Reactive ion etching for fabrication of biofunctional titanium nanostructures

www.nature.com/scientificreports OPEN Reactive ion etching for fabrication of biofunctional titanium nanostructures Mahya Ganjian1,4*, Khashayar Modaresifar1, Hongzhi Zhang2, Peter-Leon Hagedoorn Lidy E. Fratila-Apachitei1 & Amir A. Zadpoor1 3 , One of the major problems with the bone implant surfaces after surgery is the competition of host and bacterial cells to adhere to the implant surfaces. To keep the implants safe against implant-associated infections, the implant surface may be decorated with bactericidal nanostructures. Therefore, fabrication of nanostructures on biomaterials is of growing interest. Here, we systematically studied the effects of different processing parameters of inductively coupled plasma reactive ion etching (ICP RIE) on the Ti nanostructures. The resultant Ti surfaces were characterized by using scanning electron microscopy and contact angle measurements. The specimens etched using different chamber pressures were chosen for measurement of the mechanical properties using nanoindentation. The etched surfaces revealed various morphologies, from flat porous structures to relatively rough surfaces consisting of nanopillars with diameters between 26.4 ± 7.0 nm and 76.0 ± 24.4 nm and lengths between 0.5 ± 0.1 μm and 5.2 ± 0.3 μm. The wettability of the surfaces widely varied in the entire range of hydrophilicity. The structures obtained at higher chamber pressure showed enhanced mechanical properties. The bactericidal behavior of selected surfaces was assessed against Staphylococcus aureus and Escherichia coli bacteria while their cytocompatibility was evaluated with murine preosteoblasts. The findings indicated the potential of such ICP RIE Ti structures to incorporate both bactericidal and osteogenic activity, and pointed out that optimization of the process conditions is essential to maximize these biofunctionalities. It is known that micro- and nano-scale topographies have a significant impact on the behavior of both eukaryotic and prokaryotic cells1. For example, nanowires2, nanopillars3–12, nanotubes13, and nanopits14,15 with specific dimensions have been shown to exhibit antibacterial properties. Developing such structures provides a drug-free approach to combat infections which is considered as an alternative to the common antibacterial surfaces which release antibacterial agents16–18. The characteristics of such topographies (e.g., shape, height/depth, diameter, interspace, and spatial arrangement) are important factors that can be also used to influence the stem cell fate19. Therefore, harnessing nanoscale topography represents a powerful approach for achieving the required biofunctionalities of implants, such as improved osseointegration (i.e., integration into the host bony tissue) and antibacterial properties. That is why techniques that enable fabrication of controlled topographies on suitable biomaterials are of growing interest. Several techniques have been used to create topographies which discourage bacterial colonization. Examples include hydrothermal treatment3,20–24, reactive ion etching (RIE)5,8,9,12,25, pulsed plasma polymerization26, electron-beam lithography14, anodization27, and nanoimprint lithography10. Recent studies demonstrate that bacterial adhesion on nanostructured surfaces is strictly correlated with the nanoscale morphological features of the surface, indicating that the bactericidal effect is highly morphology-dependent28,29. Minor changes in the characteristics of these surfaces can therefore have a major impact on their bactericidal efficiency, as demonstrated in a recent study based on black Si (bSi) surfaces9,12. For instance, the sharper tip of bSi nanostructures compared with cicada wing and dragonfly wing, as two naturally occurring bactericidal surfaces, leads to a higher killing efficiency against both Gram-positive and Gram-negative 1 Department of Biomechanical Engineering, Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology, Mekelweg 2, 2628 CD, Delft, The Netherlands. 2Department of Materials, Mechanics, Management & Design, Faculty of Civil Engineering and Geosciences, Delft University of Technology, Stevinweg 1, 2628 CN, Delft, The Netherlands. 3Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands. *email: Scientific Reports | (2019) 9:18815 | https://doi.org/10.1038/s41598-019-55093-y 1 www.nature.com/scientificreports/ www.nature.com/scientificreports bacteria4. In another study, the highest killing efficiency was obtained when the interspace was between 130 and 380 nm10. Therefore, to harness the potential of nanotopographies for specific biofunctionalities, their morphological features should be precisely controlled and optimized. RIE can rapidly generate biomimetic, high aspect ratio nanostructures (e.g., inspired by the dragonfly wing4,30,31, damselfly wing32, and gecko skin13) on large areas without any need for masks. This process has been applied to create bSi topographies with antibacterial properties4,5,9,28. However, silicon is not a proper choice of material for orthopedic implants. Titanium and its alloys represent one of the most important groups of biomaterials33 for orthopedic and dental applications due to their combination of properties such as cytocompatibility, corrosion resistance34,35, fatigue properties34,35, low density, and relatively low elastic modulus36–38 as compared to other metallic biomaterials. Creating RIE topographies on titanium substrates is therefore clinically highly relevant. Femtosecond laser ablation33, hydrothermal treatment2,20,22,23,39, and anodizing13 are the most commonly used methods to create nanostructures on titanium substrates. Jaggessar et al. have studied the effects of different hydrothermal treatment conditions such as process temperature, time, and NaOH concentration on the resulting nanotopographies on Ti, followed by investigations of these surfaces with regard to their mechanical properties and bactericidal effects22. Although the hydrothermal method is environmentally friendly, simple, and inexpensive, it is relatively slow as compared to RIE (>2 hr as compared to a few min). The maximum killing efficiency of this work for Gram-positive Staphylococcus aureus bacteria is reported to be 54% and 33%, after 3 and 18 hr incubation times, respectively22. In another work, which used the hydrothermal technique to create TiO2 nanowires on the titanium substrate20, the bactericidal effects of the created nanostructures against S. aureus were insignificant. This can be explained by the fact that it is generally difficult to produce nanowires with a sharp tip using the hydrothermal technique. Fadeeva et al. have reported the bacterial response to superhydrophobic, self-organized titanium microstructures created by femtosecond laser ablation, but the killing efficiency has not been reported33, and there are a (...truncated)