Comparative study of cell interaction and bacterial adhesion on titanium of different composition, structure and surfaces with various laser treatment

Titanium and its alloys are commonly used in modern implantology. Cell viability on the surface of titanium implants depends on the surface topography, roughness, and wettability. Laser treatment is a successful method to control the surface morphology. The aim of this study was to comprehensively investigate the effects of laser ablation on titanium surfaces and their interactions with cells and bacteria. Cell adhesion, proliferation, and bacterial retention on smooth and laser-textured samples of commercially pure and nanostructured titanium of two grades were evaluated. Femtosecond laser treatment effectively enhances the wettability. Titanium grade four exhibits superior adhesion and proliferation rates when compared to titanium grade two. The cytotoxicity of nanostructured titanium is significantly lower, regardless of the surface treatment. Laser treatment resulted in increased short-term cell proliferation on grade two titanium and long-term cell proliferation on nanostructured grade two titanium only. Although the laser ablation has a limited effect on bacterial adhesion, the coverage of less than 1% in most samples indicates that the material itself has an antibacterial effect on the bacterial strain Streptococcus oralis. These findings provide valuable insights into how different material structures and surface treatments can affect cellular response and antibacterial properties for potential use in dental implantology.


Introduction
In modern implantology, titanium and its alloys are commonly used as materials for fabrication of dental implants and joint replacements [1].Titanium became a preferred substrate among other materials because of the combination of its outstanding biocompatible properties, corrosion resistance, high load-bearing capacity, rigidity, and lightness [2].Factors such as type and design of material, surface properties or the quality and volume of the host bone affect biocompatibility [3][4][5].Actually, the concept of biocompatibility represents the ability of the material to induce a conforming response in the surrounding tissues [6].The biocompatibility of titanium is based on its ability to form an oxide layer.This thin layer of titanium dioxide can improve its corrosion resistance and protect the surface of the implant [7].
There are several ways to improve the mechanical properties and biocompatibility of titanium implants.Recent investigations have shown that ultra-fine grained (UFG) titanium has higher strength levels compared to commercially pure titanium [8,9].Methods of severe plastic deformation are commonly used to produce UFG materials [10].One of these methods often used for grain refinement of metallic materials is equal channel angular pressing (ECAP).In the ECAP process, the material is extruded through a die consisting of two channels intersecting at a certain angle.During this process, the structure of the material changes, but the dimension of the sample remains the same [11,12].
Cell adhesion and proliferation on the surface of titanium implants depends on the surface topography, roughness, and wettability.It has been found that materials with a combination of micro-and nanostructured surfaces with a certain roughness can show improved levels of cell attachment [13].Methods such as sandblasting and acid etching [14,15], anodic oxidation [16], plasma spraying [17], magnetron sputtering [18], or meniscus-dragging deposition [19] are commonly used for surface modification.These methods assume contact with the material, so they have some limitations to their universal use such as limitations in substrate size or contamination with hazardous chemicals [20].The use of lasers can avoid the direct contact and the associated undesirable surface contamination.
Laser ablation is a successful method for creating regular morphology on the surface of materials.This contactless method is also advantageous due to its high resolution, high operating speed and low operating cost.Laser-assisted surface modifications of titanium are used to texture the material and leads to the production of highly controlled microstructures.In particular for these dental applications, avoidance of thermal effects is required [21].Long pulse or continuous wave lasers can produce a high energy at the surface and increase the temperature of the material, which is not always desirable.Femtosecond laser structuring is a perspective method for creating nanostructures on the titanium surface.Its main advantages are a wide range of nanostructures, ultra-high resolution, contactless manufacturing [22], and less thermal side effects due to its ultrashort light pulses [21].Chen et al investigated the in vitro bioactivity of bioinspired Ti-6Al-4V alloy surfaces modified by combined laser micro/nano structuring.The same micro/nanostructures were formed on the titanium implant surfaces by microsecond laser direct writing.Adhesion and proliferation of osteoblastic cell MC3T3 were studied.It was shown that adhesion and proliferation were improved on the bioinspired nanostructures formed by femtosecond laser [7].
A frequent cause of dental implants loosening and failure is bacterial colonization on the implant surface and subsequent immune response [23].Bacterial biofilms are considered to be the primary factor in the development of peri-implantitis and peri-mucositis [24].The bacterium Streptococcus oralis is a common species of the commensal oral microflora and one of the pioneer microbes involved in the process of bacterial adhesion and the early phase of biofilm formation [25].This bacterial biofilm, if not sufficiently removed, enhances the retention of pathogenic bacteria.The main bacteria causing peri-implantitis are by some investigators noted Staphylococcus aureus, Streptococcus mutans, and Porphyromonas gingivalis [26,27].
During the development of implant materials there is an effort to find such materials and their surface modifications that limit the adhesion and growth of bacteria, but at the same time support the integration of bone tissue.The roughness, topography, hydrophilicity, charge and surface free energy of the implant surface can affect bacterial adhesion.The methods to change the surface can be either additive or subtractive.The additive methods bring on the surface a new layer of material with enhanced bioactivity and antimicrobial properties [28].The subtractive techniques remove the original material and thereby modify the surface topography.Femtosecond laser ablation is considered to be a suitable method to create an antimicrobial surface [29].Ionescu et al reported an inhibitory effect of femtosecond laser treated titanium surface on bacterial biofilm development [30].Doll et al demonstrated the effect of different topographies created by nanosecond laser ablation on the development of S. oralis biofilm, where some structures had an inhibitory effect while others did not [31].The antibacterial effect of femtosecond laser modified titanium surface was also reported by other authors [32].
The objective of this study was to investigate the impact of different laser ablation of titanium surfaces on cell adhesion, proliferation, toxicity, and bacterial retention.To the best of our knowledge, what makes our investigation unique is a complex work evaluating the properties of different surfaces treated by various types of laser, including the femtosecond laser, and their impact on interactions with both bone cells and bacteria.In total, twelve sample sets were analyzed -three types of surfaces (smooth, textured with nanosecond laser, and textured with femtosecond laser), on commercially pure and UFG titanium of grades two and four.

Titanium samples
The commercially pure titanium (cpTi) of two grades (grade 2 and grade 4) was used as basic material for production of nanostructured titanium (nTi).Nanostructured specimens were produced by using the ECAP method on a continuous extrusion machine Conform 315i (BWE Ltd, Ashford, UK).The details of the ECAP technique are described elsewhere [33][34][35].The mechanical characteristics of used material are in table 1.The average grain sizes of the basic material and the nanostructured material were 54 μm and 220 nm, respectively [36].
The samples (height 4 mm, diameter 5 mm) were manufactured by turning from cpTi and nTi rods.The discs were subsequently polished under water with sandpaper P2000 on the flat side to produce smooth surfaces.
Each group was further divided into three subgroups with different surface treatment.Subgroup with smooth surface, subgroup textured with nanosecond laser, and subgroup textured with femtosecond laser.The overview of twelve different types of samples is in table 2.
Laser processing of the samples was conducted either by means of direct laser interference patterning or direct laser writing.Line-line patterns exhibiting a periodicity of 10 μm have been generated via two-beam laser interference utilizing a Nd:YAG (Quanta Ray, Spectra Physics, USA) seed laser with 10 ns pulse duration and 532 nm wavelength.Within the interference setup, the seed beam passes a condenser lens with a focal width of 1000 mm, before it gets separated by a partially reflective beam splitter whereas the two generated partial beams are overlapped on the sample surface utilizing a mirror setup.Processing was conducted at a pulsing frequency of 10 Hz and a fluence of 1.7 J cm −2 , whereas stitching of the individual 1 × 1 mm 2 laser spots was conducted avoiding a multi-pulse overlap.Pattern scales of 0.7 μm have been processed via low spatial frequency LIPSS (LSFL) generation utilizing a Ti:Sapphire (Spitfire, Spectra Physics, USA) laser system with a pulse duration of 100 fs and a wavelength of 800 nm.For direct laser writing, the seed beam was focused utilizing a f = 100 mm condenser lens.Processing was conducted by means of lateral scanning of the substrate surface at a fluence of 0.6 J cm −2 , whereas hatching was adjusted for a pulse overlap of N = 30.Each sample was then sterilized by immersion in 70% ethanol, rinsed in deionized water and left to dry in a laminar box.

Characterization of surfaces
We investigated the surface modifications induced by laser treatment using a scanning electron microscope (JSM 6380, JEOL, Tokyo, Japan).The secondary electron channel was used to visualize the surface topography at magnifications of 22×, 500×, and 1,000×.
Surface roughness was quantitatively assessed using a Keyence VHX-7000 digital microscope (Keyence, Japan).Each sample was analyzed at 500× magnification across five different areas.For each area, a line profile with a length of 0.8 mm was captured.To quantify the roughness, we calculated two common parameters: the arithmetic mean roughness (Ra), which represents the average deviation of the profile from the mean line, and the root mean square roughness (Rq), defined as the square root of the sum of the squares of the individual heights and depths from the mean line.
The measurement of the contact angle between the surface and the droplet applied by the sessile drop method was used to assess the wettability of the surface.Angles were measured on photographs taken with a Leica S9i stereo microscope (Leica Microsystems, Wetzlar, Germany).A 2 μ deionized water droplet was deposited on the surface, and the contact angle on both sides was measured after 2 s.The reported value is the average of five independent measurements.

Cell adhesion and proliferation
Cell adhesion after 24 h as well as cell proliferation after 48 h (short-term) and 168 h (long-term) from seeding was evaluated by using CCK-8 (Cell Counting Kit-8, Bimake, Munich, Germany) as stated in the manufacturer's instructions with volume modifications.Briefly, CCK-8 solution (100 μl/1 ml DMEM) was added into each well of a 48-well plate (TPP, Techno Plastic Products AG, Trasadingen, Schwitzerland), followed by 3 h incubation at 37 °C.The absorbance value was measured in quadruplicate at 450 nm using a microplate reader Synergy H1 (Biotek, Winooski, VT, USA).

Cytotoxicity of materials
The cytotoxicity of samples was evaluated by CyQUANT LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Waltham, MS, USA).The cultivation medium of each sample (50 μl) was transferred into the 96-well plate, mixed 1:1 with reaction mixture and incubated for 30 min in the dark at room temperature.Stop solution was then added and absorbance measured at 490 nm and 680 nm.To determine lactate dehydrogenase (LDH) activity, the absorbance at wavelength 680 nm (background signal from the instrument) was subtracted from the absorbance at 490 nm.

Cell staining
The nuclei of the cultured cells were stained with NucBlue ® Live ReadyProbes ® Reagent and the cell cytoplasm with CellTrackerTM Green CMFDA Dye (all Thermo Fisher Scientific, Waltham, MS, USA).Briefly, the cells were incubated for 30 min at 37 °C in a premix consisting of 4 μM CellTracker Green and 2 drops of NucBlue per milliliter of supplement-free DMEM medium.Finally, the cells were observed and photographed by using an inverted fluorescent microscope (CKX41, Olympus, Hamburg, Germany) with a magnification 40×.

Bacterial adhesion
Prior to the bacterial growth experiment, the samples were ultrasonically cleaned separately for 1 min in distilled water and 1 min in 70% ethanol.Samples representing 12 materials/surface modifications were then placed on a stand with 12 positions (figure 1(a)).The stand was designed by 3D modeling using Autodesk Fusion 360 software (Autodesk Ireland Operations UC, Dublin, Ireland) and printed on 3D printer Prusa i3 MK3S+ (Prusa Research a.s., Prague, Czech Republic) from 1.75 mm white HD-PLA filament (Fiberlab S.A., Brzezie, Poland).Following the manufacturer's instructions, the printed stand was heat treated at 80 °C for 15 min.This heat treatment increases the stability of the material at temperatures up to 140 °C, allowing sterilization in autoclave.The stands were sterilized by autoclaving (120 °C, 30 min) and then four M5 × 16 sterile stainless-steel screws were screwed into the threaded holes of the stand base from below.These screws ensured that the stand was held at the bottom of the culture vessel during cultivation.This holder allowed all samples to be exposed to identical conditions during cultivation, as well as during the washing and fixation.
After placing the samples in the positions of the stand, the entire assembly was placed in 70% ethanol and stored until the beginning of cultivation, but not less than 12 h.The experiment was performed five times under the same conditions.
S. oralis CCM 7412 was obtained from the Czech Collection of Microorganisms (CCM, Masaryk University, Brno, Czech Republic).Bacteria were pre-cultured in Brain-Heart-Infusion broth (BHI, Roth).Bacterial adhesion tests were performed in sterile 250 ml beakers covered with aluminium foil.100 ml of BHI media was inoculated with pre-cultured S. oralis to achieve a final concentration of 10 7 CFU ml −1 .The sample holder was removed from ethanol, rinsed with sterile saline solution, and placed in the beaker containing the inoculated culture medium.Cultures were incubated for 22 h at 37 °C with shaking at 80 rpm.As a contamination control, a sample of the medium was taken from each beaker at the end of the incubation period and inoculated onto blood agar plates.All of these controls revealed growth of alpha-hemolytic S. oralis colonies only.
After 22 h of incubation, stands containing samples were washed in sterile saline solution to remove nonadherent bacteria: stands were immersed three times in a beaker with saline.Samples were air dried and fixed in methanol for 1 min.
Bacterial adhesion was quantified by analyzing the bacterial coverage of the tested surface, i.e., the ratio of the area covered by bacteria to the area not covered by bacteria.For imaging, samples were stained with acridine orange (AO) acid stain (ThermoFisher Scientific, Waltham, MS, USA).AO stock solution was prepared by dissolving 50 mg of AO in 10 ml of distilled water and stored refrigerated.Working solution was prepared on daily basis by mixing of 1 ml of AO stock solution with 0.5 ml of glacial acetic acid and 50 ml of distilled water.Methanol fixed samples were stained for 2 min, rinsed with water and air dried in the dark.
AO-stained samples were observed and photographed using an Olympus BX50 fluorescence microscope (Olympus, Hamburg, Germany).The evaluation of the coverage was carried out by the method of quantitative microscopy and with respecting the principles of unbiased sampling [37,38].Images of the AO-stained surface with bacteria were photographed according to the previously prepared scheme: using an immersion objective (100×, numerical aperture 1.30), every third field of view on a vertical line passing through the center of the sample and every third field of view on a horizontal line passing through the center of the sample were photographed, representing 2 × 5 fields on each sample (figure 1(b)).
Quantitative analysis was performed using ELLIPSE 2.0.8.1 software (ViDiTo, Kosice, SK).Each image was overlaid with a grid of 3,185 points (figure 1(c)).For each point, it was determined whether it was located on a surface covered with bacteria (foreground) or not (background).Foreground/background ratios were calculated and statistically analyzed for each sample.

Statistical analysis
Five specimens of each type of titanium material were used for analysis.The normality test (Kolmogorov-Smirnov) was performed to confirm the hypothesis that the data have a normal distribution.Two-tailed unpaired t-tests were used for statistical comparisons.

Material characterization
The samples were characterized using a scanning electron microscope.Figure 2 shows three different types of evaluated surfaces at magnifications of 22×, 500× and 1,000×.There is a visible difference between smoothed (figures 2(a)-(d)) and laser-treated surfaces (figures 2(e)-(l)).A periodic micron-sized pattern generated on titanium samples by nanosecond laser treatment can be observed in figures 2(e)-(h).The line marked with a red ellipse in figure 2(h) marks the distance over five topographic maxima of the surface and corresponds to about 50 μm.This implies that the periodicity is about 10 μm.Figures 2(i)-(l) show the typical femtosecond laser induced ripples on the surface.The line marked with red ellipse in the figure 2(l) marks the distance over five topographic maxima corresponding to about 3.5 μm, resulting in periodicity about 0.7 μm.
The surface roughness quantified by the arithmetic mean roughness Ra and root mean square Rq is shown in table 3 for each sample.There was no significant difference between the samples except for Ti2 and Ti2F, where Ti2F samples had increased roughness compared to Ti2 (p < 0.0497).
Surface roughness can affect the interaction between the cells and the material.Increased roughness can simultaneously increase the surface area of the implant, change cell migration and attachment to the implant, enhance the osseointegration process, and increase bone-to-implant contact [39,40].Femtosecond laser treatment is considered to be one of the modern methods to achieve precise topography [41].In the article by Souza et al, a roughness range of about 1-10 μm was found to be most suitable for osseointegration [42].
Wettability is considered to be another important surface property.Surface wettability was estimated by measuring the contact angle of the sessile drop of distilled water.A decrease in the contact angle is associated with higher hydrophilicity.Higher wettability can be associated with air remaining trapped in the surface irregularities.As water spreads over the surface, air is expelled as the liquid penetrates and the contact angle becomes smaller.
The results of the wettability evaluation are shown in a figure 3.According to the measurements, all the samples have hydrophilic properties (contact angle less than 90°).The femtosecond laser modified surfaces showed increased wettability.
However, recent studies have shown that the femtosecond laser can create both hydrophobic and hydrophilic surfaces.Lackington et al investigated the biocompatibility of titanium-based implants with femtosecond laser modified surfaces and reported that hydrophobicity can be affected by surface topography and chemical composition [43].
In contrast to the aforementioned study, Exir and Weck reported that femtosecond ablation is associated with an increased amount of TiO and Ti 2 O 3 on the treated surface.The electron structure of Ti 2 O 3 has a large number of polar sites originating from coordinatively unsaturated titanium and oxygen atoms acting as Lewis acid and base pairs.The Ti 3+ ions on the surface are electron deficient with seven electrons in their outer orbital available to accept electrons from water oxygen, resulting in a hydrophilic hydration structure and hence hydrophilicity [44].
Cunha et al used two types of surface textures, consisting of laser-induced periodic surface structures and nanopillars.Both surfaces exhibited hydrophilic properties [32].Some authors reported that hydrophilic surfaces can also be obtained by laser texturing by creating smoother surface features, with roughness values <0.5 μm [45].

Cell adhesion and proliferation
Cell adhesion to material surfaces depends on a number of physical and chemical characteristics.The surface composition, topography and wettability are considered to be the decisive features.Distinctive surface treatment  is a way to improve hydrophilicity and to induce protein adsorption, which is of paramount importance for cell adhesion.At the same time, surfaces with low water wettability delay primary interactions with the aqueous biosystem.Recently, moderately hydrophilic surfaces have been shown to be optimal for cell adhesion [46] and show better biocompatibility [47].Both extremes, in the form of hydrophobic and highly hydrophilic surfaces, interfere with the adsorption of proteins mediating adhesion [48].
Our results in figure 4 show better cell adhesion on titanium samples with chemical composition Ti4 than Ti2.Similarly, nanostructured titanium samples nTi4 have enhanced cell adhesion than nTi2 samples.
Within the group of titanium grade 2, the samples treated with femtosecond laser (Ti2F) showed better cell adhesion than the samples treated with nanosecond laser (Ti2L).While in the group of titanium grade 4 the best adhesion is on polished samples Ti4, in the group of nanostructured titanium grade 4 there is no difference between polished and lasered surfaces.The difference between samples with treated surfaces has a different effect in the case of femtosecond and nanosecond lasers.While the Ti2F and nTi4F samples have higher adhesion against to Ti2L and nTi4L samples, respectively, the Ti4F and nTi2F samples have no difference in adhesion against to Ti4L and nTi2L samples, respectively.
Several other authors within the field of study have obtained similar results.The findings of Chen et al indicate that the micron grid topography produced by femtosecond laser irradiation was beneficial for cell colonization by anchoring the cells to the substrate surface [49].Liu et al found no significant difference in osteoblast adhesion at 24 h between polished titanium and femtosecond laser-treated titanium in their assay [22].
Additional to testing the adhesion of cells to the material, cell proliferation assays are used to evaluate the biocompatibility.Cell proliferation was compared across different chemical compositions of titanium materials.In terms of material grade, grade 4 titanium was shown to be superior to grade 2 titanium in both short-term and long-term proliferation for both cpTi and nTi.Short-term and long-term proliferation is better on titanium grade 4 materials.
Concerning the influence of surfaces on cell proliferation, there was a different behaviour of the cells on grade 2 and grade 4 materials.For cpTi4 and nTi4, both short-term and long-term proliferation was worse on both types of laser-treated surfaces.For cpTi2 and nTi2, laser treated surfaces exhibited a better short-term proliferation, while long-term proliferation was better only on the nanostructured version.Regarding the type of surface treatment, there is a difference in cell proliferation on nanostructured samples between nanosecond and femtosecond laser treatment, where nTi2F is better than nTi2L.Comparing the progress in cell proliferation between short-term and long-term cultivation, nanosecond laser treated nanostructured samples had the highest progress, 2.5× for nTi2L and 3.6× for nTi4L (figure 5).
The data obtained correspond with some of our previous findings with estimation of biocompatibility of nanolaser-treated titanium surfaces.For example, cell proliferation assessed after 48 h cultivation was better on nanostructured grade 4 titanium than on nanostructured grade 2 titanium.Further, polished surface of grade 4 nanostructured titanium showed better proliferation than laser-treated surface of the same material [50].We hypothesize that excellent proliferation on grade 4 material affects the behaviour of cells on the laser-treated surface.Therefore, it is likely that proliferation is improved only on grade 2 titanium, which has comparatively worse proliferation on the smooth surface.
In the present study, we extended the range of surface modifications by femtosecond laser treatment to estimate differences in cellular and bacterial response.Other investigators found that femtosecond laser texturing did not influence the adhesion but significantly decreased cell proliferation in comparison to the smooth polished titanium surface.These results may indicate that some elements of the surface topography can reduce the cell proliferation.More highly aligned cell extension and less cell-cell communication may reduce the cell proliferation rate [49].Similar results were obtained by Oliveira et al who concluded that laser texturing of samples did not induce cell proliferation [51].Shaikh et al investigated proliferation of human osteosarcoma cell line U2OS on titanium alloy surfaces treated with femtosecond laser.They found no difference in cell proliferation between untreated and laser-treated surfaces, but admitted more efficient cellular attachment as examined by confocal microscope [52].

Cytotoxicity of materials
Estimating cytotoxicity of materials through the LDH test is a valuable and widely used method in biomedical research and material science.The quantification of LDH release offers a sensitive, non-invasive, and quantitative method for assessing the membrane integrity, which can serve as an indicator of the potential harmful effects of materials to living cells.
The values of LDH activity at 24 h after cell seeding were generally low, so none of the materials showed a cytotoxic effect.The LDH release from cells seeded on different samples is shown in figure 6. Comparing the samples with each other, cpTi samples showed statistically significantly higher LDH activity than nTi samples (p < 0.001).The cytotoxicity of nanostructured titanium samples was even lower than that of a standard Petri dish (p < 0.001).It was found that no type of surface treatment, neither nanosecond nor femtosecond-laser treatment, affected LDH activity.This is consistent with research by other authors who also report low cytotoxicity for both cpTi and nTi materials [53,54].

Cell staining
To complement the assessment and comparison of cell proliferation after 168 h on individual samples the cell nuclei were stained with NucBlue ® LiveReadyProbes ® Reagent.Fluorescent images of the entire titanium samples surfaces were taken.The size of the stained area is comparable to the long-time proliferation values measured by the CCK-8 test.CellTrackerTM Green CMFDA Dye was used to assess cell morphology.The small inserted pictures show the cells after 48 h of cultivation.Cell morphology was not affected by any of the implant surface types evaluated (figure 7).

Bacterial adhesion
To demonstrate the effectiveness of antibacterial action of different surfaces, a quantitative experiment of bacterial growth was conducted using S. oralis as a bacterial model of pioneer microbes in oral cavity.Fluorescence microscopy of AO stained samples revealed adhesion of S. oralis and initiation of biofilm  formation on all tested samples.Bacteria appeared on the surface as single cells, chains and clusters of chains (figure 8) and their distribution on the surface was irregular.
The average value of bacterial coverage calculated according to the quantitative analysis methodology from five samples for each combination of material and its surface is shown in percentage on figure 9.The mean value of surface coverage by S. oralis on all sample types was 0.417%, ranging from 0.183% to 1.13%.
Although there was no statistically significant difference found among the individual samples, most samples exhibited a coverage percentage of below 1%.Therefore, it can be concluded that the material itself possesses antibacterial effect against the selected bacterial strain.However, as pointed out by Azeredo et al, the effect of the sample rinsing process after cultivation should be taken into account [55].This process removes bacteria from the surface by detaching and eliminating loosely attached bacteria.The proportion of bacteria eliminated depends on the intensity of liquid flow and exposure of the air-liquid interface [56].Aware of the effect of sample rinsing, to avoid unpredictable cell detachment, all specimens were washed in a common holder that exposed them to the same conditions during the washing step.The washing procedure was executed exactly as described for all five repetitions of the experiment.Surface treatment can change the wettability of titanium materials, but it depends on the chosen surface topography.Therefore, different authors report both hydrophobic and hydrophilic surfaces created by nanosecond and femtosecond lasers [57][58][59].Bacteria can adhere to materials with low hydrophilicity through hydrophobic interactions.Increased hydrophilicity of the surface causes forming of a water layer which prevents bacterial adherence and biofilm agglomeration [60][61][62].The relationship between surface wettability and biofilm formation can depend on the specific bacteria, the environment, and other conditions.The low percentage of bacterial coverage is crucial to prevent infection and maintain the longevity of the implanted material.
The chosen topography created grooves and, probably due to the capillary effect, increased the wettability of the femtosecond laser-treated surface, resulting in a more hydrophilic surface.The assumption that the grooves in a dimension comparable to the size of the bacteria would have an antibacterial effect did not appear.The interplay between surface properties and bacterial behavior is complex, and designing effective solutions requires a deep understanding of both microbiology and materials science.

Conclusions
The study comprehensively investigated the effects of laser ablation on titanium surfaces and their interactions with cells and bacteria, providing valuable insights into material surface modifications for potential medical applications.
The investigation revealed that femtosecond laser treatment effectively enhanced the wettability of titanium samples.Titanium grade 4 samples, both nanostructured and commercially pure, exhibited superior adhesion and proliferation rates when compared to titanium grade 2. Different properties between commercially pure and nanostructured titanium were demonstrated in the cytotoxicity testing, where the cytotoxicity of nanostructured titanium is significantly lower, regardless of the surface treatment.Cell proliferation also responded to titanium surface treatment.Laser treatment resulted in increased short-term cell proliferation on grade 2 titanium for both commercially pure and nanostructured titanium materials.In contrast, the long-term cell proliferation was increased only on nanostructured grade 2 titanium.Although the laser ablation had a limited effect on bacterial adhesion, the percentage of coverage in most samples was less than 1%, indicating that the material itself has an antibacterial effect on the bacterial strain S. oralis.
In conclusion, the findings contribute to the understanding of how various material types and surface treatments can influence cellular responses and antibacterial properties, providing a basis for further investigation in the field of biomaterial engineering.

Figure 1 .
Figure 1.(a) The model of holder used for achieving uniform bacterial growth in the cultivation medium.(b) The method and direction for selecting the reading fields on samples.(c) The system of quantitative sampling.

Figure 4 .
Figure 4. Comparison of cell adhesion on surfaces of samples with different type of material and surface treatment after 24 h, as assessed by CCK-8 assay.Error bars indicate means ± standard deviations.Asterisks above the bars indicate statistically significant differences ( * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5 .
Figure 5.Comparison of the cell proliferation after (a) 48 and (b) 168 h on samples with different type of material and surface treatments assessed by CCK-8 assay.Error bars indicate means ± standard deviation.Asterisks above the bars indicate statistically significant differences ( * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7 .
Figure 7. MG-63 cells were tested for proliferation after 168 h.The samples were stained with NucBlue ® LiveReadyProbes ® Reagent and viewed at a magnification of 40×.The inserted images show the cells after 48 h of cultivation.The samples were stained with CellTrackerTM Green CMFDA Dye and NucBlue ® LiveReadyProbes ® Reagent and viewed at a magnification of 400×.

Figure 8 .
Figure 8. Demonstration images of bacteria Streptococcus oralis on Ti4 surface.(a) Image taken with Keyence VHX-7000 material microscope and (b) fluorescence microscope image of acridine orange stained bacteria.

Figure 9 .
Figure 9. Average percentage of bacterial coverage of the titanium sample with different surface treatments.

Table 1 .
Mechanical properties of commercially pure and nanostructured bulk titanium materials.σ0.2: yield strength, σ UTS : ultimate tensile strength, ε f : elongation to failure, A f : reduction of area.

Table 2 .
The overview of the titanium samples.

Table 3 .
The values of arithmetic mean roughness Ra and root mean square Rq.