Development of Laser-Structured Liquid-Infused Titanium with Strong

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16159 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

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ACS Applied Materials & Interfaces

Development of laser-structured liquid-infused titanium with strong biofilm-repellent properties

Katharina Doll1*, Elena Fadeeva2,4, Joern Schaeske1, Tobias Ehmke3, Andreas Winkel1, Alexander Heisterkamp3,4, Boris N. Chichkov2,4, Meike Stiesch1, Nico S. Stumpp1*

1

Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical

School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 2

Department of Nanotechnology, 3Department of Biomedical Optics, Laser Zentrum

Hannover e.V., Hollerithalle 8, 30419 Hannover, Germany 4

Institute of Quantum Optics, Leibniz University of Hannover, Welfengarten 1, 30167

Hannover, Germany

* Corresponding authors: Lower Saxony Center for Biomedical Engineering, Implant Research and Development (NIFE), Stadtfelddamm 34, 30625 Hannover, Germany [email protected] [email protected] 1 ACS Paragon Plus Environment


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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, which 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 biofilm-repellent properties were demonstrated. To simplify prospective approval of the medical device and to avoid chemical contamination, surface structuring was performed by ultra-short pulsed laser ablation. Four different structures (hierarchical micro- and nano-sized spikes, micro-sized grooves, nano-sized ripples and unstructured surfaces) and five infusing perfluoropolyethers of different viscosities were screened; the best results were obtained with a 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 or protracted exposure. 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 bio-adhesion should support the prevention of bacterial implant-associated infections without the use of antibacterial agents.

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Keywords Slippery liquid-infused porous surface, biofilm, implant infection, Streptococcus oralis, titanium, laser structuring



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Introduction Implants - such as joint prostheses, pacemakers, stents, mechanical heart valves or dental implants - have become a standard treatment procedure in modern medicine. Their total revenue in Germany has continuously increased during the last years and the largest proportion by far are dental prostheses with about 50%1,2. The improvements in implants include implementation in drug delivery systems, application of micro- and nanotechnology and combination with tissue engineering procedures2. This is one of the fastest growing areas of medical technology1,2 and the number of implanted medical devices is steadily increasing 3. In parallel, the prevalence of implant-associated infections is also increasing4-6. This leads to follow-up treatments that are a burden to the patient and to rising health care costs. The rate of implant-associated infections is between 1-5% in orthopedics, but is as high as 20% for dental implants and catheter patients7,8. These infections are caused by bacterial biofilms - attached bacterial cells of multiple species, embedded into a self-produced polysaccharide matrix and altered in their phenotype9. 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 site10. Once developed, biofilms exhibit several resistance mechanisms11. 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 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 4 ACS Paragon Plus Environment

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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 plants13, 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 pitcher14. 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 immiscible13. Liquid-infused surfaces made of polymers or silicone have already been shown to withstand bacterial colonization of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus12,15-18. It was also possible to create these surfaces from titanium, a common biomaterial for dental implants and joint replacement19,20. 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 anti-biofilm properties have not yet been demonstrated21. The aim of this study was to create liquid-infused titanium using ultra-short 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 Staphylococcus oralis was chosen as model organism. The gram positive bacterium has been shown to be an important initial colonizer that influences biofilms formation in the human mouth22-24. Once attached, it serves as binding site for late colonizing, pathogenic bacteria which promote the development of peri-implantits24,25. 5 ACS Paragon Plus Environment


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Experimental Section

Titanium SLIPS fabrication Titanium SLIPS were generated from disk shaped titanium specimens (grade 4) and were 3 mm 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 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 a 50-fold demagnification and using a 50x objective (Leica HCX PL APO L 50x /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 about 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 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 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 6 ACS Paragon Plus Environment

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GmbH, Göttingen, Germany) with the 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 homogenous 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 minutes.

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.



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Optical coherence tomography In order to visualize 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 interest26,27. Light propagating in the sample arm is backscattered, reflected from various structures inside the sample and superimposed with light from the reference arm26,27. 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/). Since 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 lubricant-titanium distance present in the optical coherence tomography images (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 hours. Experiments were performed 8 ACS Paragon Plus Environment

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using nine specimens for each condition. For biofilm formation, pre-cultures 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 4x1010 CFU/ml for S. oralis. Specimens were incubated in the bacterial suspension and biofilm was allowed to grow for 18 hours at 37 °C under static conditions. Before evaluation, samples were gently rinsed twice with phosphate buffered saline to remove non-bound cells. For analysis of initial bacterial adhesion, pre-cultures were washed two times 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 3x1011 CFU/ml for S. oralis. Specimens were incubated with bacterial suspension and adhesion was allowed for 5 hours at 37 °C under constant agitation at 150 rpm. Non-adherent cells were removed by rinsing six times with sterile distilled water.

Confocal laser scanning microscopy analysis Bacteria were fluorescently stained using the LIVE/DEAD®BacLightTM 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 minutes at ambient temperature. Cells were fixed with 2.5% glutardialdehyde in phosphate buffered saline at 4 °C for 15 minutes. Imaging was performed by confocal laser-scanning microscopy (CLSM, Leica TCS SP2, Leica Microsystems, Mannheim, Germany) with image areas of 120x120 µm2 or 90x90 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



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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 pre-cultures were diluted in trypton soya broth and the optical density at 600 nm was adjusted to 0.1. Bacteria 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 hours at 37 °C under agitation at 500 rpm. Absorption at 600 nm was measured using a multi-mode reader (Synergy 2, BioTek, Bad Friedrichshall, Germany). Bacterial viability was quantified with BacTiter-GloTM Microbial Cell Viability Assay (Promega, Mannheim, Germany). The BacTiter-GloTM reagent was first mixed with bacterial suspension. After 5 minutes of incubation at ambient temperature, luminescence was measured using a multi-mode 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 pre-cultured for 24 hours 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 hours under constant agitation at 500 rpm. To quantify cellular viability, CellTiter-Blue® reagent (CellTiter-Blue® Cell Viability Assay, Promega, Mannheim, Germany) was added followed by 4 hours incubation at 500 rpm and 37 °C. The fluorescence was recorded using a multi-mode reader (λex = 10 ACS Paragon Plus Environment

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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 cells/well or 50,000 cells/well for fibroblasts or osteoblasts, respectively, and incubated for 24 hours 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 minutes of incubation at 37 °C, fluorescence was recorded with a Zeiss Axio Scope.A1 fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) using a 20x immersion objective.

Statistical analysis Data were documented and evaluated with GraphPad Prism 5.02 software (GraphPad Software, Inc., La Jolla, USA). The d’Agostino & 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 twoway ANOVA with Bonferroni’s test for multiple comparison. Detail analysis of spike SLIPS and their long term effect 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.



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Results and Discussion

Surface structure characterization Previously it has been shown that aluminum SLIPS based on hierarchical, micro- and nanostructures show different characteristics regarding lubricant immobilization stability28. To address these findings, hierarchical micro- and nano-sized spikes, micro-sized grooves and nano-sized ripples were generated by ultra-short pulsed laser ablation for titanium SLIPS formation. All surfaces were initially characterized by scanning electron microscopy and roughness measurement. 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 self-organizes spontaneously under femtosecond laser irradiation29 and consists of a grain-like, convex microstructure, which is additionally covered by nano-sized irregular undulations30. The groove structure (Figure 1B) is an operator-defined geometric microstructure, which was generated by the mask projection 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 nano-sized parallel features, which are additionally covered with nano-sized sub-features30. Unstructured, polished titanium served as 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 conditions31,32 and the comparison of nanostructured and hierarchically structured aluminum SLIPS (comparable to spikes) showed that the former was more stable under flow conditions28. 12 ACS Paragon Plus Environment

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Table 1. Characteristics of surface structures used for SLIPS generation.

Surface

Spikes Grooves Ripples Unstructured

Category

Main Feature Dimension [µm]

Sub Feature Dimension [µm]

Average Roughness Ra [µm]

Average Maximum Profile Height Rz [µm]

Hierarchic Microstructure Nanostructure Flat

10-20 5/10 0.7 n.a.

≤ 0.2 n.a. ≤ 0.2 n.a.

2.15 ± 0.10 0.46 ± 0.01 0.07 ± 0.00 0.16 ± 0.01

11.53 ± 0.77 1.81 ± 0.06 0.69 ± 0.15 1.02 ± 0.08

n.a. – not applicable

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.



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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 about 130°. Ripples showed superhydrophobic properties, with a static contact angle of 150°. In contrast, grooves had a slightly hydrophilic contact angle of about 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 (approximately 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 mass34. 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 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). To analyze 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. Due to differences in the refractive indices of titanium, lubricant and air, the structures can be identified in an axial view and are shown in Supplementary Figure S1. In all cases, the lubricant's thickness is approximately 60 - 85 µm, 14 ACS Paragon Plus Environment

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but varies according to the underlying structure and lubricant. The thickness of 143 AZ is always about 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 lubricants of lower viscosity (GPL 100 and 143 AZ) on the latter structures were removed to a greater extend 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, the microstructured grooves or the 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 approximately 200 nm. This variation in size and exact nano shape 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.



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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.


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Biofilm formation screening To evaluate the biofilm-repelling properties of the different titanium SLIPS, 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, 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 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. To analyze lubricant thickness under conditions of biofilm formation, differently structured titanium samples coated with 143 AZ and GPL 104 were incubated in an aqueous environment for 18 hours and exposed to optical coherence tomography. As 17 ACS Paragon Plus Environment


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shown in Supplementary Figure S2, lubricant layer on spike SLIPS was reduced by 20-30% due to immersion, but a film of approximately 60 µm thickness maintained on the surface. In contrast, on grooves, ripples and unstructured titanium a complete removal of the lubricant film was observed. Due to the hydrophobic effect, the lubricants tend to separate as spheres from a hydrophilic environment, such as the biofilm cultivation medium or PBS, in order to decrease the overall free energy35. 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 potential for stable SLIPS with biofilm-repellent properties.


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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.



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Detail 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 approximately 100-fold decreased compared to 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, category 1 and 2). This difference was statistically significant. There was no statistical 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 perfluorpolyether lubricants consist of fully saturated molecules, composed of fluorine, carbon and oxygen36. 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 neutral36. 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 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 probably cannot fully explain the results. Therefore, other mechanisms should be responsible for major reduction in biofilm volume detected on all spike SLIPS. Surface anti-biofilm 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 20 ACS Paragon Plus Environment

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hypothesized that SLIPS mainly inhibit stable bacterial attachment12,17-19,37. To address this hypothesis, 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 hours. This facilitates bacteria to sediment and adhere onto the surface, to reproduce and 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 hours only. This leads to the formation of a homogenous, single layer of adhering bacterial cells on the surface, whereas biofilm outgrowth is inhibited38-41. 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 approximately 10-fold compared to 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 and exposed to aqueous shear stress for 5 hours at 150 rpm and examined by optical coherence tomography. A lubricant layer could only be detected for the lubricants of intermediate viscosity (Supplementary Figure S3). It has already been shown that physical forces, such as dynamic flow or centrifugal forces, may remove some of the infused lubricant28,42,43. As optical coherence tomography was only performed in isolated samples, it 21 ACS Paragon Plus Environment


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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.


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Figure 4. Reduced biofilm volume and adhering cell number on spike SLIPS. Biofilm volume (per 120x120 µm2) Tukey box plot (A) and mean ± standard deviation of live/dead distribution (B) of S. oralis biofilm formation and adhering cell number (per 90x90 µm2) 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. Unstructured, uncoated titanium serves as control. * indicates statistical significance compared to control with p < 0.05.



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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. Liquid-infused 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 significant reduced by approximately 100-fold compared to the unstructured, uncoated control. The percentage of dead cells was slightly increased by 5 or 10% for GPL 104 and 143 AZ coated SLIPS, respectively (Figure 5B). These results were similar to the findings without vertical exposure (compare Figure 4A, 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.

Figure 5. Reduced biofilm volume on spike SLIPS after 15 days of vertical exposure. Biofilm volume (per 120x120 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 analyzed by CLSM. Unstructured, uncoated titanium serves as control. * indicates statistical significance compared to control with p < 0.05.


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Toxicity of lubricants To confirm 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 hours 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 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 BacTiter-GloTM 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 (Figure 4B, Figure 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 approximately 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 probably due to inhibition of initial bacterial adhesion.



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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 contend of bacterial suspensions incubated with 143 AZ (10%), GPL 104 (10%) and GDA (5, 10, 20%) compared to the control samples.


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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 143 AZ and GPL 104 - were evaluated on human gingival fibroblasts. Cellular viability was quantified using the CellTiter-Blue® assay, which measures cellular reduction capacity via resazurin color change. As shown in Figure 7A, metabolic activity was not significantly different from 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 be also 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 – in order 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. To analyze cell adhesion on liquid-infused titanium, human gingival fibroblasts and human osteoblasts were seeded on spikes coated with 143 AZ and GPL 104. After 24 hours of incubation, cells were fluorescently labeled. Microscopic observation revealed that neither cell types adhered to the liquid-infused surfaces (Figure 7B). Unstructured, uncoated titanium control surfaces, in contrast, showed 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, since in solution only a maximum concentration of 0.3% lubricant could be possible. It is probable that 27 ACS Paragon Plus Environment


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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 materials18,19,47,48. . The results of this study further support the concept that SLIPS will be preferentially applicable for biomedical surfaces which 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 influence, however tissue integration at SLIPS’ sites will not be feasible. Future biomedical application of SLIPS will address those, which necessitate cell- and microorganism-free surfaces, like catheter or endoscopes17,19,47.


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Figure 7. Growth of fibroblasts in the presence of lubricants and reduced cell adhesion on spike SLIPS. (A) Mean ± standard deviation of gingival fibroblasts 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 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.



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Conclusion The concept of liquid-infused surfaces has already been applied to numerous materials and been demonstrated to effectively withstand bacterial and cellular adhesion under in vitro and in vivo conditions21. 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 approval21. 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 ultra-short 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 typical for SLIPS. They demonstrated strong bacteria-repellent properties and maintained their functionality when exposed to shear forces or after long time exposure. Together with previous findings, the results of this study suggest that the SLIPS anti-biofilm properties are mainly due to inhibition of bacterial adhesion12,17-19,37. 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 the focus on those which do not need tissue healing or which aim at avoiding 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 a promising basis for the


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development of biomedical liquid-infused titanium, which prevents bacterial infection without the use of antimicrobial agents.

Supporting Information OCT images of lubricants on different structures after SLIPS generation, under biofilm formation conditions or under initial adhesion conditions

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

References

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TOC Graphic


Development of Laser-Structured Liquid-Infused Titanium with Strong

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