Lower Susceptibility of Laser-irradiated Ti-15Mo Surface to Methicillin-resistant Staphylococcus aureus Cells Adhesion

Extensive data reported the influence of the physicochemical properties on the bacterial adhesion in biomaterials, of which surface roughness of titanium (Ti) can dictates methicillin-resistant Staphylococcus aureus (MRSA) adhesion to orthopedic implants. Herein, we investigated the influence of the Yb:YAG laser texturing of titanium-15molybdenum (TiMo-L) surface on the MRSA (ATCC #33591) cells adhesion and viability. The physicochemical properties and antibacterial performance of TiMo-L were compared to samples of laser-irradiated pure titanium (Ti-L). Polished samples (Ti-P and TiMo-P) were used as controls. Laser textured surfaces presented a high degree of hydrophilicity, an irregular-shaped cavity and a typical microstructured pattern, compared to the polished substrates. The laser irradiation reduced the peaks of molybdenum (Mo) in the surface of Ti-15Mo alloy, which is explained, at least in part, by the difference between the melting point of Ti (1.668 °C) and Mo (2.623 °C). Laser texturing raised the MRSA cells viability and statistically increased the bacterial adhesion to pure Ti (P < 0.01; Wilcoxon-Signed rank test) and Ti-15Mo alloy (P < 0.001; Paired t test). The TiMo-L surface was significantly less susceptible to MRSA cell adhesion compared to Ti-L substrate (P < 0.001; Paired t test).


Introduction
Titanium (Ti) and Ti-based alloys have been used as implantable biomaterials in orthopedic surgery 1 and oral rehabilitation of edentulous patients 2 , because of their excellent properties such as adequate mechanical strength, resistance to corrosion 3,4 , and biocompatibility in vivo [5][6][7] .
However, in a physiological in vivo environment, oxide dissolution or micro-scale corrosion of Ti-6Al-4V alloy may result in metal ion release to the surrounding tissues 16 , and the possible occurrence of aluminum toxicity; which is associated with neurodegenerative disorders including Alzheimer and Parkinson diseases 17 . Moreover, toxicity related to vanadium was reported in vitro and in vivo 18 .
The development of multifunctional biocompatible materials with excellent mechanical properties, electrochemical stability and resistance to microorganisms adhesion, has been extensively researched in biomedical sciences. To overcome the possible adverse effects of Ti-6Al-4V, current research addressed the synthesis of new biomaterials composed of nontoxic elements, such as tantalum (Ta), niobium (Nb), zirconium (Zr) and molybdenum (Mo) 5,7 . Thus, Ti-Mo alloys received great attention due to its electrochemical stability, low elasticity modulus and higher corrosion resistance in physiological-simulated media result, indeed the ions exist in the surrounding implant tissue, particularly Ti-15Mo 3,7 .
Another important aspect to consider is the morphology and physicochemical properties of implants surface to improve bone-implant osseointegration 19,20 . Different techniques have been proposed to modify implant surface characteristics, such as ion deposition, surface coatings, sand blast and acid-etch treatments, plasma spray, laser-beam irradiation 21 and microarc oxidation 22 . Plasma electrolytic oxidation, also known as micro-arc oxidation, is similar to conventional anodizing, except that higher voltages are used to disrupt the oxide layer of Ti substrates until the grow of a thick protective oxide coating 23 . On the other hand, electropolishing is an electrochemical method used to produce smooth surfaces by controlled dissolution of Ti substrate in acid solution 24 .
Regarding implants surface modifications, it has been documented the favorable effects of laser beam irradiation on Ti implants osseointegration; which occurred in a shorter period compared to non-irradiated groups [25][26][27] . In fact, a positive correlation was observed between specific physicochemical properties and higher surface roughness, that increased removal torque values among laser irradiated implants 26 . Recently, it was demonstrated that laser irradiation greatly enhanced biomechanical properties and the osseointegration of biomedical Ti-15Mo implants in vivo 7 .
However, although increased surface roughness may improve bone-implant contact 28 , it is reasonable to assume that it may also favor the bacterial adhesion to Ti surfaces 29 . In fact, Staphylococcus aureus (S. aureus) has preferential adhesion to Ti-6Al-4V alloy compared to other orthopedic biomaterials 30 , which is partially explained by different properties such as the chemical composition of the material, surface morphometry, surface energy, degree of hydration, electrostatic charge, hydrophobicity/hydrophilicity, and Van der Waals forces among others 31,32 .
S. aureus, including methicillin-resistant S. aureus (MRSA), is a major concern in orthopedic surgery because is often associated to surgical site infections 33 , which is one of the main causes for surgical revision in total hip arthroplasties 34 . Nevertheless, there is a paucity of data regarding the influence of physicochemical properties of laser-irradiated Ti-15Mo on MRSA cells adhesion. Therefore, the purpose of this in vitro study was to evaluate the surface topography and the physicochemical properties of Ti-15Mo alloy after laser-beam irradiation and the influence of laser irradiation on MRSA cell adhesion to Ti-15Mo surface.

Samples preparation and experimental groups
Ti-15Mo (in wt %) samples (developed by the Biomaterials Group, Institute of Chemistry of Araraquara, UNESP) were casted in an arc-melting furnace with inert argon atmosphere, as described elsewhere 7 . Twelve disks (with 10-mm diameter and 2-mm thickness) made of Ti-15Mo alloy and grade 2 cpTi (Titanews, Barueri, SP, Brazil) were used. First, the samples were ground under demineralized water with SiC sandpapers until grit size 600. Thereafter, the disks were ultrasonically cleaned in distilled water, acetone and ethanol for 10 min each, and then, air dried at room temperature.
Half of the disks had their surface treated with laserbeam irradiation using a pulsed laser Yb:YAG (Pulsed Ytterbium Fiber Laser, Ominitek Tecnologia Ltda, Brazil).

Surface topography and physicochemical characterization
The surface topography of the studied samples, before and after laser irradiation of the surfaces, was analyzed by using field-emission gun scanning electron microscopy (FEG-SEM; Jeol 7500F; Jeol, Peabody, MA, USA), whereas energy dispersive X-ray (EDX) spectroscopy was used to check the chemical composition or detect any contamination of the materials.

Wettability analysis
The wettability of the samples was evaluated by contact angle measurement using a contact angle tester OCA-15 (Dataphysics, Germany). Drops of distilled water were delivered onto the specimen surface by a syringe giving the same drop size. The contact angle was measured after 20 seconds and repeated 3 times for each sample.

Surface roughness measurements
Surface roughness measurements of all specimens were performed using a profilometer (Mitutoyo SJ 400; Mitutoyo, Tokyo, Japan), with a resolution of 0.01 μm, an interval (cut-off length) of 0.8 mm, a transverse length of 2.4 mm, a stylus speed of 0.5 mm/s 35 . For each specimen, readings were performed at four points, and the average reading of surface roughness was designated as the R a value (µm) of that specimen. All measurements were recorded by a trained operator.

Microorganism and culture conditions
Methicillin-resistant Staphylococcus aureus (MRSA; American Type Culture Collection 33591) was the bacterial strain used in the present study. To prepare the inoculum, a loopful of the stock culture was streaked onto Mueller Hinton agar (Himedia, Mumbai, India) and incubated at 37°C for 48h. One loopful of this young culture were transferred to 20 mL trypticase soy broth (TSB) culture medium (Sigma, St Louis, MO, USA) and incubated at 37°C for 21h. Bacterial cells of the resultant culture were harvested, washed twice with sterile phosphate-buffered saline (PBS) (pH 6.8) at 4000 × g for 5 min, and resuspended in TSB. Staphylococcus suspensions were spectrophotometrically standardized at an OD600nm of 0.1, which corresponds to a final concentration of 10 7 cells/mL.

Adherence assay
Specimens of each experimental group were used for the adherence assay (90 min of incubation). Prior to the microbiological tests, the specimens were ultrasonicated in distilled water for 20 min and then exposed to UV light under dry conditions for another 20 min (each side) for sterilization. Two milliliters of the inoculum (10 7 cells/mL) were added to each well of a 24-well microplate containing the specimens, and then incubated at 37°C in an orbital shaker for 90 min. The non-adherent cells were removed from the specimens by gently washing twice with 2 mL PBS after 90 min (initial adherence). After washing, the cells adhered to the specimens were removed by sonication and the assays described below were performed. For all experiments, negative controls were specimens to which no cells were added. All experiments were performed in triplicate on three independent occasions. The initial adherence of MRSA cells was evaluated by counting c.f.u./mL.

CFU method
Viable microorganisms after the initial adherence of MRSA were quantified by counting the c.f.u. After washing the non-adherent cells with PBS, each specimen was transferred to a Falcon tube containing 10 mL PBS, which was vortexed vigorously for 1 min to resuspend the adhered cells. Cell suspensions obtained were serially diluted (from 10 -1 to 10 -8 ) in PBS and 10 µL of each dilution plated on Mueller Hinton agar in duplicate. After 48h of incubation at 37°C, the number of colonies was counted and expressed as c.f.u./mL.

MRSA cells morphology and spreading
MRSA cell morphology and cell distribution onto polished and laser-treated samples was determined, respectively, by FEG-SEM and confocal laser scanning microscopy (CLSM; Carl Zeiss, Germany) analysis. Briefly, MRSA cells were cultured as aforementioned, washed with PBS, and then, fixed with 2.5% glutaraldehyde for 24h at room temperature. Thereafter, cells were dehydrated through a graded ethanol series (70%, 85%, and 100%) and sputter-coated with gold for FEG-SEM analysis. For cell distribution, MRSA cells were stained with a Live/Dead BacLight Viability Kit (Invitrogen) and marked cells were visualized using Zeiss LSM 800 CLSM.

Statistical analysis
Data from microbiological adhesion was subjected to Shapiro-Wilk normality test to assess whether the data followed a normal distribution. The Bartlett's test was used to determine if the assumption of homogeneity of variance was valid. If data satisfied both criteria (normality and homoscedasticity), parametric Paired t-test was performed.
The nonparametric Wilcoxon signed rank test was used when data did not adhere to normal curve. All tests were performed using Prism 5.0 (GraphPad Software, San Diego, CA, USA) and the level of significance was set to be 5%.

Surface morphology and roughness
Laser surface modification increase roughness and produce a larger area of bone-implant contact compared to acid etching (i.e. most common surface modification of dental implants surface) 26,[36][37][38]40 (Souza et al., 2014). Also, laser beam irradiation technology for surface preparation creates a unique 3D topography and maintains a rough pattern on the surface of the implant, provides an increase in hardness, and resistance to corrosion [41][42][43] .
Surface topography analysis were carried out at high resolution using FEG-SEM. Figure 1 shows the clear differences in the surface morphology between laser-irradiated (Ti-L and TiMo-L) and polished (Ti-P and TiMo-P) substrates. Thereof, a smooth surface was observed in the Ti-P and TiMo-P samples, whereas Ti-L and TiMo-L groups presented irregular-shaped cavities and a typical microstructured surface with large depressions. This results are in accordance with previous findings in which pulsed Yb:YAG laser treatment resulted in a typical macro-and micro-roughness surface on cpTi 41,44 and Ti-15Mo 7 .
Roughness is an important property for dental implants influencing directly cellular adhesion and proliferation 42,43,45 . The laser irradiation increased the surface roughness measured by the R a values (average of the peak-to-valley measurements), in both cpTi and T-15Mo substrates. While Ti-P and TiMo-P presented 0.20 and 0.39 µm, Ti-L and TiMo-L exhibited 5.64 and 4.54 µm, respectively.

Chemical composition and surface wettability
When studying implants' surface modifications, it is extremely important to analyze the possible chemical contamination of the surface. Figure 2 shows the surface chemical composition of both cpTi and Ti-15Mo substrates.
The EDX spectra detected peaks of Ti, Mo and O. The laser irradiation reduced the peaks of Mo in the surface of Ti-15Mo alloy, which can be explained, at least in part, by the difference in the melting point between Ti (1.668 °C) and Mo (2.623 °C). Moreover, regarding the laser irradiation process, the EDX showed no trace of impurities on the surface of both Ti-L and TiMo-L samples. Similar findings were observed after the laser irradiation of the cpTi 42, 43 and Ti-15Mo 7 substrates.
To obtain these results the surfaces of the substrates were irradiated by laser beam under conditions that promote the phenomenon of ablation, which is directly correlated with the energy density used, leading to oxygen diffusion through the molten metal and oxidation of the alloy surface. Ablation process is characterized by a quick melting (melted metal zone formation) and solidification process in the presence of ambient atmosphere, forming stoichiometric and non-stoichiometric oxides.  The parameters for irradiating the surface of metal implants (cpTi and Ti alloys) by laser beam were established more than 10 years ago by the Biomaterials Group (Institute of Chemistry at Araraquara/UNESP) 41 . Also, laser beam irradiation technology for surface preparation generates a high degree of purity and produces a thick layer of oxides [41][42][43] .
The contact angle values of both cpTi and Ti-15Mo substrates were obtained in order to evaluate the influence of laser-beam irradiation on the surface wettability. A complete wetting was observed for the laser-irradiated disks, whereas polished surfaces exhibited a higher degree of hydrophobicity. While the contact angle for Ti-P and TiMo-P was 67.9° (± 13.01°) and 58.65° (± 3.18°), the contact angle from laserirradiated samples was 0° for both Ti-L and TiMo-L groups, respectively (Table 1). Our results are in accordance with previous findings that reported the enhanced wettability after laser irradiation of Ti surface 21 . The literature reports several studies that employs the technology of laser modification of cpTi and Ti alloys, with improvements in their physicochemical properties, wear and corrosion resistance, favoring the in vivo bone response to these surfaces 7,26,42,43 . Hydrophilicity is considered an important factor to early bone response 46 . Higher surface energy and increased wettability properties are correlated to enhance the interactions between surface and the surrounding bone 47 , leading to faster healing and consequently early loading of dental implants 48 . Based on the observation of the morphology of the laser groups, rougher surfaces were obtained in the present study compared to that for Nd:YAG laser irradiation on different Ti implant material 45 , showing that the laser parameters employed were able to create a controllable, reproducible and clean method to modify Ti implant surface 41 .
In this context, our study is the first to acknowledge the ability of MRSA cells' adhesion onto laser-irradiated Ti-15Mo alloy. Figure 3A shows that laser irradiation statistically increased MRSA cells adhesion to Ti (p<0.01) and TiMo (p<0.001) samples when compared to its respective controls (polished substrates). Bacterial adhesion to polished surfaces was statistically higher in TiMo alloy compared to Ti substrates (p<0.01). As shown in Figure 3B, the number of colonies on laser-irradiated samples was statistically higher in Ti compared to TiMo alloy (p<0.001). Biomedical materials made of Ti-6Al-4V has been extensively used in implant dentistry 8 and orthopedic surgery 12 to treat the lack of hard tissues, such as teeth and bone, because of their high mechanical properties, corrosion resistance, and biocompatibility 54 . However, the infection of cpTi and Ti alloys usually occurs after adhesion of pathogenic microorganisms and subsequent biofilm formation 54 .

MRSA cells morphology and spreading
Determining and understanding the most common pathogenic microorganisms present at the infected sites, as well as their behavior onto polished and modified Ti surfaces, can contribute to clinically manage contaminated surfaces of orthopedic implants. Figure 4A shows the MRSA cells morphology adhered to the Ti or TiMo substrates. FEG-SEM images showed a characteristic round-shape morphology of MRSA cells, with 1.14 µm in diameter, adhered to cpTi and Ti-15Mo samples. As shown in Figure 4B, laser-irradiated cpTi and Ti-15Mo substrates presented more adherent cells compared to polished specimens, in which TiMo-L group exhibited fewer adherent cells compared to Ti-L group. The CLSM images presented in Figure 4C showed that both substrates (cpTi or Ti-15Mo) consisted of viable (green) MRSA cells adhered to polished or laser-treated samples. Data from CLSM analysis corroborates with FEG-SEM observations. The high wettability and the unique 3D surface topography produced by laser beam irradiation might be an explanation. It is known that MRSA cells exhibit better adhesion and growth on hydrophilic surfaces 55 , and a preferential growth onto Ti substrates 30 . Analyzing separately the results of bacterial adhesion on polished and laser-irradiated groups, they seem divergent. It can be assumed that the laser beam irradiation conducted to the decrease in the number of colonies onto TiMo surface. Also, the formation of different oxides on the surface of the TiMo alloy after laser irradiation potentially influenced these findings. The irregular-shaped cavities and the unique 3D surface produced by laser irradiation conducted both cpTi and Ti-15Mo substrates to present more adherent cells compared to polished specimens. Further, the increase in the specific surface area by laser irradiation directly influenced and justify these findings.
Comparing both polished groups, additional studies are required to better elucidate the reason why MRSA bacterial adhesion was more evident at TiMo than cpTi surfaces, once when considering the laser irradiated groups, a decrease in the MRSA cell adhesion was found for TiMo surfaces. Table 1. Contact angle measurements on Ti and Ti-Mo alloy, polished (P) or modified by laser beam irradiation (L).

MRSA cells' adhesion
The rationale for the use of antibiotic-resistant S. aureus is that MRSA is one of the most common pathogens associated to persistent prosthetic joint infections in primary total hip arthroplasties 49 . Considering it was previously documented the existence of MRSA strains isolated from infected prosthetic joints 50 , the development orthopedic implants (effective against microorganisms adhesion) is highly pursuit. Previous studies reported that surface topography has a significant impact on the bacterial adhesion to implant materials 51, 52 , in which biofilm formation was found to be more evident in rougher surfaces compared to smoothie ones 53 .  Also, the development of a coating to both polished and laser irradiated Ti-15Mo alloy surfaces to decrease the MRSA cell adhesion enabling to reduce the potential of infection represents an important strategy for the biomedical application of this alloy.

Conclusions
Based on our observations, we highlight, within the limits of an in vitro study, that pulsed Yb:YAG laser-beam irradiation is a feasible clean method to generate a homogeneous micro-roughness surface on cpTi or Ti-15Mo materials, apart from increase surface wettability of these materials. MRSA cells adhesion was significantly higher in laser-treated samples, in which cpTi presented a greater number of colonies per milliliters compared to Ti-15Mo alloy.