Relevant Aspects of Piranha Passivation in Ti6Al4V Alloy Dental Meshes

  Citation: Cruz, N.; Gil, J.; Punset, M.; Manero, J.M.; Tondela, J.P.; Verdeguer, P.; Aparicio, C.; Rúperez, E. Relevant Aspects of Piranha Passivation in Ti6Al4V Alloy Dental Meshes. Coatings 2022,12, 154. https://doi.org/ 10.3390/coatings12020154 Academic Editors: Jasmina Primožiˇc and Vincenzo D’Antò Received: 28 December 2021 Accepted: 24 January 2022 Published: 27 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). coatings Article Relevant Aspects of Piranha Passivation in Ti6Al4V Alloy Dental Meshes Nuno Cruz 1, Javier Gil 1,2,* , Miquel Punset 3,4,5,6 , JoséMaría Manero 3,4,6, João Paulo Tondela 7,* , Pablo Verdeguer 1, Conrado Aparicio 2and Elisa Rúperez 3,4,6 1Bioengineering Institute of Technology, International University of Catalonia (UIC), 08195 Barcelona, Spain; [email protected] (N.C.); pablover[email protected] (P.V.) 2School of Dentistry, International University of Catalonia (UIC), 08195 Barcelona, Spain; [email protected] 3Biomaterials, Biomechanics and Tissue Engineering Group (BBT), Department of Materials Science and Engineering, Technical University of Catalonia (UPC), 08019 Barcelona, Spain; [email protected] (M.P.); [email protected] (J.M.M.); elisa.r[email protected] (E.R.) 4Barcelona Research Centre in Multiscale Science and Engineering, Technical University of Catalonia (UPC), 08019 Barcelona, Spain 5 Innovation and Technology Center (CIT), Polytechnic University of Catalonia (UPC), 08034 Barcelona, Spain 6Sant Joan de Déu Research Institute (IRSJD), 08034 Barcelona, Spain 7CIROS from the Faculty of Medicine, University of Coimbra, FMUC, 3004-531 Coimbra, Portugal *Correspondence: xavier[email protected] (J.G.); [email protected] (J.P.T.) Abstract: Passivation of titanium alloy dental meshes cleans their surface and forms a thin layer of protective oxide (TiO 2 ) on the surface of the material to improve resistance to corrosion and prevent release of ions to the physiological environment. The most common chemical agent for the passivation process of titanium meshes is hydrochloric acid (HCl). In this work, we introduce the use of Piranha solution (H 2 SO 4 and H 2 O 2 ) as a passivating and bactericidal agent for metallic dental meshes. Meshes of grade 5 titanium alloy (Ti6Al4V) were tested after different treatments: as-received control (Ctr), passivated by HCl, and passivated by Piranha solution. Physical-chemical characterization of all treated surfaces was carried out by scanning electron microscopy (SEM), confocal microscopy and sessile drop goniometry to assess meshes’ topography, elemental composition, roughness, wettability and surface free energy, that is, relevant properties with potential effects for the biological response of the material. Moreover, open circuit potential and potentiodynamic tests were carried out to evaluate the corrosion behavior of the differently-treated meshes under physiological conditions. Ion release tests were conducted using Inductively Coupled Plasma mass spectrometry (ICP-MS). The antibacterial activity by prevention of bacterial adhesion tests on the meshes was performed for two different bacterial strains, Pseudomonas aeruginosa (Gram-) and Streptococcus sanguinis (Gram+). Additionally, a bacterial viability study was performed with the LIVE/DEAD test. We complemented the antibacterial study by counting cells attached to the surface of the meshes visualized by SEM. Our results showed that the passivation of titanium meshes with Piranha solution improved their hydrophilicity and conferred a notably higher bactericidal activity in comparison with the meshes passivated with HCl. This unique response can be attributed to differences in the obtained nanotextures of the TiO 2 layer. However, Piranha solution treatment decreased electrochemical stability and increased ion release as a result of the porous coating formed on the treated surfaces, which can compromise their corrosion resistance. Framed by the limitations of this work, we conclude that using Piranha solution is a viable alternative method for passivating titanium dental meshes with beneficial antibacterial properties that merits further validation for its translation as a treatment applied to clinically-used meshes. Keywords: titanium; dental meshes; passivation; piranha; corrosion resistance; ion release; bacterial adhesion Coatings 2022,12, 154. https://doi.org/10.3390/coatings12020154 https://www.mdpi.com/journal/coatings Coatings 2022,12, 154 2 of 17 1. Introduction The amount of bone is paramount to predictably achieve success and long-term survival of implant-supported rehabilitations. Actually, implant dentistry has evolved to a prosthetically driven implant placement concept, meaning that biology, biomechanics, function and esthetics of the implant supported rehabilitation should be considered for the adequate implant position in bone. Although proper amount of bone is needed to go along with the esthetical and functional prosthetic design, variable discrepancies in the available bone are seldom found. This may occur because of prolonged tooth loss, trauma, injury or bone disease and resection, conducting to a horizontal, vertical or combined bone defect (Siebert). Hence, several techniques and materials for bone augmentation have been used concomitant with implant placement or as a staged approach [1–4]. Following the biological principles of selective cell exclusion for regenerative wound healing and guided tissue regeneration, these were later proven to be applicable to guided bone regeneration also. Techniques involve placing a mechanical barrier to protect the blood clot and to isolate the bony defect from the surrounding connective and epithelial tissue invasion. This space is needed to allow the osteoblasts to access the space intended for bone regeneration [5,6]. Titanium rigid scaffolds were successfully used for bone augmentation, even outside of the bone envelope. Presently, one mainstream direction for 3D printing is biomedical applications, specifically in creating scaffolds for medical implants such as individualized titanium meshes for bone regeneration [ 7 – 9 ]. In recent years, the development of personalized rapid prototyping medical devices based on the digital imaging and communications in medicine (DICOM) files provided by computerized tomography/cone beam computerized tomography (CT/CBCT) scans has deeply intensified [ 10 ]. Based on the patient’s bone defect and resorting to computer aided design (CAD) software, it is possible to design medical devices with the intent of recreating the lost tridimensional bone anatomy. Regardless of the production technique for any implantable devices, it is mandatory to control the characteristics such as permeability, surface topography and roughness, and optimize their biological performance [ 11 – 16 ]. High degrees of roughness represent a major risk for ionic leakage from the material [ 17 ] and the bacterial adhesion can be increased, with the consequence of implant failures [ 10 ]. Smooth surfaces are able to slow down the biological processes at the interface, keeping the titanium oxidized layer properties unaffected for longer time periods [ 9 ]. The associated correct microand nano-roughness level can stimulate osteoblast differentiation, proliferation and production of both matrix and local growth factors [ 10 ]. Furthermore, changes in roughness correlate with selective protein adsorption, collagen synthesis and the maturation of chondrocytes, which all significantly influence the implant’s osseointegration [10]. It is well known that the implant–living tissues interactions depend on the surface properties, such as roughness, wettability, surface energy and chemical composition, among others. Biomaterials research should optimize, at different scales, the surface characteristics in order to improve different functions: bioactivity, osseointegration or bactericide behavior. In addition, titanium meshes are susceptible to corrosion due to the presence of metals of different chemical nature in the mouth, as well as the release of titanium ions into the environment which must be taken into account [ 11 – 13 ]. It has been long recognized that the corrosion products formed as a result of metal–environment interactions have a significant bearing on the biocompatibility and long-term stability of the prostheses/implant. The material used must not cause any biological adverse reaction and must retain its form and properties [ 11 , 12 ] during function. Human stomatognathus is subjected to varying changes in pH and temperature owing to differences in local, systemic, environmental, economic and social conditions for each individual. Corrosion can result from the presence of a number of corrosive species such as hydrogen ion (H + ), sulfide compounds (S 2− ), dissolved oxygen, free radicals (O 2− , O − ), and chloride ion (Cl − ) resulting in the metal surface breakdown and a consequent adverse tissue reaction [ 13 ]. In addition, the effect of Coatings 2022,12, 154 3 of 17 bacteria can lead to the appearance of bacterial plaque which will affect bone regeneration and cause inflammation in the patient [14–16]. Passivation is, in general, an oxidation reaction obtained by chemical or electrochemical process which promotes the formation and increasing of the thickness of protective layers [ 10 – 13 ]. The effect of passivation and oxidative agents and the role of titanium oxide as the physico-chemical characteristics of the surface are poorly studied and understood [17–20]. In vitro studies have implied that the negatively charged and hydrophilic TiO 2 layer is, in fact, the key factor for the overall biocompatibility as it regulates the protein adsorption [ 9 ]. For the particular case of the dentistry, countless studies have already been conducted in order to guarantee the implantation safety. Usually, no inflammatory response signs are found in the oral tissue adjacent to titanium implants; however, it is important to note that for some patients, hypersensitivity can be induced [9]. In this work, the aim was to study an alternative passivation method using the socalled Piranha solution. The Piranha solution is a mixture of sulfuric acid and hydrogen peroxide. We studied the effects of Piranha solution treatment on surface physical-chemical properties, chemical degradation (corrosion and release of ions) and antimicrobial activity against Gram-positive and Gram-negative bacteria. 2. Materials and Methods 2.1. Samples One hundred twenty Grade 5 titanium alloy (Ti6Al4V) meshes (BoneEasy, Arada, Portugal) were used. Figure 1shows the mesh and its application as a membrane with calcium phosphate. We worked with 3 groups of samples: Control: as-received material. HCl passivation: The meshes were immersed in a solution of hydrochloric acid (HCl) 20% (v) for 40 s at room temperature (HCl group). This is the gold-standard passivation treatment for dental implants and prosthesis. Piranha passivation: The meshes were immersed in a solution of Piranha, which is a mixture of sulfuric acid 96% (v) and a 50:50 ratio of hydrochloric acid (HCl) 20% (v) and hydrogen peroxide 30% (v) for 2 h. Piranha solutions are a mixture of concentrated sulfuric acid with hydrogen peroxide, usually in a ratio of 3:1 to 7:1. They are used to remove trace amounts of organic residues, such as photoresist, from substrates. The mixing procedure is an exothermic reaction that can reach temperatures of 100 ◦ C or higher. The reaction of hydrogen peroxide on concentrated sulfuric acid produces highly activated and oxidizing peroxymonosulfuric acid (H 2 SO 5 ), also called Caro’s acid [ 1 ]. However, there are many different mixture ratios that are commonly used, and all are called Piranha. The addition of NH 4 OH in order to accelerate the decomposition of H 2 O 2 or the addition of HCl, as in this research, favors cleanness and increases the oxide stabilization. Piranha solution must be prepared with great care. It is highly corrosive and an extremely powerful oxidizer. Surfaces must be reasonably clean and completely free of organic solvents from previous washing steps before coming into contact with the solution. Piranha solution cleans by decomposing organic contaminants, and a large amount of contaminant will cause violent bubbling and a release of gas that can cause an explosion [21]. After treatment, all samples were cleaned a sequence of 3 ultrasonic baths (3 min each): two consecutive with distilled water, followed by one with ethanol. Coatings 2022,12, 154 4 of 17 Coatings 2022, 12, x FOR PEER REVIEW 4 of 18 Figure 1. Grade 5 titanium mesh used in this study. 2.2. Surface Characterization Roughness for all groups was determined using an Olympus LEXT OLS3100 confocal microscope (Olympus, Tokyo, Japan). Three samples per group were tested and 3 measurements per sample were taken at ×1000 magnification. The parameters Ra and Rz were determined. Ra corresponds to the arithmetic mean of the absolute values of the deviations of the profiles of a given length of the sample. Rz corresponds to the sum of the maximum peak height and the maximum valley depth within the sampling length. [21]. The water sessile drop technique was used for the measurement of the contact angle, θ, formed between the water drop and the surface. The greater the contact angle, the lower the wettability and vice versa. For angles less than 10°, the surface is considered superhydrophilic, for angles between 10° and 90° surfaces are hydrophilic and for angles greater than 90°, surfaces are considered hydrophobic. A droplet generation system equipped with a 500 μL Hamilton syringe with micrometric displacement control was used to control the volume (3 μL) and to deposit the droplet. The analysis was performed using a gonyometer with drop profile image capture (Contact Angle System OCA15plus, DataPhysics, Filderstadt, Germany) and analyzed with SCA20 software (DataPhysics, Filderstadt, Germany) [22,23]. To calculate the surface free energy, the contact angle was measured with two different liquids, water and diiodomethane. The contact angle measurements of diiodomethane were obtained following the same procedure used to measure water contact angles [22]. The surface free energy and its polar (γp) and dispersive (γd) components were then calculated using the Owens and Wendt equation [17]: ))()((2)cos1( 2/1 p S p L 2/1d S d LL  (1) Surface morphology of the samples was analyzed with a focused ion beam Zeiss Neon40 FE-SEM (Carl Zeiss NTS GmbH, Oberkochen, Germany). Images of uncoated samples were taken at a working distance of 7 mm and an accelerating voltage of 5 kV. An EDS detector (INCA PentaFETx3 system, Oxford Instruments, Abingdon, UK) was used to detect silver presence on the surface of the samples. This microscope has a resolving power of 3 nm and allows the observation of the nanotextures produced by the reaction of the Piranha solution with the Ti6Al4V alloy. Figure 1. Grade 5 titanium mesh used in this study. 2.2. Surface Characterization Roughness for all groups was determined using an Olympus LEXT OLS3100 confocal microscope (Olympus, Tokyo, Japan). Three samples per group were tested and 3 measurements per sample were taken at × 1000 magnification. The parameters Ra and Rz were determined. Ra corresponds to the arithmetic mean of the absolute values of the deviations of the profiles of a given length of the sample. Rz corresponds to the sum of the maximum peak height and the maximum valley depth within the sampling length [21]. The water sessile drop technique was used for the measurement of the contact angle, θ , formed between the water drop and the surface. The greater the contact angle, the lower the wettability and vice versa. For angles less than 10 ◦ , the surface is considered superhydrophilic, for angles between 10 ◦ and 90 ◦ surfaces are hydrophilic and for angles greater than 90 ◦ , surfaces are considered hydrophobic. A droplet generation system equipped with a 500 µ L Hamilton syringe with micrometric displacement control was used to control the volume (3 µ L) and to deposit the droplet. The analysis was performed using a gonyometer with drop profile image capture (Contact Angle System OCA15plus, DataPhysics, Filderstadt, Germany) and analyzed with SCA20 software (DataPhysics, Filderstadt, Germany) [22,23]. To calculate the surface free energy, the contact angle was measured with two different liquids, water and diiodomethane. The contact angle measurements of diiodomethane were obtained following the same procedure used to measure water contact angles [ 22 ]. The surface free energy and its polar ( γp ) and dispersive ( γd ) components were then calculated using the Owens and Wendt equation [17]: γL·(1+cos θ) = 2·((γd L·γd S)1/2 + (γp L·γp S)1/2)(1) Surface morphology of the samples was analyzed with a focused ion beam Zeiss Neon40 FE-SEM (Carl Zeiss NTS GmbH, Oberkochen, Germany). Images of uncoated samples were taken at a working distance of 7 mm and an accelerating voltage of 5 kV. An EDS detector (INCA PentaFETx3 system, Oxford Instruments, Abingdon, UK) was used to detect silver presence on the surface of the samples. This microscope has a resolving power of 3 nm and allows the observation of the nanotextures produced by the reaction of the Piranha solution with the Ti6Al4V alloy. 2.3. Corrosion Behavior A total of 60 samples, (n= 20) for each group of samples, were used for the corrosion tests. The test area for each sample was 19.6 mm 2 . The electrolyte for all tests was Hank’s solution (Table 1), which is a saline fluid that closely captures the ion composition of the human serum environment. Coatings 2022,12, 154 5 of 17 Table 1. Composition of Hank’s solution. Chemical Product Composition (mM) K2HPO40.44 KCl 5.4 CaCl21.3 Na2HPO40.25 NaCl 137 NaHCO34.2 MgSO41.0 C6H12O65.5 The electrochemical cell used was a polypropylene (PP) container with a capacity of 185 mL and a methacrylate lid with 6 holes for the introduction of the sample, the reference electrode and the counter electrode (Figure 2). For both the open circuit potential measurement tests and the potentiodynamic tests, the reference electrode used was a calomel electrode (saturated KCl), with a potential of 0.241 V compared to the standard hydrogen electrode. All tests were performed at room temperature and in a Faraday cage to avoid the interaction of external electric fields. For the open circuit potential measurement tests, only the sample and the reference electrode were placed in the electrochemical cell. Tests were carried out for 5 h for all the samples, taking measurements every 10 s. The potential was considered to be stabilized when the variation of the potential is less than 2 mV over a period of 30 min as indicated in the ASTM G31 standard [23]. This test assesses which materials are more noble (higher potential) and thus, less susceptible to corrode. The data and the E-t curves were obtained using the PowerSuite software (Schneieder Electric, Ruil-Malmaison, France) with the PowerCorr-Open circuit (Schneieder Electric, Ruil-Malmaison, France). Coatings 2022, 12, x FOR PEER REVIEW 5 of 18 2.3. Corrosion Behavior A total of 60 samples, (n = 20) for each group of samples, were used for the corrosion tests. The test area for each sample was 19.6 mm2. The electrolyte for all tests was Hank’s solution (Table 1), which is a saline fluid that closely captures the ion composition of the human serum environment. Table 1. Composition of Hank’s solution. Chemical Product Composition (mM) K2HPO4 0.44 KCl 5.4 CaCl2 1.3 Na2HPO4 0.25 NaCl 137 NaHCO3 4.2 MgSO4 1.0 C6H12O6 5.5 The electrochemical cell used was a polypropylene (PP) container with a capacity of 185 mL and a methacrylate lid with 6 holes for the introduction of the sample, the reference electrode and the counter electrode (Figure 2). For both the open circuit potential measurement tests and the potentiodynamic tests, the reference electrode used was a calomel electrode (saturated KCl), with a potential of 0.241 V compared to the standard hydrogen electrode. All tests were performed at room temperature and in a Faraday cage to avoid the interaction of external electric fields. For the open circuit potential measurement tests, only the sample and the reference electrode were placed in the electrochemical cell. Tests were carried out for 5 h for all the samples, taking measurements every 10 s. The potential was considered to be stabilized when the variation of the potential is less than 2 mV over a period of 30 min as indicated in the ASTM G31 standard [23]. This test assesses which materials are more noble (higher potential) and thus, less susceptible to corrode. The data and the E-t curves were obtained using the PowerSuite software (Schneieder Electric, Ruil-Malmaison, France) with the PowerCorr-Open circuit (Schneieder Electric, Ruil-Malmaison, France). Figure 2. Experimental set up used for assessing corrosion resistance. Cyclic potentiodynamic polarization curves were obtained for the 3 study groups following the ASTM G5 standard. In this test, a variable electrical potential is imposed by the potentiostat between the sample and the reference electrode, causing a current to flow between the sample and the counter electrode. The counter electrode used was platinum [17,24–25]. Before starting the test, the system was allowed to stabilize by means of an Figure 2. Experimental set up used for assessing corrosion resistance. Cyclic potentiodynamic polarization curves were obtained for the 3 study groups following the ASTM G5 standard. In this test, a variable electrical potential is imposed by the potentiostat between the sample and the reference electrode, causing a current to flow between the sample and the counter electrode. The counter electrode used was platinum [17,24,25]. Before starting the test, the system was allowed to stabilize by means of an open circuit test for 1 h. After stabilization, the potentiodynamic test was launched, performing a cyclic sweep from − 0.8 mV to 1.7 mV at a speed of 2 mV/s. These parameters were entered into the PowerSuite program using the PowerCorr-Cyclic Polarization function to obtain the curves. The parameters studied were: 1. icorr (µA/cm2)—corrosion current density; 2. E corr (mV)—corrosion potential: value at which the current density changes from cathodic to anodic; 3. Erep (mV)—repassivation potential: potential at which the passive layer regenerates; 4. Ep(mV)—pitting potential: value at which pitting corrosion may occur; Coatings 2022,12, 154 6 of 17 5. ip(µA/cm2)—passivation current density; 6. ip (µA/cm2)—repassivation current density. The E corr and i corr parameters are obtained by extrapolating the Taffel slopes. The Taffel slopes are also used to obtain the Taffel coefficients: anodic ( β a) and cathodic ( β c). These coefficients represent the slopes of the anodic and cathodic branch, respectively. In accordance with the ASTM G102-89 standard [ 23 – 26 ], these values are then used to calculate the polarization resistance (R p ) using the Stern–Geary expression and the corrosion rate (CR in mm/year) [24–28]. Rp =βa·βc 2.303 ·(βa+βc)·icorr (2) The polarization resistance indicates the resistance of the sample to corrosion when subjected to small variations in potential. A total of 30 potentiodynamic tests were carried out, obtaining at least 10 curves per group. CR =K1·icorr ρ·EW (3) 2.4. Ion Release Five samples from each group were used for the metal ion recovery test. After weighing the samples (m = 0.206 g) and following the ISO 10993-12 standard [ 26 ], a weight adjustment was made at the rate of 1 mL of Hank’s solution for each 0.2 g of sample, as indicated in the standard. The 5 samples of each group were placed in the same Eppendorf with 5 mL of Hank’s solution and stored at 37 ◦ C. Hank’s solution should be extracted and stored in the refrigerator after 1, 3, 7, 14 and 21 days. After each extraction, 5 mL of fresh Hank’s solution has been replenished into the Eppendorf containing the samples. All Eppendorf tubes should be cleaned with 2% Nitric Acid and dried before use. After 21 days, the concentration of released titanium ions was measured, at the test times indicated above, by inductively coupled plasma mass spectrometry (ICP-MS) with the Agilent Technologies 7800 ICP-MS. 2.5. Bacteria Analysis Two types of bacteria, P. aeruginosa (Colección española de cultivos tipo, CECT 110, Valencia, Spain) and S. sanguinis (Culture Collection University of Gothenburg, CCUG 15915, Gothenburg, Sweden), a Gram-negative and a Gram-positive strain, respectively, were used for the bacterial adhesion test. Three samples per group and bacterial strain were tested. The culture media and material (PBS) were previously sterilized by autoclaving at 121 ◦C for 30 min. Prior to the adhesion test, the samples were also sterilized. For this purpose, three 5 min ethanol washes were carried out in sterile culture plates. After removing the ethanol, the samples were exposed to ultraviolet light for another 30 min [29,30]. The agar plates were cultured at 37 ◦ C for 24 h. From this culture, the liquid inoculum was prepared by suspending the bacteria in 5 mL of BHI (Brain Heart Infusion) and incubated for 24 h at 37 ◦ C. The medium was then diluted to an optical density of 0.1 at a wavelength of 600 nm (OD600 = 0.1). For bacterial adhesion, enough solution with a concentration equivalent to OD600 = 0.1 to cover the surfaces (500 µ L/sample) was introduced into the well of the culture plate of each sample and incubated at 37 ◦ C for 1 h. After this time, the samples were rinsed with PBS for 5 min twice and the bacteria were fixed with a 2.5% glutaraldehyde solution in PBS (30 min in the refrigerator). The glutaraldehyde solution was then removed and the samples were rinsed with PBS 3 times for 5 min. For viability analysis by confocal microscopy, the LIVE/DEAD BacLight bacterial viability kit (Thermo Fisher, Madrid, Spain) was used [ 13 , 14 ]. A solution was prepared with 1.5 µ L of propidium in 1 mL of PBS. Using a micropipette, a drop of this solution (approximately 50 µ L/sample) was deposited on the study surface and after incubation at room temperature in the dark for 15 min, the samples were rinsed 3 times with PBS for Coatings 2022,12, 154 7 of 17 5 min. The surfaces were then observed under a confocal microscope. Three images per sample were taken at 630 × magnification ( × 63 objective). Wavelengths of 488 and 561 nm were used to detect bacteria with non-compromised membranes (LIVE) and compromised membranes (DEAD), respectively. Prior to the observation of the samples by scanning electron microscopy (SEM), the samples were dehydrated. For the dehydration process and the critical point drying, 10 min washes were carried out with ethanol solutions of gradual concentrations of 30%, 50%, 70%, 80%, 90%, 95% and 100%. They were then left to dry for 24 h at room temperature. Then, samples were coated with platinum for 5 s before observation under the microscope. Ten images of each sample were taken at 20,000 × magnifications for bacterial quantification on each surface. 2.6. Statistical Analysis All results were expressed as mean and standard deviation except for the bacterial adhesion test results which were expressed as median and standard error. The comparative T.TEST (with the Excel software) was carried out between the different groups at 95%, which means that for values of p< 0.05, there are significant differences. 3. Results Figure 3shows SEM images of the surfaces of the titanium alloy after passivation treatments. No significant variations between the control and HCl treatment were detected and both types of surfaces clearly showed machining marks. Machining marks in HClpassivated surfaces were lighter than in as-machined surfaces, probably due to the effect of the higher concentration of the acid. However, on the surface of the samples subjected to the Piranha passivation treatment, the acid attack almost completely removed the machining marks and, notably, produced a homogenously-distributed and commonly-obtained surface nanotexture in the form of nanocavities (Figure 4) [15]. Coatings 2022, 12, x FOR PEER REVIEW 8 of 18 Figure 3. (A) Surfaces of grade 5 Ti alloy treated with different passivation methods; (B) at higher magnifications. Figure 4. Nanotexture of titanium alloy after Piranha passivation treatment observed by high-resolution scanning electron microscopy. The different passivation treatments on the titanium alloy meshes, either with HCl or Piranha solution, did not alter the average roughness (Ra), as no statistically significant differences were observed with respect to the control group (Table 2). However, the Piranha treatment showed statistically significant lower Rz values with respect to the other groups. These results suggest that the Piranha solution treatment attacked the titanium, reducing machining failures and creating an oxide layer that reduces the differences between valleys and peaks. The large difference between the Ra and Rz values shows that we have two types of texture (Figure 5), one associated with the turning marks responsible for the high Rz values and the other the nanotexture associated with the passivation treatment. Figure 3. ( A ) Surfaces of grade 5 Ti alloy treated with different passivation methods; ( B ) at higher magnifications. Coatings 2022,12, 154 8 of 17 Coatings 2022, 12, x FOR PEER REVIEW 8 of 18 Figure 3. (A) Surfaces of grade 5 Ti alloy treated with different passivation methods; (B) at higher magnifications. Figure 4. Nanotexture of titanium alloy after Piranha passivation treatment observed by high-resolution scanning electron microscopy. The different passivation treatments on the titanium alloy meshes, either with HCl or Piranha solution, did not alter the average roughness (Ra), as no statistically significant differences were observed with respect to the control group (Table 2). However, the Piranha treatment showed statistically significant lower Rz values with respect to the other groups. These results suggest that the Piranha solution treatment attacked the titanium, reducing machining failures and creating an oxide layer that reduces the differences between valleys and peaks. The large difference between the Ra and Rz values shows that we have two types of texture (Figure 5), one associated with the turning marks responsible for the high Rz values and the other the nanotexture associated with the passivation treatment. Figure 4. Nanotexture of titanium alloy after Piranha passivation treatment observed by highresolution scanning electron microscopy. The different passivation treatments on the titanium alloy meshes, either with HCl or Piranha solution, did not alter the average roughness (Ra), as no statistically significant differences were observed with respect to the control group (Table 2). However, the Piranha treatment showed statistically significant lower R z values with respect to the other groups. These results suggest that the Piranha solution treatment attacked the titanium, reducing machining failures and creating an oxide layer that reduces the differences between valleys and peaks. The large difference between the R a and R z values shows that we have two types of texture (Figure 5), one associated with the turning marks responsible for the high Rzvalues and the other the nanotexture associated with the passivation treatment. Table 2. Roughness values, R a and R z, for titanium alloy surfaces with different passivation treatments. Different letters in the same column denote statistically significant differences (p< 0.05) between groups. Mesh Ra (µm) Rz (µm) Control 0.12 ±0.03 (a) 4.95 ±0.76 (A) HCl 0.14 ±0.08 (a) 4.87 ±0.90 (A) Piranha 0.12 ±0.05 (a) 1.90 ±0.73 (B) Coatings 2022, 12, x FOR PEER REVIEW 9 of 18 Table 2. Roughness values, Ra and Rz, for titanium alloy surfaces with different passivation treatments. Different letters in the same column denote statistically significant differences (p < 0.05) between groups. Mesh Ra (μm) Rz (μm) Control 0.12 ± 0.03 (a) 4.95 ± 0.76 (A) HCl 0.14 ± 0.08 (a) 4.87 ± 0.90 (A) Piranha 0.12 ± 0.05 (a) 1.90 ± 0.73 (B) Figure 5. Roughness parameters quantified with different passivation conditions: (a) Ra and (b) Rz. Wettability, i.e., hydrophilic/hydrophobic character of the tested surfaces, was determined measuring the water contact angle with the sessile drop technique (Table 3). Firstly, as-received control surfaces were hydrophobic with a contact angle higher than 90°. Secondly, all passivated surfaces had significantly higher hydrophilicity than untreated control surfaces. Thirdly, the surfaces passivated with Piranha solution produced a significantly higher hydrophilic material than the surfaces treated with HCl. Water contact angle, as well as polar and dispersive components of SFE, are plotted in Figure 6. Corresponding with the results for the wettability of the different surfaces, the polar component of the surface free energy in the titanium alloy passivated with Piranha solution was the highest among all tested surfaces. The differences in the dispersive and polar components of the surface free energy for all tested surfaces were statistically significant [31–35]. It is widely accepted that increasing the polar component of a material’s surface energy promotes initial adhesion and cell proliferation [17]. Table 3. Contact angles and components of the surface free energy for the differently passivated meshes. Mesh Θ Water (°) Θ Diidomethane (°) γd (mJ/m2) γp (mJ/m2) SFE (mJ/m2) Control 102.76 ± 7.00 48.40 ± 2.32 35.15 ± 1.28 0.12 ± 0.10 35.28 ± 1.35 HCl 86.37 ± 4.12 53.54 ± 0.92 32.39 ± 0.52 3.31 ± 1.28 35.70 ± 1.60 Piranha 49.05 ± 7.67 34.12 ± 3.94 42.37 ± 1.79 16.52 ± 4.22 58.90 ± 4.11 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 Control HCl Piranha Ra (μm) a) 0 1 2 3 4 5 6 7 Control HCl Piranha Rz (μm) b) Figure 5. Roughness parameters quantified with different passivation conditions: ( a ) Ra and ( b ) Rz. Coatings 2022,12, 154 9 of 17 Wettability, i.e., hydrophilic/hydrophobic character of the tested surfaces, was determined measuring the water contact angle with the sessile drop technique (Table 3). Firstly, as-received control surfaces were hydrophobic with a contact angle higher than 90 ◦ . Secondly, all passivated surfaces had significantly higher hydrophilicity than untreated control surfaces. Thirdly, the surfaces passivated with Piranha solution produced a significantly higher hydrophilic material than the surfaces treated with HCl. Water contact angle, as well as polar and dispersive components of SFE, are plotted in Figure 6. Corresponding with the results for the wettability of the different surfaces, the polar component of the surface free energy in the titanium alloy passivated with Piranha solution was the highest among all tested surfaces. The differences in the dispersive and polar components of the surface free energy for all tested surfaces were statistically significant [31–35]. It is widely accepted that increasing the polar component of a material’s surface energy promotes initial adhesion and cell proliferation [17]. Table 3. Contact angles and components of the surface free energy for the differently passivated meshes. Mesh ΘWater (◦) ΘDiidomethane (◦) γd (mJ/m2) γp (mJ/m2) SFE (mJ/m2) Control 102.76 ±7.00 48.40 ±2.32 35.15 ±1.28 0.12 ±0.10 35.28 ±1.35 HCl 86.37 ±4.12 53.54 ±0.92 32.39 ±0.52 3.31 ±1.28 35.70 ±1.60 Piranha 49.05 ±7.67 34.12 ±3.94 42.37 ±1.79 16.52 ±4.22 58.90 ±4.11 Coatings 2022, 12, x FOR PEER REVIEW 10 of 18 Figure 6. θ values (a) and SFE values (b) of cpTi treated with different passivation conditions. Table 4 shows that the highest open circuit corrosion potential values (EOCP) were obtained for titanium alloy surfaces treated with HCl. Therefore, HCl passivation produces the surfaces with the least tendency for corrosion, and therefore the best corrosion behavior. Conversely, surfaces treated with the Pirahna solution showed the lowest values in open circuit, which indicated the highest tendency for corrosion. The potentiodynamic studies confirmed that the treatment that produced surfaces with the best corrosion resistance was using HCl, as these passivated surfaces showed the lowest values of corrosion current density (icorr) and corrosion rate (Vc). In addition, the HCl-treated samples show the highest resistance to polarization (Rp). The Piranha solution should produce the thickest protective TiO2 layer; however, surfaces passivated with Piranha did not have an improved corrosion behavior with respect to the control samples. Moreover, only in samples treated with Piranha solution pitting corrosion could be observed after the potentiodynamic tests (Figure 7). Table 4. Electrochemical and corrosion parameters assessed for Ti alloy meshes with different passivation treatments. Mesh EOCP (mV) icorr (μA/cm2) Rp (MΩ/cm2) Ecorr (V) Vc (μm/year) Control −196 ± 01 0.027 ± 0.008 2.428 ± 0.390 −361 ± 14 0.233 ± 0.066 HCl −145 ± 11 0.018 ± 0.005 2.479 ± 0.083 −536 ± 39 0.176 ± 0.048 Piranha −206 ± 27 0.056 ± 0.006 1.102 ± 0.149 −447 ± 26 0.488 ±0.047 0 20 40 60 80 100 120 Control HCl Piranha θ, (o) a) 0 10 20 30 40 50 60 70 Control HCl Piranha SFE Total (𝒎𝑱/𝒎𝟐) ϒsd ϒsp b) Figure 6. θvalues (a) and SFE values (b) of cpTi treated with different passivation conditions. Table 4shows that the highest open circuit corrosion potential values (E OCP ) were obtained for titanium alloy surfaces treated with HCl. Therefore, HCl passivation produces the surfaces with the least tendency for corrosion, and therefore the best corrosion behavior. Conversely, surfaces treated with the Pirahna solution showed the lowest values in open circuit, which indicated the highest tendency for corrosion. 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