*3.3. Surface Morphology Dimensions*

Three laser-textured surface patterns were chosen, based on their morphologies and uniformity, for the study of wettability properties, tribological performance and anti-bacterial behavior, see Figure 5. The laser parameters used to create these structures are listed in Table 3.


**Table 3.** Surface structures to be functionally evaluated processed with a pulse frequency of *f* = 400 kHz and a laser scanning velocity of 2 m/s.

The roughness parameters of these surface textures are listed in Table 4. As can be concluded from this table, the roughness parameters of these surfaces vary. Hence, significant differences in the functional properties (wetting, wear, biocompatibility) of these textures are expected. The higher value of *R*a indicate that Grooves are more rough than a surface covered with only LSFL. Compared to the polished CoCrMo surface, the square root surface roughness *R*<sup>q</sup> increases significantly due to laser-texturing. Quantitavely, 23 times *R*polished <sup>q</sup> in the case of nanopillars and up to 130 times *<sup>R</sup>*polished q in the case of grooves.

**Figure 5.** AFM micrographs of a CrCoMo surface: unprocessed (**a**), LSFL (**b**), grooves with superimposed LSFL (**c**) and triangular nanopillars (**d**). (**a**) AFM micrograph of polished CrCoMo sample. (**b**) AFM micrograph of LSFL. (**c**) AFM micrograph of Grooves + LSFL. (**d**) AFM micrograph of Triangular Nanopillars.


**Table 4.** Geometrical properties of the of the surface structures to be functionally evaluated.

As can be observed in Table 4, the dimensions of the chosen surface structures are indeed in the range of the sizes of the bacterias *S. aureus* and *E. coli*, which potentially gives these structures anti-bacterial properties [3].

## *3.4. Wetting Properties*

When anti-bacterial properties of surfaces found in nature are studied, a correlation between hydrophobicity and anti-bacterial behavior is found [2,31,32]. Since LIPSS have been found to be hydrophobic [10–15] and also anti-bacterial [15–18], hydrophobicity is used in this paper as an indication of anti-bacterial behavior.

The three surface textures (see Figure 5) show hydrophobic behavior compared to the untextured, mirror polished surface, which shows a water contact angle of (82.7◦ ± 0.7◦, see Table 5).

**Table 5.** Contact angles of chosen surface structures.


The LSFL surface is superhydrophobic for water, whereas the contact angle of the mirror polished CoCrMo substrate is hydrophilic. The surfaces denoted Grooves and TNP are both hydrophobic, but less so than LSFL, as is shown in Table 5. The hydrophobicity of the textured surfaces is subject to variability due to the formation of oxide layers after laser micromachining over time. The polished sample will oxidize rapidly to a protective layer of CoO, Cr2O3 and MoO3 [33–36]. For example, it was shown by Huerta–Murillo et al. [37], that the contact angles of laser textured titanium alloys increase over a time period of five weeks from about 90◦ to 130◦. The contact angles in this study were measured after seven weeks for LSFL and Grooves and after 12 days for TNP. However, a positive effect of surface texturing (irrespective of morphological class) was seen on the hydrophobic behavior of the surfaces, in line with results found in literature [12,13,15].

The influence of the surface roughness on the contact angle can be described by either Wenzel [38], where it is assumed that the total surface will be in contact with the liquid, or by Cassie [39], where different materials or a combination of trapped air and a solid will be in contact with the liquid. In case of Wenzel the relation between the apparent contact angle (CA) *θ*<sup>a</sup> and the intrinsic CA *θ*<sup>i</sup> is given by

$$\cos(\theta\_\mathbf{i}) = \sigma(\cos \theta\_\mathbf{i}),\tag{1}$$

where *σ* is the ratio between the true surface area and the projected area. In case of hydrophilic surfaces an increase of the roughness will result in a decrease of the CA and in the case of hydrophobic surfaces an increase of the roughness will result in an increase of the CA.

In case air might be trapped due to surface morphology the contact angle according to Cassie–Baxter [39] is defined as

$$\cos(\theta\_{\text{CB}}) = \sigma\_{\text{CB}} f(\cos \theta\_{\text{i}}) + f - 1. \tag{2}$$

In this equation, *θ*CB is the apparent contact angle, *f* is the fraction of the projected area of the surface that is wet by the liquid and *σ*CB is the roughness ratio of the wet area. This shows, that an increasing amount of trapped air, which means a smaller ratio *f* , will lead to an increase of the apparent contact angle.

This indicates that the measured contact angles on the CrCoMo samples due to laser processing can be explained by the increase of the ratio between the real surface area and the projected area *σ* and a reduction of the wetted area due to LIPSS [40,41]. Nonetheless, the contact angles are highly dependent on the formation of additional oxide layers. Interpretation of the origin of the hydrophobic properties would require a more thorough study of this surfaces.

#### *3.5. Tribological Properties*

The measured coefficient of friction (CoF) of the textured CoCrMo surfaces are listed in Table 6. The CoF of TNP with the hexagonal TNP is significantly lower than those of LSFL and Grooves. The friction coefficient of polished CoCrMo with 0.5 N (18 MPa), 11 mm/s and BCS lubricant was 0.22 ± 0.07. The friction coefficients of the textured surfaces are thus significantly higher than the CoF of the polished surface, due to the surface topograhphy changes.


**Table 6.** Coefficient of friction and polyethylene (PE) wear diameter of chosen surface structures.

Figure 6 shows SEM micrographs of the LSFL structure after the wear test. From this figure it can be observed that the surface morphologies on the CoCrMo surface remain intact during the given wear test. After 104 min of sliding the PE sphere over the LSFL textured surface with a 18 MPa load, 11 mm/s speed and BCS lubricant, the PE ball had a volume loss of 43 mm3. This is nearly 9.5% of the total sphere volume. Hence, it can be concluded that LSFL cannot be used as a bearing surface of a hip joint, since in the end of high loading, it reduces the durability of the hip joint significantly. The other two textures lead to noticeably less wear on the PE ball, see Table 6. The CoF of LSFL was also higher than that of Grooves and TNP. However, the difference between the CoF LSFL and Grooves is much smaller than the wear PE experiences against LSFL and Grooves. The fact that LSFL show a higher hydrophobicity (see Table 5) may influence the wear rate as well. High friction in a joint will lead to more heat generation, which may cause performance degradation of the joint. However, no maximally defined CoF is stated for a hip joint. The wear recorded for TNP is actually very close to that found on the polished surface.

**Figure 6.** SEM micrographs of LSFL structure after wear test. (**a**) SEM image LSFL. (**b**) SEM image LSFL.

The wear conditions of the UMT, which are 18 MPa, 11 mm/s, reciprocal movement, are not comparable to the wear conditions in a natural hip joint, approximately 7.8 MPa and 21 mm/s during normal gait and rotational movement in all directions [21]. Since the surface structure TNP shows a periodicity in three directions (see Figure 4), instead of one in the cases of LSFL and Grooves, and also shows the lowest CoF and PE wear very close to the polished surface, TNP is the most promising candidate for a potential anti-bacterial surface structure on an artificial hip-joint.

#### *3.6. Biocompatibility*

Lutey et al. [15] showed that LSFL and TNP performed best on anti-bacterial properties regarding *E. coli* and *S. aureus* on stainless steel. A bacterial count reduction of 99.8% and 99.2% was found for *E. coli* and 84.7% and 79.9% was found for *S. aureus*, for the LSFL and TNP, respectively. Grooves (in [15] defined as Spikes) on the other hand, do not show improvement in anti-bacterial properties. However, to estimate the leaching of hazardous elements of the CoCrMo alloy into the human body, LSFL textured CoCrMo samples were used to perform a leaching test.

Release of Cobalt (Co) ions from the CoCrMo substrate may have an adverse affect on the patient's health. The Medicines and Healthcare products Regulatory Agency recommended a 7 μg/L threshold. Concentrations above that threshold can be toxic for the patient [42]. Due to the increased surface area of the textured samples, when compared to the polished samples, textured samples may cause a higher ion release rate of Co and Ni ions. Chromium (Cr), Molybdenum (Mo) and Nickel (Ni) are also toxic in certain concentrations, but to the best of the authors knowledge no medical standardized regulations exist on acceptable concentration levels. The release of ions can be studied by means of a leaching experiment.

To that end, polished CoCrMo as well as LSFL textured CoCrMo samples were immersed in simulated body fluid (SBF, see Section 2.2.5) for nearly four weeks. Ion release was measured after 1, 7, 21 and 26 days. All samples were analyzed for the presence of Co, Cr, Mo and Ni elements by means of atomic emission spectroscopy analysis. No significant concentrations of Cr, Mo and Ni were found for any of the samples. Traces of Co were found in the SBF samples of the polished and the textured CoCrMo samples, see Figure 7. A gradual release of Co can be observed during the first two weeks for both polished and textured CoCrMo. After one day of immersion, the Co ion concentration is slightly higher for textured CoCrMo. Interestingly, there is no significant difference between the textured and untextured sample observed after one week and three weeks of immersion. After two weeks of immersion a larger Co concentration is found for polished CoCrMo and after four weeks of immersion the textured samples give a higher concentration, 27 ± 3 ppb vs. 17 ± 1 ppb. It was expected that the concentration of cobalt in the SBF would increase in time as more and more cobalt leaches from the surface into the fluid, until the equilibrium state is reached. The decrease in cobalt concentration of the untextured sample after 14 days could be explained by a change in pH due to a change in ion concentration in the SBF. The pH change could influence the equilibrium of Co ions. No precipitation of any element was observed at any point during and after the experiment. Unfortunately, the pH was not measured after the experiment. The difference in cobalt concentration after four weeks of immersion between polished and textured CoCrMo could be explained by the difference in surface area. According to Leyssens et al. [42], levels of Co lower than 300 μg/L will not cause health complications for individuals. The levels of Co in this study measured during 26 days of immersion, are well below this threshold. In the body the CoCrMo surface will be slightly larger. However, in the patients body, larger amounts of bodily fluids are present, and the human body does process low concentrations of Co [43]. However, it is questionable if this test can be compared with levels measured in patients. There are many factors which effect the leaching behavior of surfaces. To the best of our knowledge, no research on leaching of CoCrMo in SBF or similar circumstances has been conducted so far.

**Figure 7.** ICP-AES analysis of cobalt ion release of polished and LSFL textured CoCrMo samples in ppb as a function of time.

#### **4. Conclusions**

In this study, surface textures of nano and micrometer scale were produced on polished Cobalt–Chrome–Molybdenum alloy (CoCrMo) surfaces, using an infrared picosecond pulsed laser source. It was shown that the shape and size of the surface features can be controlled by adapting the laser fluence, the number of overscans of the laser spot over the surface and the type of polarization. To evaluate the wetting, tribological and leaching properties of laser-textured surfaces, three different types of textures were homogeneously produced on a large area (larger than the laser beam diameter), namely: low spatial frequency LIPSS, hierarchical grooves with superimposed low spatial frequency LIPSS, and triangular hexagonally packed nanopillars. The tribological behavior and the wettability of these three textures on CoCrMo were compared to a polished (i.e., untextured) CoCrMo surface. It was found that the textured surfaces caused higher friction in a CoCrMo-against-PE reciprocating contact compared to a polished reference. Moreover, only the LSFL textured surface showed a significantly higher wear of the PE counter surface. Furthermore, it was found that the hydrophobicity of the surface increases significantly due the micro-machined textures. Additionally, the biocompatibility of a LSFL textured surface on CoCrMo was compared to a polished CoCrMo surface. Both polished and textured surfaces release cobalt ions over a period of four weeks, but are still well below critical threshold levels reported in literature. Although, long term leaching experiments are recommended.

Based on the experimental conditions and results of this study, it is concluded that the laser textured surfaces on CoCrMo are not suitable for bearing surfaces in a metal-on-plastic contact. It is recommended to repeat the wear experiments at lower contact pressures, comparable to the conditions found in the hip joint, to study the friction and wear of PE under realistic conditions. The wear resistance, the antimicrobial activity and the effect on human cells of the processed surface textures would have to be investigated more thoroughly. It is recommended to look into other, possibly static, applications for antibacterial LIPSS surface textures on CoCrMo, e.g., dental implants.

**Author Contributions:** Conceptualization, S.H.v.d.P.; methodology, M.M. and S.H.v.d.P.; software, M.M.; validation, M.M., E.G.d.V. and S.H.v.d.P.; formal analysis, M.M., D.T.A.M. and S.H.v.d.P.; investigation, S.H.v.d.P.; resources, all authors; data curation, M.M. and S.H.v.d.P.; writing—original draft preparation, M.M. and S.H.v.d.P.; writing—review and editing, all authors; visualization, M.M. and S.H.v.d.P.; supervision, D.T.A.M. and G.-w.R.B.E.R.; project administration, S.H.v.d.P.; funding acquisition, D.T.A.M. and G.-w.R.B.E.R.

**Funding:** Parts of this study was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 675063 (Laser4Fun project, www.laser4fun.eu).

**Acknowledgments:** We thank Soheyla Ostvar Pour, University of Manchester School of Materials, for assistance with the execution of the leaching experiment, in particular the ICP-AES analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

The following abbreviations are used in this manuscript:

