Bioactivity of Ti6Al4V alloy with bioglass and corrosion protection by silane coating

The Ti6Al4V alloy is usually employed as a biomaterial, however, when in use, exhibits a few drawbacks such as corrosion, caused by the release of aluminum and vanadium ions besides the bioinert behavior. Bioactive coatings offer a barrier effect and bioactivity, promoting biocompatibility and osseointegration processes. The present work aims to study the biocompatibility behavior of a bioglass-containing silane film deposited on a titanium alloy (Ti6Al4V) substrate. The effect of the surface roughness of the metallic substrate was also evaluated. Film/substrate systems were characterized as their morphological, chemical, physical, electrochemical behavior, and cell cytotoxicity and cell viability. The main results pointed out that silane films augment corrosion resistance of titanium alloy substrates. The biological results indicated a growth of osteoblast cells (MG-63), for all the test conditions. The bioglass film deposited on the ground substrate exhibits the highest cell density.


Introduction
The Ti6Al4V alloy is widely employed as a biomaterial in the orthopedic field. It exhibits higher values of fracture and tensile strength and lower Young's modulus compared to other metallic materials (Chen & Thouas, 2015;Chiu et al., 2007;Fu et al., 2012). Ti6Al4V alloy has some disadvantages, including the release of aluminum (Al) and vanadium (V) ions as a consequence of corrosion and wear as well as the lack of bioactivity (Chen & Thouas, 2015;Cremasco et al., 2011;Harada et al., 2016;Punt et al., 2008;Veronesi et al., 2017).
The relative movement between bone and implant surfaces causes severe wear of the metallic material and promotes the release of metallic ions within the human body. Concomitantly, the harsh conditions of the physiological environment can result in metal corrosion. In this case, the corrosion mechanism is characterized by the occurrence of anodic and cathodic processes at metal/solution and oxide/solution interfaces (Mathew et al., 2012;Silva-Bermudez & Rodil, 2013). Corrosion and wear can cause inflammatory and allergenic reactions in the host body (Ahmed et al., 2016;Ibrahim et al., 2017;Rodríguez-Cano et al., 2013).
The Ti6Al4V alloy exhibits a bioinert behavior when inserted into the human body and hinder the interaction of the implant with bone tissue, making it difficult to form fibrous tissue around the implant. The formation of fibrous tissue around the implant is a fundamental condition for the implant not to come loose. Thus, the material to be employed for bone tissue reconstruction should provide tissue cicatrization, complying with biological requirements such as biocompatibility, biofunctionality, and osteocondutibility (Andrade et al., 2007;Asri et al., 2017;Chen & Thouas, 2015;Pires et al., 2015;Romagnoli et al., 2013;Su et al., 2018).
The use of protective barrier films has been proposed to increase the corrosion resistance of metallic surfaces. Such films can be deposited by different coating techniques such as physical vapor deposition, electrochemical deposition, plasma spraying, and sol-gel immersion (Aydınoğlu & Yoruç, 2017;Owens et al., 2016). Sol-gel immersion of alkoxide precursors by dip-coating is considered one of the most promising low-cost methods for the deposition of functional coatings. Among silicon, aluminum, zirconium, and titanium alkoxides, silicon alkoxide has called the attention because they are relatively easy to produce (Owens et al., 2016). Bioactive particles may be incorporated through this process (Aydınoğlu & Yoruç, 2017;El-Ghannam & Ducheyne, 2017;Pires et al., 2015). Examples of bioactive materials are hydroxyapatite, bioglasses, and vitroceramics (El-Ghannam & Ducheyne, 2017;Hallab et al., 2004;Pires et al., 2015;Rasouli et al., 2018;Sepulveda et al., 2002).
The topography and chemical composition of implants influence the morphology, the viability, and the proliferation of the cells (Baxter et al., 2002;Rosa et al., 2013). It is believed that cell behavior can be modulated by adjusting the topographic characteristics in micro-and nanometric scales. The diameter of individual cells is typically dozens of micrometers, while the dimensions of the subcellular structure tend to the nanoscale. In this way, the nanometric scale roughness can directly influence cellular interaction (Biggs et al., 2010;Huang et al., 2016;Sjöström et al., 2013).
In this context, the goal of this study was to investigate the corrosion and biocompatibility of a silane film deposited on a Ti6Al4V alloy substrate by dip-coating. Changes in biocompatibility were also evaluated when the silane film was modified by the incorporation of bioglass particles and when the roughness of the metallic substrate was changed by grinding processes. The film biocompatibility was assessed for osteoblastic cells, considering the adhesion ability, viability/proliferation, and cytotoxicity. The present work adopted the case study as a scientific methodology, described by Pereira et al. (2018) and supported by Bacon's fundaments (Köche, 2011). These authors indicate the following steps searching for scientific knowledge: (a) experimentation, (b) formulation of hypotheses based on the analysis of the results obtained from the various experiments, (c) repetition of the experimentation by other scientists, and (d) repetition of the experiment to test the hypotheses, seeking to obtain new data and new evidence to confirm them.
Ti6Al4V samples were manually ground with silicon carbide paper starting with 300-grit and following with 600-and 1200-grit. After each grinding step, the metallic substrates were washed with an alkaline detergent. Subsequently, the samples were immersed in an ultrasonic bath of acetone and alcohol (Vetec) for 10 min in each solvent (Wang et al., 2013). Following the cleaning stage, the substrates were submitted to alkaline treatment in a sodium hydroxide solution (NaOH) (Neon) 0.01 mol.L -1 (pH = 11.5) for 3 min at 24 °C and dried in cold air. This process activates the substrate surface for better film adhesion (Salvador et al., 2018).
Then, the hydrolysis was carried out at 24 °C for 24 h without agitation (Kunst et al., 2013). The sol-gel application on the metallic surfaces was conducted by the dip-coating method, keeping the samples in the solution for 5 min. Afterward, the samples were removed from the solution and exposed to air for 1 h at 24 °C (Kunst et al., 2015). The film/substrate systems were cured in an oven at 90 °C for 1 h (Certhoux et al., 2013;Sakai et al., 2012;Salvador et al., 2017). F18 Bioglass particles were also incorporated into the silane film between the drying at 24 °C and cure at 90 °C steps. For this purpose, the dissociation of 0.001 g.mL -1 F18 Bioglass was first conducted in alcohol under agitation, according to the ISO 10933-14 standard. The suspension was subsequently dropped on one of the faces of the coated surface using a Pauster pipette (Certhoux et al., 2013;Sakai et al., 2012). In this way, silane films, without (MAP/TEOS) and with the addition of F18 Bioglass particles (MAP/TEOS/F18) were prepared and deposited on Ti6Al4V alloy substrates. Samples sterilization was conducted by Esterilizare Esterilização de Produtos Ltda (Caxias do Sul, RS, Brazil).
The chemical bonding characterization of cured MAP/TEOS and MAP/TEOS/F18 films was conducted by Fourier Transformed Infrared Spectroscopy (Nicolet iS10 -Thermo Scientific -England), employing Attenuated Total Reflectance mode (FTIR-ATR). The infrared spectra were recorded from 4000 to 700 cm -1 at a resolution of 4 cm -1 .
The morphology of the uncoated and coated surfaces was evaluated using a Field Emission Gun Scanning Electronic Microscopy (FEGSEM) equipment (Tescan, model Mira3, Czech Republic) and an Energy Dispersion X-Ray Spectroscopy (EDS) detector (Oxford, model X-Max50, Czech Republic) coupled to the FEGSEM equipment. The adhesion of the films to the substrates and the adhesion rate (percent of film area removed) were evaluated by the tape test following the ASTM D3359-09 standard (Salvador et al., 2018), with the aid of the FEGSEM technique.
The surface topography of both the substrates and the film/substrate systems was measured by Atomic Force Microscopy (AFM) (SHIMADZU, model SPM-9500J3, Japan) using non-contact mode and scan size of (10 × 10) μm. The quantification of the roughness irregularities was conducted using the amplitude parameters Ra (arithmetic average surface roughness), Rz (ten-point height), and Rt (maximum height of the profile) (Gadelmawla et al., 2002).
The surface wettability was assessed by contact angle measurements by the sessile drop method using a Yale 3D B-D syringe and SBF solution. Data treatment was conducted with the aid of the Surftens 4.5 Software. Surfaces with a contact angle lower than 80 o were considered hydrophilic and those with contact angle higher than 80 o , hydrophobic.
The formation of protective films on the uncoated and coated surfaces was studied by Open Circuit Potential (OCP) measurements carried out in a potentiostat/galvanostat (IviumStat of Ivium Technologies, software IviumSoft, Netherlands). A conventional three-electrode cell was used to monitor the evolution of the OCP values as a function of time, which consisted of the sample (working electrode), the saturated calomel reference electrode (SCE), and the platinum counter electrode. The measurements were carried out in the absence of agitation at 24 °C with SBF as the electrolyte (Salvador et al., 2018). The evolution of OCP values was monitored during the first hour of immersion. OCP curves were obtained at 1 mV.s -1 scanning rate by sweeping the potential from 200 mV below to 600 mV above with respect to the open circuit potential.
The biological properties of the samples were evaluated considering cell adhesion ability, viability/proliferation, and cytotoxicity. MG-63 osteoblastic cells were used for these analyses and were acquired from the Bank of Cells of Rio de Janeiro from ATCC. The samples were first exposed to a cell Dulbecco's Modified Eagle's medium (DMEM) according to ISO 10993-12 standard procedures (Savaris et al., 2016). The extraction solution was prepared using the samples (6.0 cm 2 .mL -1 ) in DMEM culture medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin (P/S). The culture medium was placed in contact with the plates for 24 h at 37 °C in a 5 % CO2 humidified atmosphere to obtain the extraction.
MG-63 cells were seeded in 6-well plates at a density of 1.5×10 5 cells with 2000 μL of DMEM culture medium supplemented with 10 % FBS and 1 % P/S on the samples for 1 and 7 days, respectively. Cells were then fixed with a 3 % glutaraldehyde solution in PBS (v/v) for 15 min, followed by a dehydration process with 30, 50, 70, 90, and 100 % (v/v) ethanol for 10 min. Afterward, the samples were kept in a desiccator until the biological analysis was performed. The samples were coated with gold in sputtering equipment and then examined by FEGSEM in high vacuum mode at a maximum beam voltage of 15 kV.
Research, Society and Development, v. 10, n. 6, e23310615308, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15308 5 For the cell viability MTT test, MG-63 cells were seeded at a density of 1.5×10 4 cells.mL -1 in 100 μL of DMEM culture medium supplemented with 10 % FBS and 1 % P/S. After 24 h, the cells were treated with the extract solution of the samples for 1 and 7 days, respectively. DMEM was used as the negative control, and 5 % dimethyl sulfoxide (DMSO) was used as the positive control. All samples were incubated at 37 °C in a humidified atmosphere with 5 % CO2. The culture medium was removed, and 1.0 mg.mL -1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in FSB-free medium and P/S was added to the wells. The samples were incubated at 37 °C for 2 h in a humidified atmosphere with 5 % CO2. MTT solution was withdrawn, and the formazan crystals were dissolved in 100 μL of DMSO. Spectrophotometric measurements at 570 nm were conducted on a microplate reader (Max190 spectra, Molecular Devices, USA), and the results were expressed as percent viability. The absorbance of the negative control represented 100 % viability, and values for the treated cells were calculated as a percentage of the control.
A direct method to investigate apoptosis events was conducted by dual staining technique using acridine orange and ethidium bromide in 6 well plates. The samples (20 cm 2 ) with and without coating were arranged in the different wells (Liang et al., 2014). MG-63 cells at a density of 1.5×10 5 cells/well were then seeded in 2000 µL of DMEM culture medium supplemented with 10 % fetal bovine serum and 1 % P/S, followed by incubation at 37 °C in 5 % CO2, for 1 and 7 days. The culture medium was then washed with PBS with acridine orange (100 μg.mL -1 ) and ethidium bromide (100 μg.mL -1 ) dye staining technique. Cells were counted using a fluorescence microscope at 100× magnification.
Statistical significance was assessed using the one-way analysis of variance (ANOVA) with the multiple mean comparison test (Turkey) to evaluate statistical differences in the case of normal distribution. Significance was accepted for p < 0.05 using the Statistical Package for Social Sciences (SPSS, version 19.0) software for Windows. Figure 1 shows the FTIR spectra of cured and uncured MAP/TEOS and MAP/TEOS/F18 films. The FTIR technique makes it possible to evaluate the functional groups present in each sample and, in this study, also to evaluate the cure promoted in the film. The curing process of silane films is essential to avoid the migration of aluminum and vanadium ions from the metallic substrate to the body fluid. The observed bands for each functional group numbered from 1 to 8 can also be seen (Kurella & Dahotre, 2005;Liang et al., 2014). A broad absorption band related to the -OH group can be seen at 3445 cm -1 (1), which is derived from the non-condensed silanol groups (Si-OH) during the curing step of the films. The 1730 cm -1 (3) band represents the C=O functional group originated from the acetic acid used to acidify the distilled water used for preparing the sol-gel. The band between 1130 and 1043 cm -1 (6) is assigned to the Si-O-Si bond. In comparison to the cured films, the band (1) in IR spectra of the uncured films exhibits higher intensity, whereas the band (6) shows a lower intensity. These band features confirm, respectively, that the uncured films did not yet undergo the condensation process and that crosslinking between silane molecules effectively occurred during film condensation in the curing process at 90 °C (Liang et al., 2014;van Ooij et al., 2005). The crosslinking process is responsible for forming a network of Si-O-Si siloxane chains, which results in a silane film with enhanced densification, low permeability, and barrier properties (De Graeve et al., 2007;Romano et al., 2011;van Ooij et al., 2005).

Results and Discussion
Comparing the IR spectra of the cured films (red and blue lines), the intensity of the band (6) of the bioglasscontaining cured film appears higher than that of the bioglass-free cured film, which relates to the contribution of Si-O-Si stretch coming from the bioglass particles added to the silane film (Chen & Thouas, 2015;Sepulveda et al., 2002).  The MAP/TEOS films deposited on the metallic substrates seemed thin since the surface irregularities (grinding grooves) of the metallic substrates were still visible from the observation of Figure 2(c,d). Moreover, light and dark regions can be observed, which can be attributed to the variation in film thickness (Salvador et al., 2018). The dark regions corresponded to the microcavities and grooves regions observed on the surface of the metallic substrate, where there was probably more film accumulation (Zhao et al., 2017). Figure 2(e,f) shows the surface morphology of the MAP/TEOS/F18 films where it is possible to notice dark regions located at the perimeter of the F18 particles, which can be associated to the affinity between the silicon-containing F18 Bioglass particles and the silane film. However, microcracks were observed at the dark regions of the bioglass-containing film deposited on the as-received metallic substrate [ Figure 2(e)], which can be associated with the mechanical anchorage of the film in the substrate. When compared with the TS/GCF18 sample, the TS/BCF18 sample, i.e., the as-received substrate, coated with MAP/TEOS/F18 had a smoother surface with less anchoring spots, which might have favored the nucleation and propagation of microcracks in the regions where the bioglass-containing silane film was thicker. Thus, it should be understood that a surface with controlled and uniform roughness, such as the ground one, provides anchoring spots that promote the adhesion of the film to the substrate (Kurella & Dahotre, 2005;Shoucheng et al., 2018). Table 1 shows the results of Ra, Rz, and Rt roughness parameters for the six samples, including the uncoated asreceived bare and after grinding Ti6Al4V alloy, and the MAP/TEOS and MAP/TEOS/F18 coated. The grinding process significantly increased the asperities amplitude of the substrate since, for example, Ra value increased from 7.60 nm for the asreceived substrate to 14.07 nm for the ground substrate. The ground substrate coated with the MAP/TEOS film had a Ra roughness (11.30 nm) lower than that of the uncoated ground sample (14.07 nm) and higher than the as-received coated substrate (10.12 nm). This behavior also agrees with the FEGSEM analysis of the coated ground surface [ Figure 2 Figure 2(e, f)], increased the roughness of the film for the two substrate conditions.
Under a biological point of view, studies have reported that roughness is a key factor for bioactivity since cells require anchoring spots at the surface of the implants to start their proliferation (Slepička et al., 2017). According to the obtained results, the arithmetic average surface roughness of all the samples ranged from 7.60 nm to 20.71 nm. Nanometric scale structures can modulate cell behavior in vivo since the subcell structuresincluding elements of the cytoskeleton, transmembrane proteins, and filopodiatend to the nanometric scale. In this way, extracellular support tissues also typically present a complex signal network in the nanometric scale, made up of a complex mixture of nanometric sized (5-200 nm) pits, grooves, pores, protrusions, and fibers, suggesting that the nanoscale roughness has a promising role in cellular interaction. In this context, a few studies report that protrusions in the range from 10 nm to 350 nm and Ra between 4 nm and 60 nm provide suitable bio-fixation sites and, therefore, were qualified as possible promising ranges for cell viability (Hotchkiss et al., 2016;Huang et al., 2016;Rosa et al., 2013;Zareidoost et al., 2012).  Figure 3 also exhibits the EDS mapping of silicon element, complementing the adhesion test results. Figure 3(a,b) shows that the cut region resultant from the adhesion test appears black, and the original coated surface appears like a deep blue-green color that corresponds to silicon element EDS mapping. This result indicates that the cut region of the adhesion test scarcely contained silicon element and, hence, was a lack of film, confirming the occurrence of the MAP/TEOS film detachment. Additionally, it can also be verified that bioglass addition enlarges film flaking, as shown in EDS silicon element mapping shown in Figure 3(c). Table 1 also shows the contact angle of the film/substrate systems. All the samples had a contact angle lower than 90°, which characterized such systems as hydrophilic surfaces (Kota et al., 2014;Quéré, 2008). Hydrophilic coatings are suitable for cell growth conditions (Pandiyaraj et al., 2010). Despite the significant variation in the surface roughness of the metallic substrate after grinding, this roughening process did not influence the surface wettability result, as can be observed in Table 1.
Moreover, there was no meaningful change in contact angles among as-received and roughened coated surfaces. Changes in contact angles were thought to be attributable to chemistry, roughness, and nanostructural characteristics of the surfaces (Chowdhury et al., 2017;Hotchkiss et al., 2016).
The hydrophilic character observed for the as-received and ground substrates may be related to hydroxyls on the titanium dioxide passive film spontaneously formed on the surface of the Ti6Al4V alloy (Tengvall & Lundström, 1992). The negative charge of the hydroxyl groups can promote several chemical interactions between the surface and the SBF drop, such as hydrogen and Van der Waals bonds, resulting in a higher surface hydrophilia (Kuscer et al., 2008).
Hence, the contact angle and the wettability levels changed as the surfaces were coated with MAP/TEOS and MAP/TEOS/F18 films. The MAP/TEOS film/substrate systems exhibited the highest contact angle (lowest wettability) compared with the uncoated metallic substrates and the MAP/TEOS/F18 film/substrate systems. This result may be related to the variation of surface topography, those inherent of peak or asperity heights (surface roughness), and of that arising from the presence of bioglass nanoparticles (protrusion heights) (Chowdhury et al., 2017).
In this sense, the application of bioglass-free films resulted in reduced protrusion heights and increased peak heights that probably promoted an increase in contact angle and hence a reduction in wettability degree. On the other hand, bioglasscontaining films contributed to the introduction of taller and distant protrusions of bioglass particles that may act as reservoirs for a liquid drop, decreasing contact angle values (Chowdhury et al., 2017). The corrosion resistance of the uncoated substrate and film/substrate systems The analysis of open circuit potential curves of the uncoated samples ( Figure 4) showed that the ground sample had more negative potential as compared with the as-received sample, which can be to the protective action of the passive oxide film covering up the surface of the as-received sample (Mohammadloo et al., 2012). The open circuit potential makes it possible to assess corrosion resistance of the uncoated substrate and film/substrate systems. The grinding process could have   On day 7, most of the surface was covered by cell cytoskeleton protrusions, indicating that the samples provided cell anchoring (Huang et al., 2016;Wennerberg & Albrektsson, 2009). Adhered cells exhibited different morphologies, having a more elongated and starred shape, characteristic of viable and adapted cells (Huang et al., 2016). The lamellipodia and filopodia are protuberant extensions originating from the membrane of a cell. Lamellipodia is a membrane extension with several filopodia joined by actin, and the actin is a protein that forms microfilaments. It is due to the presence of actin that the filopodia become sealed as in a sheet-like with network structure. According to Mattila and Lappalainen (2008) Generally, MG-63 cells tend to be rounded on the metallic surface and elongated on the film-containing surface (Huang et al., 2016). In this way, the protrusion heights and the surface nano roughness may specifically modulate cell morphology. This fact relates to membrane projections, which allow the cells to peruse the surface to find suitable adhesion spots (Dalby et al., 2014;Gittens et al., 2011;Huang et al., 2016;Olivares-Navarrete et al., 2015;Zareidoost et al., 2012). Roughness and the nanoscale topography influenced cell interaction since the dimension of the cell structure, such as the cytoskeleton and transmembrane proteins are of nanometric scale (Biggs et al., 2010;Gittens et al., 2011;Huang et al., 2016;Olivares-Navarrete et al., 2015;Zareidoost et al., 2012).
For the ground substrate, the morphological behavior of MG-63 cells in 1 day [Figure 5(d)] was similar to that observed in the same period for the as-received samples group. On the seventh day, the morphology of the adhered cells cytoplasm seems starred and elongated, and the cells appear longitudinally spread throughout the whole surface of the samples.
Through the TS/G sample micrograph [Figure 5(d)], a huge amount of extracellular matrix can be observed, as defined by darker regions, suggesting that the cells are at the surface adaptation step. Cells cultivated at the TS/GCF18 surface exhibited less elongated morphology than the TS/G and TS/GC samples, which was probably related to increased cell density, which limited the ground for extracellular matrix elongation (Huang et al., 2016).
Besides roughness, surface hydrophilia is a further decisive factor for determining adhesion and cell proliferation, since hydrophilic surfaces provide better interactions with biological fluids, protein absorption, cells, and tissues, favoring cell adhesion and dissemination (Malaval et al., 1999). Under this point of view, all the tested systems are hydrophilic, with the TS/BCF18 and TS/GCF18 samples showing lower contact angles. Thus, the higher wettability of these two film/substrate systems, as corroborated in the literature, improved cellular growth since they exhibited higher cell density after seven days test [ Figure 5(c,f)].
Adding further comments concerning the effect of Bioglass F18 incorporation into the MAP/TEOS film deposited on both the as-received and ground samples, it can be observed in Figure 5(c,f) that the cells had affinity by bioglass particles since the higher cell density regions are located close to or on the particles. The cell membrane protrusions are elongated on the Bioglass F18 particles, indicating that the cell adapted itself and identified Bioglass F18 as an integral part of its biological system.
Viability cell results were expressed in percentage. The negative control absorbance represented 100% viability, and the values for treated cells were calculated as percentages of the control. Figure  From Figure 6, it is important to highlight that the grinding process (TS/G, TS/GC, and TS/GCF18 samples) did not show any significant difference among the periods but provided higher percent values of cell viability than those of the asreceived samples.   Counting of viable cells, in initial apoptosis and final apoptosis/necrosis for all the systems at 7 contact days with MG-63 cells.
Results obtained for seven days are qualitatively displayed in Figure 7(b). It can be observed that there was a statistically significant difference by comparing the TS/B, TS/BC, TS/BCF18 samples with the positive control for the initial apoptosis and final apoptosis/necrosis, corroborating the non-cytotoxicity of the samples. This behavior was expected since few cells in apoptosis were observed in Figure 7(a). Moreover, there were no statistically significant differences among the TS/G, TS/GC, and TS/GCF18 samples (uncoated and coated ground substrates) for the final apoptosis/necrosis.
The number of cells in final apoptosis/necrosis for the TS/G, TS/GC, and TS/GCF18 samples (after grinding) was lower than those of the TS/B, TS/BC, and TS/BCF18 samples (as-received), which is possibly related to the topography and roughness introduced by the grinding process. The higher asperity heights of the ground samples may have promoted viability and cell proliferation (Biggs et al., 2010;Gittens et al., 2011;Huang et al., 2016;Malaval et al., 1999;Olivares-Navarrete et al., 2015;Zareidoost et al., 2012).

Conclusions
The surface of a Ti-6Al-4V alloy was modified by a grinding process and MAP/TEOS coating. Besides roughness, the effect of F18 bioglass particles added to the film on surface characteristics was also investigated. Substrates and film/substrates systems were characterized by a variety of techniques to evaluate the roughness, wettability, and chemistry of the surfaces, the film adhesion to the substrate, the formation of a passive film, as well as the behavior of osteoblastic cells (viability, spreading, proliferation and cytotoxicity). The results show that surface roughness directly influences the adhesion of the silane-based film to Ti-6Al-4V alloy. Coated ground substrates (TS/GC and TS/GCF18 sample) exhibit the lowest percent values of the film area removed. This corroborates that roughness promotes anchorage spots for better film adhesion to the substrate.
Coating the as-received titanium alloy with bioglass-free and bioglass-containing silane-based films provides the formation of a protective surface layer against corrosion in the SBF medium. The as-received titanium alloy substrate covered with MAP/TEOS film presents the highest open circuit potential. Although the grinding process causes a detrimental effect in open circuit potential due to the removal of the protective surface layer, nobler potential values can be reestablished to a maximum level by coating the ground substrate with an F18 bioglass-containing MAP/TEOS film. Increasing amplitude roughness parameters, Ra, Rz, and Rt, of the titanium alloy surface by grinding process and by coating the surface with MAP/TEOS and MAP/TEOS/F18 films promote higher viability and better adaptation of cells. The incorporation of F18 bioglass particles into the MAP/TEOS films enhances surface wettability regardless of the substrate roughness. The higher cellular density near bioglass particles sites indicates the affinity of cells for F18 Bioglass particles. Furthermore, the results suggest that surface chemistry modification by silane-based coatings and, especially, the incorporation of bioglass particles dominates surface wettability, with the substrate roughness acting as a secondary factor. However, concerning film adhesion, protective properties against corrosion, and cytotoxicity of osteoblastic cells, roughness plays an important role. Thus, to achieve better film adhesion to the substrate, enhanced wettability, good protective properties against corrosion, good cell adaptation, and low cytotoxicity, the use of both the grinding process and the application of an F18 bioglass-containing MAP/TEOS coating are suggested among the studied film/substrate systems. The authors suggest as future research, bioglass mixed with other materials or different forms of deposition to improve biocompatibility.