Surface quality tests

Surface quality tests were carried out using:

optical digital microscope

Macroscopic assessment of the implant’s surfaces was performed using a Leica DVM6 optical digital microscope at magnifications of 3 ÷ 100x.

scanning electron microscope

Surface assessment was carried out using a TESCAN VEGA Scanning Electron Microscope (SEM) (TESCAN, Brno, Czech Republic). Observations were carried out using an SE detector at 15 keV energy at x100, x500 and 2000x magnifications.

optical profilometer

Surface topography studies were carried out using a Leica DCM 8 optical profilometer using the confocal method using green light at 20x magnification. The surface analysis was performed using Leica Map Premium 9 software. The area of the analyzed surface was 3.5 x 2.6 mm. Profiles in two directions were determined for each surface (upper and lower). The parameters Sq, Ssk, Sku, Sp, Sv, Sz and Sa were determined in accordance with the ISO 25178 standard and the parameters Rq, Rsk, Sku, Rz, Ra in accordance with the ISO 21920 standard. The tests were carried out for 6 randomly selected places, 3 from each implant side.

Evaluation of the alloy structure

To assess the structure, longitudinal and transverse metallographic sections were made as part of the research. The samples were hot embedded in MultiFast resin and then mechanically polished using Struers equipment. The samples were then polished using an aluminum oxide suspension. The etching process was carried out in the 8% HF solution. Metallographic examinations were carried out on the Observer Z1.m light microscope from ZEISS at magnifications of 100 ÷ 1000x.

Hardness and hardness profile measurements

The hardness of the analyzed plate was tested using the Vickers method. The measurements were carried out using a DuraScan hardness tester from Struers at a load of 5 N. The tests were carried out on longitudinal and transverse sections, with 5 tests performed on each.

The hardness profile of the coatings on the surface of the tested implant was carried out on an open platform equipped with a Micro-Combi-Tester from CSM Instruments using a Vickers indenter using the Oliver&Pharr method. The aim of the test was to determine the instrumental hardness as a function of the penetration depth. The speed of loading and unloading was 3000 nm/min, and the time of holding the sample under maximum pressure was 5 s. The value of the indenter load was the resulting one. Microhardness measurement was carried out at the following penetrator depth values: 200 nm, 300 nm, 400 nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, 6000 nm, 7000 nm.

Testing the adhesion of the coating to the substrate

The adhesion of the TiO₂ layer to the substrate was tested using a scratch test using an open platform equipped with a Micro-Combi-Tester from CSM in accordance with the recommendations of the PN-EN ISO 20502:2016-05 standard. Using a diamond Rockwell indenter, a scratch was made with a length of l = 3 mm, gradually increasing the load at a rate of v_s = 10 N/min and a table travel speed of v_t = 10 mm/min, with an increasing loading force F_c in the range of 0.03 ÷ 30 N The minimum normal force causing the delamination of the tested coating to the substrate was determined as the critical force. Changes in acoustic emission, friction force and friction coefficient, as well as microscopic observations, were used to assess the critical force L_c. There were three scratches on the upper and lower surfaces.

Pitting corrosion resistance tests

Pitting corrosion resistance tests were carried out using the potentiodynamic method. A PGP201 potentiostat from the Radiometer was used. The reference electrode was a saturated calomel electrode, and the auxiliary electrode was a platinum wire. Before testing, the samples were cleaned in ethyl alcohol in an ultrasonic cleaner for 10 minutes. The tests began by determining the E_OCP opening potential for 60 minutes. Polarization curves were then recorded, starting from the initial potential E_st = E_OCP − 100 mV. The rate of change of potential was 3 mV/s. The tests were carried out up to a potential value of +4000 mV or an anode current density of 1 mA/cm². The tests were carried out in a PBS solution (Chem Solve; chemical composition: NaCl 0.14M, KCl 0.0027 M, phosphate ions PO₄^-3 0.010 M). Based on the polarization curves using the Stern method, characteristic values ​​describing the resistance to pitting corrosion were determined, i.e.: corrosion potential E_corr (mV) and polarization resistance Rp (kΩcm²). The tests were performed on 5 samples obtained from implants.

Surface wettability tests

Wettability tests were carried out by the sessile drop method using Attention Theta Flex (Biolin Scientific). The tests were carried out at room temperature using 1.5 µl droplets of distilled water. The applied droplets were analyzed using OneAttesion software. Contact angle measurements were started 3 s after droplet deposition. Each measurement lasted 60 s with a sampling frequency of 1 Hz. Tests were performed for 6 randomly selected areas, 3 on each side of the implant.

Tests of the chemical composition of the surface layer

Energy Dispersive Spectroscopy

The analysis of the chemical composition using the EDS method was carried out using a TESCAN VEGA Scanning Electron Microscope (SEM) (TESCAN, Brno, Czech Republic) equipped with an Xplore EDS Electron Dispersive Spectroscopy (EDS) detector (Instruments OXFORD, Oxford, England). The tests were carried out point-wise at 2000x magnification and on a surface area of 110 x 110 µm, for which maps of the distribution of individual elements on the tested surface were created. Based on the measured spectra, the chemical composition of the sample's surface layer was determined in t%. The spectrum of carbon (C) was omitted in the analysis, considering it an organic and atmospheric pollutant deposited on the surface of the tested sample.

X-Ray Photoelectron Spectroscopy

The surface chemical state examination was done with the use of a multi-chamber ultra-high vacuum experimental setup equipped with an X-ray photoelectron spectroscopy (XPS) setup. For the XPS investigation, monochromatic Al Kα radiation (PREVAC, dual-anode source XR-40B; energy 1486.60 eV) was utilized for sample excitation. The binding energy (BE) axis of the analyzer was calibrated to the Ag 3d5/2 (368.27 eV) [1] region of the silver sample kept at the same sample stage as an examined sample. Additionally, the BE scale was monitored with control of the C-C signal in the C 1s region with respect to the described procedures [7]. The electron pass energy was set to 200 eV (scanning step, 0.9 eV) and to 100 eV (scanning step, 0.05 eV) for the survey spectra and the individual high–resolution regions, respectively. For the highest energy resolution and limitation of energy dispersion effects, the curved analyzer exit slit (C 0.8 mm x 25 mm) was utilized. The peaks were decomposed using CasaXPS software with the use of Shirley's background and the LA line shapes. The full-width at half-maximum (FWHM) values were slightly varying within a narrow range The spin–orbit splitting was taken into account, where applicable. The estimated uncertainty of the energy position of components was 0.08 eV.

Ar⁺ ion etching (for in-depth chemical analysis) was performed using a grazing–incidence–oriented PREVAC IS40A1 ion gun equipped with a differential pumping stage. The sputtering process was performed with energy 1.5 keV with a spot size of 3.0x2.0 mm² for 5 min per etching step. The ultimate scan was done after 45 min. of Ar⁺ etching. The partial pressure of Ar was kept at the level of 5·10^-8 mbar during the sputtering.

Surface quality tests

Surface quality tests using:

optical stereoscopic microscope,

The surface of the implants is characterized by a heterogeneous, matte, blue color (Figure 2 a), the intensity of which changes slightly within the zones subjected to permanent deformation during the shaping of its structural form (Figure 2 b). The variation in color was also influenced by the heterogeneous topography of the implants, created during vibration processing - Figure 2 c, d, e. Moreover, scratches remaining after grinding are visible on the surface of the implants (Figure 2 e, f). They occur mainly on the side surfaces of the implants (Figure 2 f). No unwanted machining residues were observed in the threaded holes and screw sockets. These surfaces are also characterized by a blue color (Figure 2 g, h).

scanning electron microscope

Microscopic examinations were performed on the upper and lower surfaces of the implant adjacent to the bone. Both surfaces were characterized by similar topography. There were numerous, evenly distributed dents on the surface, which were remnants of vibration processing - Figure 3.

optical profilometer

Analysis of the test results indicates varied surface roughness. The lower surface adjacent to the bone was characterized by greater roughness - Tables 1, 2. An example image of the external surface (3D) and profile of the implant is shown in Figures 4 and 5, respectively.

Alloy structure

Based on the observations made, the correct structure of the material from which the plate was made was observed. Observations showed the existence of a two-phase structure α + β - Figure 6. It was characterized by a homogeneous microstructure consisting of equiaxed grains of the α phase and the β phase located at the grain boundary of the α phase.

Hardness and hardness profile

The measurements performed did not show significant differences in hardness on the longitudinal and cross-sections of the tested samples. It was found that the average hardness value was in the range of 307 ÷ 328 HV0.5 - Figure 7.

Based on the analysis, different hardness values were shown depending on the distance from the implant surface. Higher hardness was observed in the surface layer to a depth of approximately 4000 nm, compared to the core of the material. This effect may be the result of the strengthening of the material caused by the use of vibration treatment preceding the anodic oxidation process - Figure 8.

Adhesion of the coating to the substrate

The analyzed coating was characterized by good adhesion to the substrate. Based on the measurements performed, it was found that the coating was chipped at a value of Lc₂ = 1.27 ± 0.05 N, and a complete break in its continuity was observed at a value of Lc₃ = 3.46 ± 0.1 N. Based on the macroscopic assessment of the scratch, discontinuous perforation of the coating was found. Examples of sample surface scratches and the curves recorded during the tests are shown in Figure 9.

Pitting corrosion resistance tests

Polarization curves of samples obtained from the implant are characterized by the absence of hysteresis loops. This indicates effective passivation of the implant surfaces in the entire measurement range E = + 4000 mV. In this range, no sudden increase in current density was observed, which would indicate the initiation of the corrosion process - Figure 10. This was confirmed by microscopic observations of the surfaces of samples after corrosion tests.

The average values ​​of the corrosion potential E_corr and the polarization resistance R_p determined using the Stern method were E_corr = -255.5 ± 10mV and R_p = 480.17 ± 55 kΩcm², respectively.

Surface wettability

The value of the contact angle Θ of the tested surfaces was in the range of 56.4±58.8°. The average value of the contact angle was Θ = 57.5±2.4°. The obtained results demonstrate the hydrophilic nature of the surface. Figure 11 shows an example droplet. Figure 12 shows the water contact angle as a function of time.

The chemical composition of the surface layer

Analysis of the chemical composition of the implants' surface layer showed that they consist mainly of Ti, O, Al and V. Moreover, in the lower surface layer of the implants (adjacent to the bone), the presence of Si and Na, which are remnants of vibration processing, was found. The surface image, overview spectrum and element distribution of the upper and lower surface layers are shown in Figures 13 and 14, respectively.

In order to check the chemical composition of the examined films the XPS analysis was performed. The XPS measurements were assisted with ion etching for detailed in-depth description of examined layers. In order to reduce possible preferential oxygen etching as well as other ion beam-induced distortions grazing incidence of ion beam was employed.

Figure 15 a) presents survey spectra of the surface scan as well as the consecutive scans upon ion depth profiling. Main and expected core lines are clearly visible, i.e. O 1s at ~530 eV, Ti 2p at ~460 eV, C 1s at ~284 eV. The trace N and Ca lines (at ~400 eV and 350 eV respectively) can be observed. However, together with carbon signal one can treat them as residual contamination since together with in-depth profile the mentioned signals are disappearing.

Figure 15 b) and c) presents the magnification of the regions of interest, i.e. o 1s and Ti 2p respectively. In O 1s spectrum, one can observe the significant decrease of the high binding energy branch and shift of the peak’s center towards lower binding energies along etching procedure. In case of Ti 2p core level the single distinct component at ~458.5 eV (with its spin orbit splitting counterpart at ~465 eV) can be clearly observed for surface scan. The energy position points clearly the Ti(IV) oxide with near-stoichiometric composition [19]. For in-depth signals, however, one can notice additional contributions appearing below ~459 eV indicating sub – stoichiometric composition or other Ti oxidation states.

For precise description of the oxide composition these two regions (i.e. O 1s and Ti 2p) underwent detailed decomposition. Figure 16 a) and b) presents decomposition results for the spectra acquired for the initial (non-etched) surface for o 1s and Ti 2p regions respectively. Next, panels c) and d) in Figure 16 shows the decomposition results of the bulk – related core levels after last ion etching procedure. One can see that the O 1s region (surface one) can be decomposed with four components representing (from lowest binding energy) O-Ti configuration, O=C, O-C/O-C-O and -OH configurations (14). The bulk signal (Figure 16 c)) is represented with the same components, however with significant decrease of the carbonaceous and -OH related species. This is consistent with decrease of the carbon signal along etching procedure (see Figure 15 a)). The main component (O-Ti), even if relatively narrow (i.e. ~1.35 eV full width at half maximum) does not however allow on titanium oxide composition description. Therefore Ti 2p analysis was also done. For the surface related Ti 2p region (Figure 16 b)) presents distinct Ti 2p 3/2 signal with its spin-orbit splitting ½ counterpart. The ½ to 3/2 energy distance as well as the 3/2 energy position points near-to stoichiometric Ti (IV) oxide with no suboxides (the FWHM is below 1.18 eV). After the etching process, for the bulk region (Figure 16 d)) two additional components appears at ~456.8 eV and at ~455.2 eV. The energy positions indicates that the most probable configurations are Ti (III) and Ti (II) oxides respectively [19]. However, the dominating one (>80 % of the overall signal) is still Ti (IV) oxide with main component at ~458.7 eV [2, 3] which is important, no Ti0 state was detected.