLIPS, contact

Figure 2. Uniform static contact angle and low contact angle hysteresis of liquid-infused titanium made of different structure/lubricant combinations. Mean ± standard deviation of static water contact angle (A) and difference between advancing and receding water contact angle (contact angle hysteresis, B) on differently structured and lubricant-coated titanium. “x” shows structure/lubricant combinations, where water droplets were pinned on the surface and no contact angle hysteresis could be measured. “*” indicates statistical significance with p < 0.05 compared to all other structures with the same coating. (C) and (D) show typical images of static contact angle (C) and contact angle hysteresis (D) of spike SLIPS coated with GPL 104.

angle hysteresis was only measurable for more viscous lubricants (GPL 104, GPL 105, and GPL 106). In this case, contact angle hysteresis was likewise below 5° (Figure 2B). Groove, ripple, and unstructured SLIPS coated with GPL 100 and 143 AZ showed pinning of the water droplet as also found for the uncoated control samples (Figure 2B). For analyzing whether lubricant thickness was the cause of the differences in water droplet behavior, titanium of different structures was infused with 143 AZ or GPL 104 and subjected to optical coherence tomography. Because of differences in the refractive indices of titanium, lubricant, and air, the structures can be identified in an axial view and are shown in Figure S1. In all cases, the lubricant’s thickness is approximately 60−85 μm but varies according to the underlying structure and lubricant. The thickness of 143 AZ is always ∼20 μm lower than the corresponding thickness of GPL 104. Furthermore, the lubricant’s thickness on spike-structured titanium is approximately 10 μm greater than that on the corresponding groove, ripple, or unstructured samples. It may be assumed that the 9362


Research Article

ACS Applied Materials & Interfaces lubricants of lower viscosity (GPL 100 and 143 AZ) on the latter structures were removed to a greater extent during the spin-coating process. The resulting lubricant layer is probably too thin to avoid water droplets contacting the underlying surface, which leads to droplet pinning. Kim et al.28 observed that nanostructured liquid-infused surfaces were more efficient in capturing lubricants of low viscosity during application of centrifugal force than hierarchical, microstructured, or flat surfaces. In this study, the hierarchical spike structurebut not the nanostructured ripples, microstructured grooves, or unstructured surfaceswas able to trap lubricants of lower viscosity, too. Kim et al.28 generated their nanostructures via formation of boehmite nanostructures, resulting in rhombohedral bipyramid crystals of 50−100 nm. In contrast, in this study, the nanostructure generated by femtosecond laser ablation consists of undulations of ∼200 nm. This variation in size and exact nanoshape is probably responsible for the differences in lubricant-capturing capability. According to the results of this study, liquid-infused titanium with characteristic low contact angle hysteresis was formed from all structures when using lubricants of higher viscosity, but only hierarchical spike structures were also able to form stable SLIPS using lubricants of lower viscosity. Biofilm Formation Screening. For the biofilm-repelling properties of the different titanium SLIPS to be evaluated, they were subjected to screening of S. oralis biofilms. The recorded data were divided into five surface coverage categories: (1) nearly no biofilm, (2) only some very small biofilm fragments, (3) medium biofilm fragments all over the surface, (4) surface partially covered by intact biofilm, and (5) entire surface covered by biofilm. Typical images of all categories are shown in Figure 3. The frequency of each category was counted for each combination of structure/lubricant. For spike SLIPS, marked reduction in biofilm surface coverage was observed (Figure 3, spikes). Their median category of surface coverage was 2 (interquartile range IQR 0) for spike SLIPS coated with GPL 100, GPL 105, and GPL 106 and 1 (IQR 1) for those coated with 143 AZ and GPL 104. For the groove, ripple, and unstructured/lubricant combinations, nearly the entire surface was covered by biofilm (Figure 3, grooves, ripples, unstructured). Their median category of surface coverage was 4 (IQR 0) for all combinations tested. The structured control surfaces without coating displayed full biofilm coverage (Figure 3). Their median surface coverage was 5 (IQR 0). Statistical analysis revealed that, for all SLIPS, biofilm surface coverage was significantly reduced compared to their corresponding uncoated surfaces. Furthermore, spike-structured SLIPS displayed a significant reduction in biofilm surface coverage compared to that of all other SLIPS. Spike SLIPS were only covered by some small biofilm fragments, whereas all other SLIPS were at least partially overgrown by biofilm. As mentioned for the measured contact angle hysteresis, grooves, ripples, and unstructured titanium appeared to be less effective in trapping lubricants of lower viscosity. For biofilm formation screening, they were also found to be less effective using lubricants of higher viscosity. For lubricant thickness under conditions of biofilm formation to be analyzed, differently structured titanium samples coated with 143 AZ and GPL 104 were incubated in an aqueous environment for 18 h and exposed to optical coherence tomography. As shown in Figure S2, the lubricant layer on spike SLIPS was reduced by 20−30% due to immersion, but a film of ∼60 μm thickness was maintained on the surface. In contrast, on grooves, ripples, and

Figure 3. Biofilm formation screening on titanium SLIPS made of different structure/lubricant combinations. Biofilm formation on different titanium SLIPS was analyzed by CLSM. One image was taken at each sample’s center (see typical images at the top; scale bar: 300 μm). Biofilm level was classified into five groups according to biofilm surface coverage with 1 (white), showing nearly no bacteria, up to 5 (black), corresponding to biofilm on the entire surface. The frequency of each group was counted for each structure/lubricant combination.

unstructured titanium, complete removal of the lubricant film was observed. Because of the hydrophobic effect, the lubricants tend to separate as spheres from a hydrophilic environment, such as the biofilm cultivation medium or PBS, to decrease the overall free energy.35 The resulting uncoated titanium surfaces are prone to bacterial attachment and subsequent biofilm formation. It can be assumed that only the spike structure thermodynamically favors lubricant spreading on the surface over hydrophobic sphere formation in a hydrophilic environment, probably due to its hierarchical nature. Therefore, only the spike-structured titanium possesses the potential for stable SLIPS with biofilm-repellent properties. Detailed Analysis of Spike SLIPS. Following the biofilm screening results, the spike SLIPS were subjected to a more detailed analysis of biofilm formation. For each spike/lubricant combination, the biofilm volume and the live/dead distribution were evaluated. As shown in Figure 4A, biofilm volume on all spike SLIPS was ∼100-fold decreased compared to that of the unstructured, uncoated titanium control. The control surfaces showed a typical S. oralis biofilm under the microscope (compare Figure 3, category 5), whereas the SLIPS surfaces were only incompletely covered by very small, separated biofilm fragments (compare Figure 3, categories 1 and 2). This difference was statistically significant. There was no statistical 9363


Research Article


initial adhesion of S. oralis on liquid-infused titanium was analyzed. For the previous biofilm experiments, specimens were incubated with bacteria suspended in nutrient broth supplemented with glucose under static conditions for 24 h. This facilitates bacteria to sediment, adhere and, reproduce to build up a three-dimensional biofilm surrounded by an extracellular polysaccharide matrix. In contrast, for initial adhesion experiments, bacteria were suspended in buffer solution without nutrients, and samples were incubated under agitation for 5 h only. This leads to the formation of a homogeneous single layer of adhering bacterial cells on the surface, whereas biofilm outgrowth is inhibited.38−41 The number of adhering bacteria and the live/dead distribution was determined for each spike/ lubricant combination. As depicted in Figure 4C, only the lubricants 143 AZ, GPL 104, and GPL 105 gave a statistically significant reduction in adhering cells of ∼10-fold compared to that of the unstructured, uncoated control. For GPL 100 and GPL 106, a slight reduction in adhering cell number was observed, but this did not reach statistical significance. For live/ dead distribution, no statistical differences compared to the control could be detected (Figure 4D). These results support the hypothesis that SLIPS prevent initial cell attachment rather than inhibiting biofilm growth. The experimental setup further revealed that the lubricants behave differently under rotational forces. A statistically significant reduction in bacterial initial adhesion was only found for the lubricants of intermediate viscosity, whereas GPL 100 and GPL 106 showed only slight effects. This was consistent with the results obtained with spike SLIPS coated with different lubricants, exposed to aqueous shear stress for 5 h at 150 rpm and, examined by optical coherence tomography. A lubricant layer could only be detected for the lubricants of intermediate viscosity (Figure S3). It has already been shown that physical forces, such as dynamic flow or centrifugal forces, may remove some of the infused lubricant.28,42,43 As optical coherence tomography was only performed in isolated samples, it is possible that GPL 100 and GPL 106 sometimes stayed on the surface, whereas GPL 105 was sometimes removed. This is depicted in the slight reduction in initially adhering bacteria on surfaces coated with GPL 100 and GPL 106 and the increased standard deviation detected for GPL 105 (Figure 4C). According to the results of this study, there seems to be a defined range where the dimensions of the surface structure and the viscosity of the lubricant lead to the most stable SLIPS. It is probable that lubricants of very low viscosity are not inert enough to withstand physical and energetic forces, whereas lubricants of very high viscosity are not fully trapped by the surface caves. For titanium SLIPS screened in this study, the spike structure combined with lubricants of intermediate viscosity (20−60 cSt at 37 °C, 143 AZ, and GPL 104) provide the greatest stability and effectively prevent initial adhesion of the bacteria to the surface. Long-Term Stability. According to the previous results, spikes coated with 143 AZ and GPL 104 showed the highest effect on bacterial adhesion. Therefore, these combinations were subjected to further analysis of long-term stability. Liquidinfused titanium was kept vertically for 15 days at ambient conditions prior to biofilm cultivation. Biofilm volume and live/ dead distribution were quantified. As shown in Figure 5A, biofilm volume on 143 AZ- and GPL 104-coated SLIPS was statistically significantly reduced by approximately 100-fold compared to the unstructured, uncoated control. The percentage of dead cells was slightly increased by 5 or 10%

Figure 4. Reduced biofilm volume and adhering cell number on spike SLIPS. Biofilm volume Tukey box plot (A) and mean ± standard deviation of live/dead distribution (B) of S. oralis biofilm formation, and adhering cell number Tukey box plot (C) and mean ± standard deviation of live/dead distribution (D) of S. oralis initial adhesion on spike-structured SLIPS coated with different lubricants analyzed by CLSM (per 190 × 190 μm2). Unstructured, uncoated titanium serves as control. “*” indicates statistical significance compared to the control with p < 0.05.

difference between the individual lubricants. For live/dead distribution, the fraction of dead cells increased moderately up to 20% with decreasing lubricant viscosity (Figure 4B). This is probably due to slight impairment of the bacterial cell membrane caused by the lubricants. Krytox perfluoropolyether lubricants consist of fully saturated molecules composed of fluorine, carbon, and oxygen.36 The covalent bond of fluorine and carbon has a high binding energy and is therefore extremely stable. This makes the Krytox lubricants chemically inert and therefore biologically neutral.36 The increase in dead cells found in this study can possibly be attributed to the direct contact between bacterial cells and lubricants. The viscosity dependence may be explained by the greater dispersive capacity of lubricants of lower viscosity. Their hydrophobicity leads to multiple small spheres with a greater contact area compared to lubricants of higher viscosity, which tend to form only a few larger spheres. The slight increase in dead cells could have further supported the reduction in biofilm volume, but most likely cannot fully explain the results. Therefore, other mechanisms should be responsible for the major reduction in biofilm volume detected on all spike SLIPS. Surface antibiofilm properties can be reached in two ways: (1) by inhibiting biofilm growth or (2) by inhibiting initial bacterial adhesion on the surface. Recent studies have already hypothesized that SLIPS mainly inhibit stable bacterial attachment.12,17−19,37 For this hypothesis to be addressed, 9364


Research Article


Figure 5. Reduced biofilm volume on spike SLIPS after 15 days of vertical exposure. Biofilm volume (per 190 × 190 μm2) Tukey box plot (A) and mean ± standard deviation of live/dead distribution (B) of S. oralis biofilm formation on spike-structured SLIPS coated with 143 AZ and GPL 104 and kept vertically for 15 days as analyzed by CLSM. Unstructured, uncoated titanium serves as control. “*” indicates statistical significance compared to control with p < 0.05.

for GPL 104- and 143 AZ-coated SLIPS, respectively (Figure 5B). These results were similar to the findings without vertical exposure (compare Figure 4A and B). Therefore, 143 AZ- and GPL 104-infused spike SLIPS resist gravitational force and ambient condition influences for up to 15 days without losing their biofilm-repellent properties. Toxicity of Lubricants. For it to be confirmed that titanium SLIPS biofilm-repellent properties are not mainly due to the toxic effects of the components, they were analyzed for effects on S. oralis growth. After 24 h of incubation with up to 20% of lubricants or Antispread, the medium’s optical density at 600 nm was recorded to measure bacterial reproduction. Figure 6A shows that the measured absorption for cultivation with up to 20% lubricants or Antispread was not significantly different from the that of the water control samples. In contrast, for the fixative glutardialdehyde, which serves as toxic control, statistically significantly decreased optical densities were detected for all concentrations. As a measure of bacterial viability, cellular ATP content was quantified by the BacTiterGlo assay’s luciferase reaction. A slight reduction in bacterial viability was measured for all lubricants at all concentrations, but which was only statistically significant for 143 AZ and GPL 104 at 10% (Figure 6B). For the glutardialdehyde toxicity control, a nearly complete, statistically significant loss of viability was observed at all concentrations. If the results of biofilm live/dead staining are considered (Figures 4B and 5B), there seems to be some impairment of bacterial cell integrity in direct contact with Krytox lubricants. However, indisputable bacterial growth and a bacterial viability of ∼70% in the presence of up to 20% lubricant or Antispread could be detected. During biofilm experiments, a maximum concentration of only 0.3% lubricant in solution was used. Therefore, the strong biofilm-repellent properties of titanium SLIPS cannot be attributed to general toxic effects of lubricants or Antispread but are most likely due to inhibition of initial bacterial adhesion. Cytocompatibility and Cell Adhesion. Besides resistance to biofilm attachment, an essential characteristic of potential implant materials is good compatibility with human body cells. In an initial analysis of biocompatibility, the cytotoxicity of the components of the most effective SLIPS, spikes coated with

Figure 6. Bacterial growth in the presence of lubricants. Mean ± standard deviation of S. oralis suspension’s optical density at 600 nm (OD600) (A) and ATP content (B) after growth in the presence of differently concentrated lubricants, Antispread, or 2.5% glutardialdehyde (GDA). Two-way ANOVA showed statistically significant differences in (A) the optical density of bacterial suspensions incubated with GDA (5, 10, 20%) and (B) the ATP content of bacterial suspensions incubated with 143 AZ (10%), GPL 104 (10%), and GDA (5, 10, 20%) compared to that of the control samples.

143 AZ and GPL 104, were evaluated on human gingival fibroblasts. Cellular viability was quantified using the CellTiterBlue assay, which measures cellular reduction capacity via resazurin color change. As shown in Figure 7A, metabolic activity was not significantly different from that of the control cells when incubating with 5% 143 AZ, GPL 104, or Antispread. With increasing lubricant concentration, metabolic activity clearly decreases. This effect is more prominent for GPL 104 than for 143 AZ. In contrast, metabolic activity slightly increases for 10% Antispread. This may also be a result of cellular stress. In summary, incubation with lubricants and Antispread up to 5% has almost no influence on cellular viability. Therefore, it can be concluded that these concentrations are cytocompatible. For dental or orthopedic implants, such as knee and hip prostheses, biomedical materials must ensure good tissue compatibility, especially in bone, to ensure healing and unrestricted functionality. Cochlea implants or cardiac pacemakers, on the other hand, suffer from fibrosis44−46 and aim to avoid cell adhesion. For cell adhesion on liquid-infused titanium to be analyzed, human gingival fibroblasts and human osteoblasts were seeded on spikes coated with 143 AZ and GPL 104. After 24 h of incubation, cells were fluorescently labeled. Microscopic observation revealed that neither cell type adhered to the liquid-infused surfaces (Figure 7B). Unstructured, uncoated titanium control surfaces, in contrast, showed 9365



Research Article

■

CONCLUSIONS The concept of liquid-infused surfaces has already been applied to numerous materials and demonstrated to effectively withstand bacterial and cellular adhesion under in vitro and in vivo conditions.21 It therefore possesses enormous potential for several medical applications. However, the principle obstacle for all these surfaces will be to fulfill all requirements for medical device approval.21 One possibility to simplify the validation process would be to remove toxic substances from the manufacturing process. This study focuses on titanium, a common material in modern implantology, and generated liquid-infused surfaces by physical surface structuring using ultrashort laser pulse ablation. Screening four different structures and five different perfluoropolyether lubricants revealed that biomimetic spike structures combined with lubricants of intermediate viscosity (143 AZ and GPL 104, 20−60 cSt) yielded very satisfactory results. They showed extremely low contact angle hysteresis as is typical for SLIPS. They demonstrated strong bacteria-repellent properties and maintained their functionality when exposed to shear forces or after a long time exposure. Together with previous findings, the results of this study suggest that the SLIPS antibiofilm properties are mainly due to inhibition of bacterial adhesion.12,17−19,37 However, to date, only indirect evidence exists of the exact SLIPS mechanism. A more detailed analysis of this mechanism could be addressed in further studies. Besides microorganisms, human cells are also unable to adhere to titanium SLIPS surfaces. This sharpens the field of potential medical applications with a focus on those that do not need tissue healing or that aim to avoid tissue healing at all. Furthermore, Krytox lubricants are not accredited medical grade and showed some impairment of human cells when exposed to higher concentrations. Before medical application, the liquid-infused titanium system developed in this study needs to be adapted to biocompatible, medical-grade lubricants. However, this study serves as a promising basis for the development of biomedical liquid-infused titanium, which prevents bacterial infection without the use of antimicrobial agents.

Figure 7. Growth of fibroblasts in the presence of lubricants and reduced cell adhesion on spike SLIPS. (A) Mean ± standard deviation of gingival fibroblast metabolic activity in the presence of differently concentrated lubricants or Antispread. Two-way ANOVA showed statistically significant differences in metabolic activity of cells incubated with 10 and 20% lubricants and 10% Antispread compared to that of the control samples. (B) Fluorescence microscopy images of Calcein/PI-stained fibroblasts and osteoblasts seeded on spike SLIPS coated with 143 AZ and GPL 104. Unstructured, uncoated titanium serves as control. Scale bar: 100 μm.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b16159. OCT images of lubricants on different structures after SLIPS generation, under biofilm formation conditions, and under initial adhesion conditions (PDF)

adhesion of fibroblasts and osteoblasts with typical morphology. Live/dead fluorescence staining also indicated that the cells are viable. As regards the cytocompatibility results, the absence of cells on liquid-infused titanium cannot be a result of general toxic effects because in solution only a maximum concentration of 0.3% lubricant could be possible. It is probable that bacteria and human cells are not able to attach to SLIPS surfaces. These results are in line with previous findings. It has already been shown that fibroblasts, platelets, and erythrocytes do not adhere to SLIPS made of different materials.18,19,47,48. The results of this study further support the concept that SLIPS will be preferentially applicable for biomedical surfaces that do not require soft tissue sealing or osseointegration. According to the cytocompatibility results, tissue healing in close proximity to functionalized surfaces will probably not be negatively influenced; however, tissue integration at SLIPS sites will not be feasible. Future biomedical applications of SLIPS will address this, which necessitate cell- and microorganism-free surfaces, like catheter or endoscopes.17,19,47

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Katharina Doll: 0000-0002-4885-9821 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was carried out as an integral part of the BIOFABRICATION FOR NIFE initiative, which is financially supported by the Ministry of Lower Saxony and the 9366


Research Article


(20) Wang, J.; Kato, K.; Blois, A. P.; Wong, T. S. Bioinspired Omniphobic Coatings with a Thermal Self-Repair Function on Industrial Materials. ACS Appl. Mater. Interfaces 2016, 8, 8265−8271. (21) Sotiri, I.; Overton, J. C.; Waterhouse, A.; Howell, C. Immobilized Liquid Layers: A New Approach to Anti-Adhesion Surfaces for Medical Applications. Exp. Biol. Med. (London, U. K.) 2016, 241, 909−918. (22) Diaz, P. I.; Chalmers, N. I.; Rickard, A. H.; Kong, C.; Milburn, C. L.; Palmer, R. J., Jr; Kolenbrander, P. E. Molecular Characterization of Subject-Specific Oral Microflora during Initial Colonization of Enamel. Appl. Environ. Microbiol. 2006, 72, 2837−2848. (23) Li, J.; Helmerhorst, E. J.; Leone, C. W.; Troxler, R. F.; Yaskell, T.; Haffajee, A. D.; Socransky, S. S.; Oppenheim, F. G. Identification of Early Microbial Colonizers in Human Dental Biofilm. J. Appl. Microbiol. 2004, 97, 1311−1318. (24) Ata-Ali, J.; Candel-Marti, M. E.; Flichy-Fernandez, A. J.; Penarrocha-Oltra, D.; Balaguer-Martinez, J. F.; Penarrocha, M. PeriImplantitis: Associated Microbiota and Treatment Med. Oral. Patol. Oral Cir. Bucal 2009, 16, e937−e943. (25) Huang, R.; Li, M.; Gregory, R. L. Bacterial Interactions in Dental Biofilm. Virulence 2011, 2, 435−444. (26) Drexler, W.; Fujimoto, J. G. Optical Coherence Tomography: Technology and Applications; Springer, 2008. (27) Chinn, S. R.; Swanson, E. A.; Fujimoto, J. G. Optical Coherence Tomography using a Frequency-Tunable Optical Source. Opt. Lett. 1997, 22, 340−342. (28) Kim, P.; Kreder, M. J.; Alvarenga, J.; Aizenberg, J. Hierarchical Or Not? Effect of the Length Scale and Hierarchy of the Surface Roughness on Omniphobicity of Lubricant-Infused Substrates. Nano Lett. 2013, 13, 1793−1799. (29) Fadeeva, E.; Truong, V. K.; Stiesch, M.; Chichkov, B. N.; Crawford, R. J.; Wang, J.; Ivanova, E. P. Bacterial Retention on Superhydrophobic Titanium Surfaces Fabricated by Femtosecond Laser Ablation. Langmuir 2011, 27, 3012−3019. (30) Fadeeva, E.; Koch, J.; Chichkov, B. N. Metallic Superhydrophobic Surfaces; Crawford, R. J., Ivanova, E. P., Eds.; Superhydrophobic Surfaces, 2015; pp 87−112. (31) Howell, C.; Vu, T. L.; Johnson, C. P.; Hou, X.; Ahanotu, O.; Alvarenga, J.; Leslie, D. C.; Uzun, O.; Waterhouse, A.; Kim, P.; Super, M.; Aizenberg, M.; Ingber, D. E.; Aizenberg, J. Stability of SurfaceImmobilized Lubricant Interfaces Under Flow. Chem. Mater. 2015, 27, 1792−1800. (32) Kim, P.; Wong, T. S.; Alvarenga, J.; Kreder, M. J.; AdornoMartinez, W. E.; Aizenberg, J. Liquid-Infused Nanostructured Surfaces with Extreme Anti-Ice and Anti-Frost Performance. ACS Nano 2012, 6, 6569−6577. (33) Vogel, N.; Belisle, R. A.; Hatton, B.; Wong, T. S.; Aizenberg, J. Transparency and Damage Tolerance of Patternable Omniphobic Lubricated Surfaces Based on Inverse Colloidal Monolayers. Nat. Commun. 2013, 4, 2167. (34) Schellenberger, F.; Xie, J.; Encinas, N.; Hardy, A.; Klapper, M.; Papadopoulos, P.; Butt, H. J.; Vollmer, D. Direct Observation of Drops on Slippery Lubricant-Infused Surfaces. Soft Matter 2015, 11, 7617− 7626. (35) Blokzijl, W.; Engberts, J. B. F. N. Hydrophobe Effekte Ansichten und Tatsachen. Angew. Chem. 1993, 105, 1610−1648. (36) Costenoble GmbH & Co. KG Krytox Product Booklet, 2016. (37) Tesler, A. B.; Kim, P.; Kolle, S.; Howell, C.; Ahanotu, O.; Aizenberg, J. Extremely Durable Biofouling-Resistant Metallic Surfaces Based on Electrodeposited Nanoporous Tungstite Films on Steel. Nat. Commun. 2015, 6, 8649. (38) Ringenberg, L.; Winkel, A.; Kufelt, O.; Behrens, P.; Stiesch, M.; Heuer, W. The Effectiveness of Poly-(4-Vinyl-N-Hexylpyridiniumbromide) as an Antibacterial Implant Coating: An in Vitro Study. Int. J. Dent. 2011, 2011, 859140. (39) Pfaffenroth, C.; Winkel, A.; Dempwolf, W.; Gamble, L. J.; Castner, D. G.; Stiesch, M.; Menzel, H. Self-Assembled Antimicrobial and Biocompatible Copolymer Films on Titanium. Macromol. Biosci. 2011, 11, 1515−1525.

Volkswagen Foundation. The authors thank Marcus Stollhans and Costenoble GmbH & Co. KG for providing Krytox lubricants.

■

REFERENCES

(1) Farkas, R.; Becks, T.; Schmitz-Rode, T.; Dössel, O. Studie zur Situation der Medizintechnik in Deutschland im Internationalen Vergleich im Auftrag des BMBF 2005, 1. (2) Bundesverband Medizintechnologie Branchenbericht Medizintechnologien, 2016. (3) Ahmed, S.; Darouiche, R. O. Anti-Biofilm Agents in Control of Device-Related Infections; Donelli, G., Ed.; Biofilm-Based HealthcareAssociated Infections, 2014; pp 137−146. (4) Braun, B. Hkk Gesundheitsreport: Knie- und Hüft-(Total-) Endoprothesen 2008 bis 2012 2008, 1−48. (5) Romanos, G. E.; Javed, F.; Delgado-Ruiz, R. A.; Calvo-Guirado, J. L. Peri-Implant Diseases: A Review of Treatment Interventions. Dent. Clin. North Am. 2015, 59, 157−178. (6) Tonetti, M. S. Risk Factors for Osseodisintegration. Periodontol 2000 1998, 17, 55−62. (7) Mombelli, A.; Müller, N.; Cionca, N. The Epidemiology of PeriImplantitis. Clin. Oral Implants Res. 2012, 23, 67−76. (8) Darouiche, R. O. Device-Associated Infections: A Macroproblem that Starts with Microadherence. Clin. Infect. Dis. 2001, 33, 1567− 1572. (9) Donlan, R. M.; Costerton, J. W. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. Clin. Microbiol. Rev. 2002, 15, 167−193. (10) Franz, S.; Rammelt, S.; Scharnweber, D.; Simon, J. C. Immune Responses to Implants - a Review of the Implications for the Design of Immunomodulatory Biomaterials. Biomaterials 2011, 32, 6692−6709. (11) Davies, D. Understanding Biofilm Resistance to Antibacterial Agents. Nat. Rev. Drug Discovery 2003, 2, 114−122. (12) Epstein, A. K.; Wong, T. S.; Belisle, R. A.; Boggs, E. M.; Aizenberg, J. Liquid-Infused Structured Surfaces with Exceptional Anti-Biofouling Performance. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 13182−13187. (13) Wong, T. S.; Kang, S. H.; Tang, S. K.; Smythe, E. J.; Hatton, B. D.; Grinthal, A.; Aizenberg, J. Bioinspired Self-Repairing Slippery Surfaces with Pressure-Stable Omniphobicity. Nature 2011, 477, 443− 447. (14) Bohn, H. F.; Federle, W. Insect Aquaplaning: Nepenthes Pitcher Plants Capture Prey with the Peristome, a Fully Wettable WaterLubricated Anisotropic Surface. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14138−14143. (15) Li, J.; Kleintschek, T.; Rieder, A.; Cheng, Y.; Baumbach, T.; Obst, U.; Schwartz, T.; Levkin, P. A. Hydrophobic Liquid-Infused Porous Polymer Surfaces for Antibacterial Applications. ACS Appl. Mater. Interfaces 2013, 5, 6704−6711. (16) Howell, C.; Vu, T. L.; Lin, J. J.; Kolle, S.; Juthani, N.; Watson, E.; Weaver, J. C.; Alvarenga, J.; Aizenberg, J. Self-Replenishing Vascularized Fouling-Release Surfaces. ACS Appl. Mater. Interfaces 2014, 6, 13299−13307. (17) MacCallum, N.; Howell, C.; Kim, P.; Sun, D.; Friedlander, R.; Ranisau, J.; Ahanotu, O.; Lin, J. J.; Vena, A.; Hatton, B.; Wong, T. S.; Aizenberg, J. Liquid-Infused Silicone as a Biofouling-Free Medical Material. ACS Biomater. Sci. Eng. 2015, 1, 43−51. (18) Yuan, S.; Luan, S.; Yan, S.; Shi, H.; Yin, J. Facile Fabrication of Lubricant-Infused Wrinkling Surface for Preventing Thrombus Formation and Infection. ACS Appl. Mater. Interfaces 2015, 7, 19466−19473. (19) Leslie, D. C.; Waterhouse, A.; Berthet, J. B.; Valentin, T. M.; Watters, A. L.; Jain, A.; Kim, P.; Hatton, B. D.; Nedder, A.; Donovan, K.; Super, E. H.; Howell, C.; Johnson, C. P.; Vu, T. L.; Bolgen, D. E.; Rifai, S.; Hansen, A. R.; Aizenberg, M.; Super, M.; Aizenberg, J.; Ingber, D. E. A Bioinspired Omniphobic Surface Coating on Medical Devices Prevents Thrombosis and Biofouling. Nat. Biotechnol. 2014, 32, 1134−1140. 9367


Research Article

ACS Applied Materials & Interfaces (40) Winkel, A.; Dempwolf, W.; Gellermann, E.; Sluszniak, M.; Grade, S.; Heuer, W.; Eisenburger, M.; Menzel, H.; Stiesch, M. Introducing a Semi-Coated Model to Investigate Antibacterial Effects of Biocompatible Polymers on Titanium Surfaces. Int. J. Mol. Sci. 2015, 16, 4327−4342. (41) Doll, K.; Jongsthaphongpun, K. L.; Stumpp, N. S.; Winkel, A.; Stiesch, M. Quantifying Implant-Associated Biofilms: Comparison of Microscopic, Microbiologic and Biochemical Methods. J. Microbiol. Methods 2016, 130, 61−68. (42) Wexler, J. S.; Grosskopf, A.; Chow, M.; Fan, Y.; Jacobi, I.; Stone, H. A. Robust Liquid-Infused Surfaces through Patterned Wettability. Soft Matter 2015, 11, 5023−5029. (43) Wexler, J. S.; Jacobi, I.; Stone, H. A. Shear-Driven Failure of Liquid-Infused Surfaces. Phys. Rev. Lett. 2015, 114, 168301. (44) Alexiades, G.; Roland, J. T., Jr; Fishman, A. J.; Shapiro, W.; Waltzman, S. B.; Cohen, N. L. Cochlear Reimplantation: Surgical Techniques and Functional Results. Laryngoscope 2001, 111, 1608− 1613. (45) Mase, H.; Tamura, K.; Hiromoto, A.; Hotta, M.; Hotomi, S.; Togashi, M.; Fukuda, Y.; Yajima, T.; Nitta, T.; Tanaka, S.; Sugisaki, Y. Histopathological Study of Tissue Reaction to Pacemaker Electrodes Implanted in the Endocardium. J. Nippon Med. Sch. 2005, 72, 52−59. (46) Araki, S.; Kawano, A.; Seldon, H. L.; Shepherd, R. K.; Funasaka, S.; Clark, G. M. Effects of Intracochlear Factors on Spiral Ganglion Cells and Auditory Brain Stem Response After Long-Term Electrical Stimulation in Deafened Kittens. Otolaryngol.–Head Neck Surg. 2000, 122, 425−433. (47) Manabe, K.; Kyung, K. H.; Shiratori, S. Biocompatible Slippery Fluid-Infused Films Composed of Chitosan and Alginate Via Layer-byLayer Self-Assembly and their Antithrombogenicity. ACS Appl. Mater. Interfaces 2015, 7, 4763−4771. (48) Yao, X.; Dunn, S. S.; Kim, P.; Duffy, M.; Alvarenga, J.; Aizenberg, J. Fluorogel Elastomers with Tunable Transparency, Elasticity, Shape-Memory, and Antifouling Properties. Angew. Chem., Int. Ed. 2014, 53, 4418−4422.

9368


Development of Laser-Structured Liquid-Infused Titanium with Strong

Recommend Documents