Influence of multi-wavelength ultrafast laser texturing and autoclave sterilization on titanium alloy-based surface wettability

Titanium alloys are widely used in bio-medical applications for their excellent bio-compatibility and superior mechanical strength. On the other hand, femtosecond laser processing is a robust versatile and industrial method widely used for surface functionalization. In this work, laser-induced periodic surface structures (LIPSS) on titanium alloy (Ti6Al4V) using different laser wavelengths of 1030 nm (IR), 515 nm (Green) and 257 nm (UV) were created and their surface wettability (physical parameter correlated with cell adhesion) was studied, in relation to sterilization process. The contact angle over LIPSS was smaller than over polished samples, with Green and UV LIPSS presenting contact angles inferior to 20∘\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ$$\end{document} and a superior stability to sterilization process.


Introduction
Femtosecond laser processing is a robust and industrial method widely used for surface functionalization allowing to enhance some properties of the materials [1][2][3][4]. Titanium and its alloys on the other hand are of great interest for processing. They exhibit excellent bio-compatibility and superior mechanical strength. Among the alloys, Ti6Al4V is of special interest as it is currently used for biological/ biomedical applications, such as dental implants. This makes of high interest the laser texturing of such alloy; particularly, for aforementioned implants, where gingival fibroblasts adhesion is essential not only for implant support but also for its role as a physical barrier against bacterial infiltration, main reason of dental implant failure [5]. The inscription of laser-induced periodic structures (LIPSS) on metals increases surface functionalization [3,[6][7][8][9] which can enhance osseointegration of these titanium dental implants [10][11][12][13]. This is achieved thanks to the sensitivity of biological cells to nanoscale texturing, which makes them sensitive to LIPSS, as shown in the literature [4,14]. There are other techniques, such as sandblasting or chemical treatment; however, they are not precisely controllable and require more complex and longer steps than in laser processing.
Wettability on the other hand is crucial to the cell adhesion properties, as cells are mostly known to prefer hydrophilic surfaces for a higher adhesion [15,16]. In this work, the effect of ultrafast laser texturing and sterilization by autoclave over titanium based surface wettability is studied. Three different wavelengths of 1030, 515 and 257 nm were employed for LIPSS formation on Ti6Al4V surfaces, generating LIPSS periodicities of 760, 400 and 180 nm, respectively. Then, wettability tests were performed prior and post wet sterilization of the samples.

Setup
To inscribe LIPSS patterns with different periodicity, a classic processing setup composed of a femtosecond laser and a galvometric scanner was employed. A schematic of the setup is depicted in Fig. 1. The employed laser source for LIPSS at laser wavelength = 1030 nm was a "Tangor HP" from Amplitude lasers with = 400 fs. The 515nm wavelength was obtained with the same laser and a nonlinear crystal to produce second harmonic generation. Finally, for 257 nm wavelength, a PHAROS and an HIRO Harmonic generation from light conversion were employed. The pulse duration was = 150 fs. A f − lens was attached to the galvo scanner (intelliScan 14 from Scanlab for 1030 and 515 nm, and Thorlabs Dual-Axis Scanning Galvo Systems for 257 nm) to get homogeneous focal distance. The scan direction is also depicted in Fig. 1, where pulses were delivered with a separation Λ x and a hatching distance Λ y . The entire sample surface (10 × 10 mm) was texturized in this way. To achieve high quality and homogeneous LIPSS, a parametric optimization was performed. Table 1 summarizes the optimal parameters obtained that were employed for LIPSS inscription at each different wavelength. Within the scope of the current study, laser process parameters far apart from the ones listed above were observed to produce LIPSS with undesired effects or incomplete coverage.
Titanium alloy (Ti6Al4V) samples were purchased from Goodfellow. For surface roughness evaluation, both confocal microscope (Stil, from Altimet) and Atomic Force Microscope (AFM, from Brucker) were used, for large (10 × 10 mm) and localized (0.1 × 0.1 mm) surface topography measurements. The initial arithmetical mean height (Sa, the value of which varies in dependence of the surface area being considered) measured with the confocal microscope was 0.052 µm on the large surface area, and was 8.8 nm with AFM on the smaller and localized area.
One month after samples texturing, they were cleaned with an ultrasonic bath before wettability measurements were conducted. Wettability measurements were carried out with a laboratory-developed system within the MANUTECH-USD consortium. They were performed in a controlled atmosphere (temperature (T) = 22.95 ± 0.45 • C, relative humidity (RH) = 40 ± 8% ). A 3 μ L droplet of demineralized water was deposited on the surface, and the evolution of the droplet shape was visualized with a camera and a sample rotation stage enabling 360 • inspection and contact angle measurements. The platform moved at a speed of 0.1 rad s −1 . The droplet profile and especially the contact angle (CA) were extracted from the complete droplet 360 • rotation leading to approximately 80 measurements per droplet. The first CA measurements were carried out 1 month after laser processing and after an ultrasonic bath cleaning (demineralized water for 15 min). The second step of CA measurements was performed on the same samples, 24 h after a sterilization procedure in which the samples received an ultrasonic bath cleaning (demineralized water for 15 min), and an autoclave treatment at 134 • C for 19 min in specific bags, in accordance with dental implant industrial standards. The data represent the measurements from 4 consecutive, individuals and contact-less droplets on each surface.
Scanning electron microscopy (SEM) was used to visualize and characterize the different laser-induced patterns. A Tescan VEGA3 SB, Brno Czech Republic electron  microscope was used operating at 20 kV and using the secondary electron detector.

Results and discussion
SEM micrographs of both control (polished) and laser processed (LIPSS) surfaces are shown in Fig. 2. An almost featureless morphology is observed from the polished sample surface. Traces of low contrast scratches are present due to the supplier polishing process. On the other hand, the LIPSS-covered surfaces show marked contrast due to the presence of surface topography variation of the periodic surface nanostructures. Since the scale is identical for all the 4 SEM micrographs in Fig. 2, the difference in periodicity of the LIPSS linked to the wavelength of the laser used is clearly observable: more precisely, the periodicity is 760 nm when the laser wavelength is 1030, 400 nm when the laser wavelength is 515, and 180 nm for a laser wavelength of 257 nm. For the sake of simplicity, the laser wavelengths shall be referred to as IR (1030 nm), Green (515 nm) or GR, and UV (257 nm) in the following text. All laser-treated surfaces are relatively smooth (measured using the confocal microscope, the surface roughness Sa being 0.082, 0.058 and 0.158 µm for IR, Green and UV, respectively). The slightly raised value of Sa for the surface with UV LIPSS is attributed to the presence of a microscale undulation on the surface, which is presumably due to a large hatching distance Λ y between successive laser scan tracks (that can be further optimized in the near future). To compare the polished, IR LIPSS and GR LIPSS surfaces (the Sa values of these surfaces are of tiny difference, revealed by confocal measurements) in further details, smaller areas of these surfaces are measured using an AFM. The 2D surface profiles obtained from AFM assessment are also presented in Fig. 2. One can see from these AFM measurements that surface Sa values derived from smaller areas (0.1 × 0.1 mm) are slightly different from those of the larger surface area measurement (10 × 10 mm) made by confocal microscope. It is also meaningful to mention that the amplitude of the LIPSS varies, depending on the laser wavelength (approximately 300 nm for IR LIPSS, 200 nm for GR LIPSS, and 100 nm for polished surface).
Once wettability evaluation protocol was performed, polished sample showed a high contact angle, as depicted in Fig. 3, resistant to sterilization process. It almost qualifies the polished Ti6Al4V as an hydrophobic (CA close to 90 • ) surface [17]. On the other hand, laser processing substantially reduces this angle by more than 60 • presenting CAs inferior to 20 • , especially with the lowest periodicity LIPSS (UV) showing the high hydrophilicity acquired by the surface due to femtosecond laser texturing process. There is  a seeming tendency that lower CA values are achieved at LIPSS surface produced at shorter wavelength.
After sterilization process, CA of IR LIPSS was drastically increased by 30 • reducing the wettability of the surface. By comparison, after same sterilization process, Green and UV LIPSS CAs were only increased by ≈ 3 • each, showing the wetting stability of those two surfaces. It is worthwhile to mention that the water droplets do not appear to elongate along the LIPSS directions in all cases. Given the small periodicity and peak-to-valley amplitude of the LIPSS (compared to the mm-scale dimension of the droplets), the drops measured do not spread anisotropically.
In the current study, aside from the apparent surface morphology change induced by ultrafast laser irradiation at various laser configurations, the surface chemistry is also expected to be modified at the very same time, probably in different manners depending on laser conditions applied [18]. Superficial oxidation, thin amorphous layer formation Ti x O y , and microstructural defects formation in the underlying area are expected to occur [19,20]. The formation and distribution of such phases are probably quite variable, depending on the wavelength deployed to form the LIPSS. Apart from surface microstructure modification itself, the surface reactivity after femtosecond laser irradiation may also play a role in the wettability modification process. Especially, the surfaces irradiated by ultrafast laser pulses at different wavelengths may have different affinity to surface adsorption of polar, non-polar C-O bonds containing species in the air, and hence great potential of behaving differently in surface wettability [21]. Dedicated surface chemistry analysis has not been pursued to de-correlate possible interplay between surface morphology change, surface chemistry change and resulting surface wettability, but this is definitely one of the key elements to investigate in the near future.
Despite the correlation between wettability and cell adhesion, biological experiments must be realised to confirm this behavior and to evaluate the impact of LIPSS periodicity on cell adhesion. As the most hydrophilic surfaces (Green and UV LIPSS) are not necessarily the best in terms of cell adhesion, an intermediate in terms of CA (IR LIPSS) may be a better compromise [22].

Conclusions
Laser-induced periodic surface structures were generated with three different laser wavelengths, namely, 1030 nm (IR), 515 nm (Green) and 257 nm (UV), presenting periodicity variation ranging from 760 to 180 nm. Wettability studies before and after clinical sterilization showed an enhanced wettability of Ti6Al4V samples after laser processing, for all LIPSS periodicity tested. Post-sterilization wettability tests also showed a stability of those hydrophilic properties for both Green and UV LIPSS which exhibited a minimal CA and negligible hysteresis for autoclave process. According to known preference of hydrophilic surfaces for cellular adhesion, ultrafast laser LIPSS texturing is of special interest for dental implant application.