Ceramic composite materials are increasingly used in dental restoration procedures, but current ceramic surface de- signs do not yet achieve the osseointegration potential of state-of-the-art titanium implants. Rapid bone tissue integra- tion of an implant is greatly dependent on its surface characteristics, but the material properties of ceramic composite materials interfere with classical surface modiﬁcation techniques. Here, ultra-short pulsed laser machining, which of- fers a deﬁned energy input mitigating a heat-a ected zone, is explored for surface modiﬁcation of ceramic composites. Inspired by surface textures of clinically relevant titanium implants, dual roughness surfaces are laser patterned. Ra- man mapping reveals a negligible e ect of ultra-short pulsed laser ablation on material properties, but a laser-induced change in the wetting state is revealed by static contact angle measurements. Laser patterning of surfaces also inﬂu- ences blood coagulation, but not the attachment and spreading of osteoblastic cells. The presented laser machining approach thus allows the introduction of a rational surface design on ceramic composites, holding great promise for the manufacturing of ceramic implants. Keywords: Ultra-short pulsed laser ablation, Surface functionalization, Bio-ceramics, Dental implant, in vitro test, XPS depth proﬁling, Raman mapping 1. Introduction Since the development of dental restoration in the 1960s, the number of inserted implants increased ev- ery year. While initially the primary goal of a restora- tion with dental prosthetics was to gain full functional- ity, aesthetic aspects related to the color of implants and concerns over possible allergic reactions to metal im- plants have found increasing interest. To this end, ad- vances in material science concerning novel alloys and engineered surface structuring have greatly contributed to enhanced implant performance [1, 2]. Generally, the requirements for a biomaterial as tooth replacement are manifold: the mechanical properties have to match the application, the material should be cytocompati- ble, show no corrosion in physiological environments, should be highly wear resistant, and ultimately, it should provide rapid osseointegration [3]. Pure titanium and ti- tanium alloys have been the standard materials for den- tal implants for decades [4], with their properties being Corresponding author Email address: ackerl@ethz.ch (Norbert Ackerl)well studied [5] and cost-e ective production being well established. Importantly, titanium implants have proven long-term clinical survival rates [6]. Surface structuring is a well known approach to enhance osseointegration of implants and can be employed to achieve distinct sur- faces at the body and neck to tailor hard and soft tissue interaction [7, 8, 9]. The most suitable surface design is dicult to specify in detail due to the complex interplay of surface characteristics and biological processes tak- ing place after implantation [10, 11, 12]. A commonly used routine treatment of titanium and its alloys is sand- blasting followed by a wet-chemical etching step to gen- erate a moderately rough surface, which is known to inﬂuence cell behaviour and ultimately to promote os- seointegration [13]. To this end, it has been speculated that engineered surfaces with hierarchical elements in the micro- and nanometer range possibly enhance the biomaterial activity via controlling cell proliferation and di erentiation [14]. Providing excellent control over process parameters, laser-induced surface structuring on titanium alloys utilizing short and ultra-short pulsed (USP) lasers has been explored [15, 16, 17, 18]. A Preprint submitted to tba November 12, 2020 commercially available surface generated by laser struc- turing revealed a high potential by a micro-channel ar- rangement combined with nanostructures in preclinical studies [19, 20]. However, introducing a deﬁned hierar- chical surface structure on metallic dental implants has yet to prove a signiﬁcant advantage over state-of-the-art surfaces and is often accompanied by surface oxidation changing the chemical potential [21]. In recent years, metal-oxide ceramics and compos- ites thereof have been proposed for several applica- tions including dental and hip implants [22]. Ceram- ics are especially promising materials for dental im- plants because of their high hardness, tunable tough- ness [23], and the ability to generate colors that more closely match the natural teeth. Furthermore, a lower risk for allergic reactions in comparison to titanium and metallic alloys has been reported for ceramics [4]. A large number of ceramics and composites are avail- able, where the functionality can be speciﬁcally de- signed and tuned [24]. Especially, metal-oxide ceram- ics give the unique opportunity to design the mechanical properties [25], corrosion resistance [26], and wear be- havior via alloying and adjusting the composition [27]. Today zirconia, alumina and alloys thereof with addi- tives for stabilization of speciﬁc phases are the main ceramics used. However, these materials are hard-to- machine in the sintered state. The prevalent industrial manufacturing process for ceramic implants is grinding with diamond tools or machining in the green state be- fore sintering [28] followed by an adapted sandblasting and etching step to generate a desired surface rough- ness. USP laser manufacturing is a promising tech- nique to machine even ultra-hard materials pointing to a minimal heat-a ected zone, no wear, and high geo- metric ﬂexibility. This technology has shown high ap- plicability to alter the wettability [29, 30], tribological performance [31], and reaction paths in electrocataly- sis [32]. Recently, a rapid-prototyping approach us- ing USP lasers to manufacture complex 3 Dalumina- toughened zirconia (ATZ) dental implants with control- lable roughness and high precision was presented [33]. However, the use of laser manufacturing for dental im- plants is still an emerging ﬁeld, wherein its poten- tial impact on ceramics has still to be unraveled. To date, little has been investigated on USP laser ablation of technical ceramics, with the choice of laser source and strategy still being under discussion [34, 35] and the research is largely focused on cutting and drilling processes [36, 37]. Studies concerning the impact of USP laser marking on hydro-thermal aging of ATZ and zirconia-toughened alumina (ZTA) pointed to a negli- gible inﬂuence of orthogonal laser ablation [38]. Re-cently, the applicability of deﬁned surface patterns on complex ATZ ceramic implants was demonstrated [39] and a femtosecond pulsed laser patterning of ATZ was reported [40]. However, the inﬂuence of these surface patterns on cell responses was not explored. In this study, rationally designed hierarchical struc- tures inspired by state of the art implant surfaces are introduced on ATZ and tetragonal zirconia polycrystal (TZP) ceramics by USP laser machining. A system- atic ablation study presents the characteristics allowing well-deﬁned and reproducible laser machining and the introduced micro- and nanostructures are assessed in vitro for their inﬂuence on blood coagulation and cell adhesion, representing the early steps of implant inte- gration. Following, the surface state is analyzed and conclusion drawn on the impact of chemistry and laser- induced surface potential changes on the observed bio- response. 2. Material and experimental routines 2.1. Zirconia composite characteristics Ceramic materials are potential candidates for den- tal implants, for which increasing the fracture tough- ness is one of the main challenges. The most important systems are metal-oxide based ceramics like alumina- toughened zirconia (ATZ), zirconia-toughened alumina (ZTA), partial stabilized zirconia (PSZ) and tetragonal zirconia poly-crystal (TZP). Yttria stabilizes the cubic or tetragonal zirconia phase depending on stoichiome- try and rectiﬁes the corrosion resistance in aqueous en- vironments being an ideal candidate to tailor the mate- rials characteristics for dental restoration [22]. These ceramics exhibit an indirect wide-band gap depending on the material and phase, where ZrO 2is monoclinic at room temperature with two transitions to a tetragonal and cubic phase in the high-temperature regimes. The gap in the tetragonal phase is about 5:5 eV [41, 42] and therefore non-linear absorption at high power density enabled by USP laser ablation is necessary for material removal. Alumina has a gap of about 8:7 eV in the alpha phase existent in ATZ for toughening and TZP as by- product [43, 44]. Moreover, pure alumina in the -phase is studied to compare the laser ablation characteristics to zirconia investigating a possible inﬂuence between ATZ and TZP. These gaps play a role in the electron gener- ation and inﬂuence the ablation behavior and following threshold. All specimens are supplied by Metoxit AG Switzerland in the used geometry and the surfaces pre- pared reaching a mean roughness Ra<0:2 µm . Gener- ally, HfO 2up to 2 wt % is present in the ceramic as con- taminant. The ATZ composition in weight percentage is 2 Adsorption & CoagulationAdhesion & ProliferationLaserstructured CeramicsBlood PlateletProteinsFibrin-network HBC ErythrocyteFigure 1: Routine for the biological assessment, where the laser-structured ceramic surface is ﬁrst tested for blood coagulation characteristics after 1315min followed by studying the adhesion and proliferation of HBC after 24 h, adapted from [12]. 76%ZrO 2, 20%Al 2O3, 4%Y 2O3and TZP is constituted by 94% ZrO 2, 5%Y 2O3, and 0.25%Al 2O3with a mean grain size of 0:4 µm and0:35 µm accordingly. The alu- mina specimen is of a 99.8% purity with a mean grain size of 5 µm . 2.2. In vitro assessment Blood coagulation and the attachment of osteoblast progenitor cells was assessed as reported previ- ously [12], with an schematic overview being provided in ﬁgure 1. In brief, all samples have been cleaned ultra-sonically in pure ethanol and sterilized at 160 °C dry heat for 2 h. Human whole blood gathered by standard venipuncture technique is used and partially heparinized. The specimens are placed in a round- shaped polytetraﬂuoroethylene holder and each cham- ber is ﬁlled with fresh blood, closed and placed on an orbital shaker for 13- 15min to allow for blood coagu- lation. Subsequently, the blood on the samples is re- moved before rinsing them with pre-warmed phosphate- bu ered saline. Primary human bone progenitor cells (HBC) from di erent donors were used to study cell ad- hesion and proliferation as well as osteogenic di erenti- ation on the blood pre-incubated surfaces. HBC attach- ment and spreading is assessed by confocal laser scan- ning microscopy (CLSM) of the samples after 24 h of culture. The samples are then rinsed with PBS, ﬁxed, and stained for the actin cytoskeleton with AlexaFluor 488-conjugated phalloidin and nuclei with DAPI for 1 h at room temperature. Fibrinogen is labelled by incu- bation with a mono-clonal anti-human ﬁbrinogen an- tibody followed by incubation with the corresponding anti-mouse Alexa Fluor 555 antibody for 1 heach. The samples are then evaluated by CLSM with a Carl Zeiss LSM780.2.3. Laser machining conﬁguration The principal conﬁguration of the used laser setup is depicted in ﬁgure 2. An USP laser source Amphos XYU V Z Scanhead SourceGalvoL/4 L/2 Amphos 200 GR 515nm / 120W <1ps pulses 1kHz - 40MHzTelescope Figure 2: Experimental laser test bed constituted by a source, beam guidance, optical (U, V), and mechanical (X, Y , Z) axes for fast beam steering plus positioning. 200 in a master oscillator pulse ampliﬁcation conﬁgura- tion realized by Innoslab technology with a second har- monic unit emits highly intense sub 1 ps 515 nm pulses with a maximal pulse energy of 300 µJ . High-precision X, Y , and Z mechanical axes (ANT180, Pro165) from Aerotech allow a distinct positioning of the specimen and adjusting the focus of the 163mm telecentric f-theta lens. Using a telescope with threefold magniﬁcation and divergence compensation enables a focal spot diameter of about 11 µm , where wave plates set the circular po- larization state in the focal plane for deﬁned ablation. A galvo scanhead (Scanlab Hurryscan II) enables a fast beam steering and the complete conﬁguration is con- trolled by Aerotech drives and the A3200 software for synchronous axes movement. The laser machining uti- lizes a 2.5 Dstrategy, where the surface design is gener- ated using CAD tools and a developed CAM software for the laser hatch and path computations [45]. This 3 conﬁguration allows a straightforward laser machining of distinct structures with high geometric ﬂexibility. 2.4. Analysis routines The laser machined work-pieces are inspected by light microscopy and measured by confocal microscopy (Leica DCM3) using a 50x objective lens with a stitch- ing routine. Scanning electron microscope (SEM) studies (Hitachi SU-70) allow to resolve the micro- and nanostructures with high resolution. Raman spec- troscopy (Thermo Scientiﬁc DXR2) at 455 nm excita- tion wavelength and a penetration depth of few hun- dred nanometers identiﬁes the crystalline phase of the ceramic and x-ray photon emission spectroscopy (XPS) assesses the quantitative surface-near chemical con- stituents. A PHI Quantum 2000 spectrometer (Physical Electronics) using a spherical capacitor energy analyzer with Al K radiation at 1486:68 eV working at a vac- uum of 5107Paenables an automated measurement routine. Following, the assessment of all samples under investigation runs in one measurement cycle with the XPS calibrated at the gold 4f line at ( 84:00:1)eV. 3. Results and discussion 3.1. Laser ablation characteristics of ATZ and TZP To generate deﬁned microstructures 2.5 Dablation is utilized and the material removal rate (MRR) is attained by machining pockets. A total of 45 layers of the re- spective material are removed using a hatching distance of5 µm and23deg rotation of each subsequent layer reducing the accumulation of deviations. The ﬂuence regime is kept below 1:4 J cm2and ﬁgure 3 presents the scaled speciﬁc MRR. Assuming the laser pulses to be Gaussian in space and time allows to identify the threshold Fthand optimal ﬂuence values Fopt. If the MRR is divided by the used average power Pavg, the speciﬁc MRR describing the ablation e ciency can be determined [46]. Keeping the ablation strategy and size of the pockets unchanged reduces the dependency from a volume removal rate to the ablation depth zablor layer thickness zlayer. Figure 3 graphs the modiﬁed speciﬁc MRR following the functionality zabl=Pavg/opt Fln2 F Fth! (1) being proportional to the optical penetration depth opt, ﬂuence F, and threshold ﬂuence Fth. A least-square ﬁt- ting allows to identify the threshold and optimal ﬂu- ence from the experimental study presented in ﬁg- ure 3. Hence, the functionality allows to estimate the 0.20 .40 .60 .81 .01 .21 .40 .10.51.01.52.02.50 .1zlayer / Pavg [µm/W]F [Jcm-2] ATZ Al2O3 TZP LSQ FitFigure 3: Speciﬁc material removal rate given by the ablation layer depth zlayerscaled by the average power Pavg. To determine the multi- pulse threshold and optimal e cient ﬂuence a least square curve ﬁt- ting following (1) and (2) is applied. multi-pulse Fthby extrapolation to zero layer thickness and assuming a full incubation reaching the saturation regime [47, 48]. Setting the derivation of (1) to zero leads to an proposed optimal ﬂuence for highest power eciency of ablation to Fopte2Fth: (2) Here, the optimal ﬂuence depends solely on the thresh- old ﬂuence Fthand inherently the focal beam radius w0. Keeping the pocket-size and laser strategy un- changed leads to the same processing time for all ex- periments [49, 50]. The attained multi-pulse threshold of ablation from ﬁtting and extrapolation to equation (1) for sub-picosecond pulsed ablation reveals 0:13 J cm2 for ATZ, 0:18 J cm2for alumina, and 0:21 J cm2in case of TZP. Moreover, the maximum at the optimal ﬂuence is found at 0:9 J cm2for ATZ, 1:3 J cm2for alumina, and 1:56 J cm2for TZP respectively. Gen- erally, the proposed trend for the ablation character- istic is followed by the experimental data allowing to estimate the anticipated optimal and threshold ﬂuence. Hence, the power density necessary for excitation de- ﬁnes a small parameter window near the threshold value enabling a deﬁned microstructuring by multi-photon ab- sorption without crack initiation. In addition to the mi- crostructures a second hierarchical roughness is gener- ated as depicted in ﬁgure 4, tailored for an enhanced osseointegration. To study the evolution of ablation fo- cusing on nano-roughness, the laser repetition rate is set to10 kHz and the pulse-to-pulse overlap altered by ad- justing the galvo scan speed leading to separate ablation 4 10μm 10μm 1μm 1μm 1μm 100μm a b d fc eFigure 4: SEM micrographs presenting the surface structure of the laser machined ATZ (a-c) and TZP (c-f) specimen. The pulse and hatch distance is optimized (a) leading to a homogeneous nano-roughness (b). A deﬁned ablation is a necessity mitigating cracks (e) and generating a comparable surface on both substrates in the magniﬁed graphs presented in (c) and (f). events up to high overlap. This parameter study leads to the ablation behavior of the ceramics, where a compa- rable surface has to be introduced to both materials. A sharp onset of ablation is observed in contrast to metals, where up to the threshold no obvious modiﬁcation of the surface is observed followed by a layer thickness of about 1 µm in agreement with literature [51]. Clearly, the ablation characteristics are governed by non-linear e ects and multi photon absorption processes for these wide-band gap materials [52]. Figure 4 presents the ap- proach for achieving a nano-roughness, where the min- imum pulse-to-pulse and hatch distance is attained for ATZ in the upper row (a-c) and TZP at the bottom (d- f) shown at di erent laser parameters summing up a broader assessment. The aim is reaching a reproducible roughness in short processing time, where the pulse- to-pulse overlap and hatching distance is varied in the range between 1 µm to10 µm . Figure 4a presents the distance set to 10 µm , where the single ablation events can be observed (at the rim marked with the red el- lipse). Setting the distance to 4 µm leads to a rough- ness comparable to the one achieved by 2.5 Dablation with multiple layers presented in ﬁgure 4b. Ceramics are in principle brittle and thermal gradients or local melting leads to cracking if the local energy input is too high [53]. Therefore, the maximal MRR is limited by the usable ﬂuence, where ﬁgure 4e corroborates this assumption revealing small cracks at a ﬂuence of only1:3 J cm2below the found optimum for multi-pulse ab- lation, where parts of the surface shown in ﬁgure 4d are not completely ablated. After this qualitative study on surface roughness altering the laser parameters between near the threshold ﬂuence up to the optimum and adjust- ing the pulse and hatch overlap, a ﬂuence of 0:5 J cm2 for ATZ and 0:7 J cm2for TZP is identiﬁed. The laser- induced nano-roughness on both specimens is compa- rable, as depicted in the magniﬁed graph of ﬁgure 4c and ﬁgure 4f. It is anticipated, that the coupling of the laser light starts at the grain boundaries and testing the same set of parameters near the threshold ﬂuence re- veals an inhomogeneous ablation state, where only parts of the pocket is ablated. The origin might be the slightly di erent ablation threshold observed between the alu- mina, ATZ and TZP ceramic, where the threshold of the additional alumina particles is in between the two compounds while additionally introducing more grain boundaries compared to the TZP with the zirconia ma- trix. 3.2. Rational surface design and laser machining In this study, textured surfaces inspired by success- ful implant designs reported in literature [54, 55] are designed and generated by laser machining. Consid- ering the knowledge of osseointegration with titanium implants in contact with human osteoblastic cells points to a random structure with hills and grooves of a pitch 5 with about 50 µm and peak-to-valley height of approx- imately 30 µm [8]. Additionally, a second hierarchi- cal morphology in the sub-micrometer range was sug- gested to enhance implant-tissue interactions [24]. In order to assess the impact of the structuring on the ini- tial steps of implant integration, three groups of sur- faces from each material are studied. An unprocessed surface (blank), rough surface (nano), and a combina- tion of random microstructures with the same rough- ness (micro&nano) are explored. A pseudo-randomly designed 3 Dstructure was generated by using a ran- dom number generator and scaling the height between 15 µm keeping the pitch at 50 µm with a measurement of the surface presented in ﬁgure 5a. For the laser man- ufacturing the Amphos 200 SHG radiation at 515 nm wavelength with sub-picosecond pulses at a repetition 500μma b 500μm 20 -20h[μm] -10010h[μm] 2.5 -2.5-1012 -2 Figure 5: Surface design and confocal measurement of the ATZ ce- ramic. (a) The rough surfaces is laser-structured by cross-hatching and (b) the superposition with microstructures and random height with spacing of 50 µm and peak to valley height of 40 µm . rate of 200 kHz , average power of 0:45 W for ATZ and0:55 W for TZP, a focal beam diameter of 11 µm , beam distance l b=5 µm , and scan speed of 300 mm s1was used. With this set of parameters, 12 layers were ablated to reach the premeditated depth and each adjacent layer was rotated by 29°mitigating the accumulation of ge- ometrical errors. Figure 5a shows the confocal optical measurement of the surfaces after laser machining the designed features. The nano surfaces are generated with a di erent set of laser parameters in a two-step pass with a second orthogonal hatched layered ablation step. The average power was altered to 0:2 Wfor ATZ and 0:3 Wfor TZP with a reduced repetition rate of 10 kHz to diminish the inﬂuence between the adjacent pulses. The beam dis- tance was set to 4 µm with a scan speed of 200 mm s1 leading to a processing time of below 1 min per sam- ple. The micro&nano hierarchical surfaces are manu- factured within 7min on the disc-shaped specimen with an area of 20 mm2and ﬁgure 5a shows a central re- gion thereof comparable for both specimen. A qualita- tive SEM assessment reveals the surface topography of the laser generated surfaces in more detail focusing on the micro&nano structure in ﬁgure 6. The magniﬁed micrographs reveal the hierarchical roughness, where microstructures constitute the peak-valley morphology, while featuring a nano-scale roughness similar to the de- signed roughness shown in ﬁgure 4, thereby staying true to the laser parameter optimization. The black appear- ing alumina particles shown in ﬁgure 4a are contained and Raman measurements are carried out to determine possible phase changes. Stabilized zirconia ceramics can undergo a phase transition from tetragonal to monoclinic upon heating, leading to a volume increase and stressed surface or even to the development of cracks [33, 53]. There, con- focal Raman mapping is utilized to measure a map of an area spanning 400 mm2attaining 100 spectra of the blank and laser patterned nano surfaces. This allows a sound statement on the phases and the representative measurements are presented in ﬁgure 7 for both ATZ (a) and TZP (b) samples. For both samples, viz. ATZ and TZP, the spectra reveal a good signal-to-noise ra- tio and the four characteristic peaks (T1 - T4) for the tetragonal conﬁguration are located at the expected po- sitions as reported in literature [56]. Indeed, the mea- surement conﬁrms the tetragonal phase of both ATZ blank and nano (7a). In some spots of the blank sample, an impurity monoclinic phase is observed by the dou- ble peak structure centered at 180 cm1and the small peak at 378 cm1. Interestingly, the amount of the mon- oclinic phase is reduced for the ATZ specimen after laser patterning in agreement with literature comparing 6 a d 50μm 10μm 1μm 1μm 100μm 500μmc fb eFigure 6: SEM graphs of the micro&nano hierarchical structures on ATZ (a-c) in the upper row tilted by 40deg and TZP (d-f) at the bottom. The rational designed micro features (a,b,d,e) are superimposed by the designed nano-roughness (c,f). The quality of the structures on both materials is comparable shown with higher magniﬁcation in (c) and (f). 1002 003 004 005 006 007 00I [a.u.]k [cm-1] ATZ blank ATZ nanoam onoclinicT 1T 2T 3T 4 1002 003 004 005 006 007 00I [a.u.]k [cm-1] TZP blank TZP nanobT 1T 2T 3T 4 Figure 7: Raman measurements of the blank, nano ATZ (a) and TZP (b) composite revealing the persistence of the tetragonal phase with an o set for clarity. The spectra for the blank (solid) and nano (dashed) point to a negligible impact of USP ablation reducing the monoclinic phase for ATZ. ceramic implants subjected to di erent manufacturing techniques [33]. Therefore, USP laser machining can be used with a distinct set of laser parameters allowing a deﬁned patterning of both ceramic specimens. More- over, there is no observable shift in the peak positions apparent, which points to an unstressed state that under- lines the negligible impact of USP laser machining on the material.Structuring the TZP with the adapted laser param- eters allowed machining comparable nano and mi- cro&nano structures, where a comparison of statistical roughness measurements are given in table 1. As- sessing a set of surface and proﬁle roughness measures, the small structures presented in ﬁgure 6 are not ad- equately covered by the confocal measurement. This nano-roughness is crucial for the wetting behavior and 7 Table 1: Statistical roughness moments from confocal measurements of the surfaces presented in ﬁgure 5. Nine samples per material are laser machined in one batch (No.) and analyzed, leading to mean and standard deviation of S q,a,pand R q,a,z. Sample No. S q[µm] S a[µm] S p[µm] R q,RMS [nm] R a[nm] R z[µm] ATZ nano 1 1.6 0:1 1.30:1 4.80:7 60035 47530 2.90:3 2 1.50:2 1.20:1 5.10:9 61525 49025 2.50:2 TZP nano 1 1.9 0:6 1.40:5 4.90:8 59515 48010 2.80:1 2 1.60:4 10:2 4.40:6 59015 47515 2.90:2 ATZ micro&nano 1 7 0:3 5.80:2 211:4 - - - 2 6.90:3 5.70:2 19.51:9 - - - TZP micro&nano 1 6.6 0:2 5.40:2 17.51:4 - - - 2 6.70:2 5.50:3 18.61:2 - - - subsequent bio-response, and is assessed by a qualita- tive SEM study. For the analysis of the confocal area measurements, ﬁve parallel line proﬁles and additional the statistical surface moments root mean square height Sq, arithmetical mean height S a, and maximum height Szare used measuring two surfaces per manufactured batch. Using a proposed cut-o c=80 µm from ISO standard 4288-1996 and an evaluation length longer 0:4 mm allows to decouple roughness and waviness of the surface. The values presented in table 1 prove a high reproducibility of the laser generated surfaces on both ceramics rounded to 100 nm respecting the precision of the optical measurement. 3.3. Blood- and cell-material interaction Blood material interaction of the laser-structured specimens is assessed by SEM analysis in ﬁgure 8. Attachment and activation of platelets as well as ﬁbrin network formation is could be observed, with blank sur- faces showing the highest degree of coagulation. Com- pared to blank, nano as well as micro&nano surfaces show a greatly reduced blood coagulation where ﬁbrin network formation has not yet started. However, blood coagulation serves only as a ﬁrst assessment and the surface coverage with blood components and a ﬁbrin network strongly depends on the incubation time. In order to investigate the inﬂuence of laser-induced sur- face structures on cell attachment, primary HBC seeded on blood pre-incubated samples are studied by confo- cal laser scanning microscopy after 24 h, presented in the bottom graphs of ﬁgure 8a-f. Generally, the cells show good attachment and spreading on all surfaces. While no di erence in cell attachment and spreading could be observed between the di erent surfaces, the cells showed a preferential alignment along the groove structure on the nano surfaces that originate from laser processing with the hatch distance lbmost prominently observed in ﬁgure 8e. While the observed impact ofthe designed surface structures on cell adhesion is small after 24 h, future studies on cell proliferation and di er- entiation over longer time periods should reveal surface- dependent changes in cell behavior. 3.4. Surface chemistry and state The ﬁrst blood contact is greatly inﬂuenced by the wetting characteristics at the surface altering the rate of coagulation, which depends on surface chemistry and topography [57]. Measuring the contact angle therefore allows a ﬁrst assessment of the surface state. A Wen- zel state is expected to act as a surface state ampliﬁer. Hence, a hydrophilic behavior will reveal a reduced CA and vice versa a hydrophobic one an increased CA by laser patterning. Figure 9 presents the attained CA of the specimen, where at least two samples with more than three independent measurements are considered. After dispersing a 1 µldroplet of distilled water with a rate of 1 µl s1on the surface the CA is measured di- rectly after reaching an equilibrium shape and after 30 s. The pristine samples are hydrophilic with a measured CA around 50°and all laser-machined surface reveal a hydrophobic characteristic with CA well above 120° . In contrary to the proposed behavior of a Wenzel state the laser ablation process modiﬁes not only the topog- raphy, but also the surface-near chemistry. This may be attributed to the manufacturing in ambient condition evidenced by an observed slight color change of the ce- ramics to yellow, which is a sign of defects and espe- cially oxygen vacancies [58]. The anticipated origin of this color change will have an impact on the total chemi- cal potential leading to a modiﬁed CA and subsequently, altered blood coagulation. To assess the impact of USP laser ablation on sur- face chemistry in more detail, an XPS study is carried out on the surface structured samples. The XPS sur- vey scan reveals the elements as expected and the quan- titative analysis is given with table 2. With a ﬁrst in- spection at the surface showing no clear trend between 8 10μm 10μma b 50μm 50μmblank nano micro & nanoATZ - blood coagulationcTZP - blood coagulation ATZ - HBC adhesion TZP - HBC adhesiond e f Figure 8: Evaluation of the ATZ (a-c) and TZP (d-f) laser-induced surfaces for blood coagulation by SEM (upper graphs) and HBC adhesion by CLSM (bottom graphs). The adhered blood platelets and evolving ﬁbrin network after 1315 min shows the impact of laser structuring the ceramic surfaces on blood coagulation. Staining for cell nuclei (blue) and the actin cytoskeleton (green) demonstrates cell attachment and spreading on blood pre-incubated surfaces. Fibrinogen adsorption and formed ﬁbrin network is shown in red. Representative images from n =2 (SEM) and n =3 (CLSM) independent experiments are shown. the pristine and laser structured surfaces, depth proﬁl- ing is carried out by Ar ion sputtering removing square ﬁelds of 4 mm2. The depth is calibrated by removing 100 nm of Ta 2O5deposited on pure tantalum, within atotal sputter time of 13:5 min . This allows to estimate the sputter rate to 7:39 nm min1. Figure 10 presents the attained depth proﬁles for TZP and ATZ accordingly for the main constituents and carbon surface contamina- 9 Table 2: Atomic percent concentration of the di erent constituents from XPS measurement for the TZP and ATZ ceramic, respectively. For each surface from the blank, nano-roughness, and micro&nano an XPS determination is carried out. Sample C 1 sO 1sNa 1 sAl 2pSi 2pY 3dZr 3dHf 4 f ATZ blank 66.3 24.0 0.9 3.1 1 0.2 4.4 0.1 ATZ nano 50.5 34.8 0.7 3.0 0.4 0.6 9.7 0.1 ATZ micro&nano 54.7 32.4 0.6 2.6 0 0.6 8.7 0.1 TZP blank 65.4 23.9 - - - 0.6 9.8 0.0 TZP nano 43.8 37.7 - - - 0.9 15.8 0.1 TZP micro&nano 50.9 31.9 - - - 1.1 15.2 0.1 4 91 281 494 91 441 37A TZ blankA TZ nanoA TZ micro&nanoT ZP blankT ZP nanoT ZP micro&nano0501001509 0static Contact Angle [°] n>4 Figure 9: Static contact angle of two samples per surface at more than two locations. For both ceramics laser machining induces a transition from hydrophilic to -phobic wettability. tion. Additionally, the laser-patterned TZP sample with nano-roughness reveals 1.19 atomic percent concentra- tion of Cu impurities attributed to the laser manufac- turing, where the experimental testbed is used for dif- ferent materials. A contamination during storage is un- likely, considering that the same conditions were used for all samples. Clearly, the carbon content due to stor- age or processing in ambient conditions decreases after the ﬁrst removed layer for both ceramics being mainly existent on the surface. ZrO 2is partially reduced upon Ar sputtering [59] resulting in a decreased O /Zr ra- tio. The small yttrium fraction stays constant after the ﬁrst removed layer showing no sign of degradation and hafnium shows no apparent features. In contrast, the sputter depth proﬁle of the ATZ ceramics reveals a de- crease in oxygen and aluminum accompanied by an in- crease in zirconium. Comparing the two laser structured surfaces with the blank surface, an aluminum depletion evolves observable with the increase in atomic concen- 01 2 3 4 5 6 7 05 1 01 52 02 53 03 50 10203040506070depth [nm]atomic concentration [%]t sputter [min]Zr content increasedA l depletion less oxygen a 01 2 3 4 5 6 7 05 1 01 52 02 53 03 50 10203040506070depth [nm]atomic concentration [%]t sputter [min] C 1s O 1s Y 3d Zr 3d Al 2p Hf 4f Cu 2p3 blank nano micro&nanoZr reducedbFigure 10: XPS depth proﬁles following the main constituents of the ATZ (a) and TZP (b) specimen revealing a clear change in chemistry for ATZ correlated with the laser machining for the nano surface struc- tures. For TZP Zr is reduced in the ﬁrst 10 nm . 10 tration up to the maximal layer depth studied. Equiva- lent to TZP, the carbon concentration decreases and only covers the surface in a thin layer. Nevertheless, no de- tailed explanation of the change in wettability and asso- ciated bio-response can be stated with the attained data. One e ect concerning the aluminum depletion could be correlated with the observed ablation characteristics of the ATZ ceramic. The sharp onset of the material re- moval ablates clusters of material and the alumina par- ticle density seems to decrease after ablation. However, for a sound interpretation cross-sections inspected by high-resolution SEM or using transmission electron mi- croscopy should be attained. Hence, the small trends in the depth proﬁling do not allow a consistent reasoning and more detailed studies using surface-near techniques like photo-luminescence would allow measuring band gap variations induced by defects and oxygen vacancies in more detail. 4. Conclusions and outlook Laser machining with USP has proven to be a vi- able approach for rapid prototyping minimizing the total time for geometrical changes on hard-to-machine ma- terials such as ceramics. A distinct parameter study and choice of strategies enabled a straight-forward pat- terning of complex 3 Dsurface geometries with custom- tailored structures. Rational designed topographies with hierarchical features have been introduced to ATZ and TZP ceramics and following an in depth material char- acterisation, the blood- as well as cell-material interac- tion was studied in vitro. The studied USP laser pro- cesses using orthogonal incidence condition unraveled a negligible impact on the materials phases assessed by electron microscopy, Raman mapping and XPS proﬁl- ing. A detailed investigation of the surface using XPS showed a slight change in atomic oxygen concentra- tion and depth proﬁling points to an aluminum deple- tion in the surface near region of the ATZ accompa- nied by an increasing zirconium portion. In contrast, the TZP only showed a slight decrease in zirconium, where both ceramics are covered by about 1 nm car- bon. The introduced change in chemical potential al- tering the static contact angle to hydrophobic interferes with blood coagulation, but has no e ect on cell attach- ment and spreading. Further studies employing the laser patterning in deﬁned ambient conditions of vacuum and inert gas would allow to decouple the impact of induced chemistry change and topography on the bio-response, whereas longer term studies on cell proliferation and di erentiation as well as animal studies would allow to assess the tissue integration potential of the presentedsurfaces. Overall, the introduced routines reveal a high potential using a fast structuring route for surface devel- opment altering the blood- and tissue-material interac- tion to potentially enhance tissue integration of implants after surgery. CRediT authorship contribution statement Norbert Ackerl: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Vi- sualization, Writing - original draft, Writing - review & editing. Alexander Hansen Bork: Resources, Data curation, Formal analysis, Writing - review & edit- ing. Roland Hauert: Formal analysis, Investigation, Re- sources, Writing - review & editing. Eike M ¨uller: Con- ceptualization, Data curation, Formal analysis. Markus Rottmar: Conceptualization, Methodology, Project ad- ministration, Resources, Supervision, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known com- peting ﬁnancial interests or personal relationships that could have appeared to inﬂuence the work reported in this paper. Acknowledgments The funding of the Swiss National Science Foun- dation (SNSF) [ grant number 169654 ] is gratefully acknowledged. All authors thank Prof. Christoph M¨uller for the access to Raman microscopy acquired under the SNSF R’Equip program [ grant number 206021 144986 /1 ], Prof. Konrad Wegener, and Prof. J¨org F. L ¨oer from ETH Z ¨urich for using their re- sources carrying out the laser machining and parts of the SEM assessment accordingly.