Osteogenesis and biofilms formation on titanium surfaces submitted to oxygen plasma immersion ion implantation

The objectives of this study were to characterize titanium (Ti) surfaces treated by ion implantation by immersion in oxygen plasma (O-PIII) at different temperatures, correlating these implanted layers with therapeutic effects and with osteogenesis, as well as the formation of monotypic biofilms microbial. The groups were divided into: a) Ti (pre-treatment) b) Ti O-PIII at 400 ° C. c) Ti O-PIII at 500 ° C. d) Ti O-PIII at 600 ° C. The properties and surface characteristics were evaluated according to the roughness, texture, corrosion resistance, formation of new phases and the identification of chemical compounds present. Cellular analyzes investigated cell interaction, viability, total protein content, alkaline phosphatase and quantification of mineralized nodules using MG-63 cells. The formation of monotypic microbial biofilms, including P. aeruginosa, S. aureus, S. mutans and C. albicans were evaluated. The increase in surface roughness, corrosion resistance and oxygen content, leading to the formation of TiO2-rutile with more intense peaks and in greater numbers according to the increase in the substrate temperature, ionic implanted Ti samples was observed. There was also a significant increase in cell viability, total protein production, alkaline phosphatase activity and formation of mineralization nodules for the group treated with O-PIII at 600ºC compared to other groups, in addition to a reduction of microorganisms in the groups treated with O- PIII. Therefore, treatment with O-PIII at 600ºC in Ti grade IV showed favorable results for its use.


Introduction
Titanium (Ti) stands out as a material of interest for orthopedics and dentistry due to a range of outstanding characteristics such as biocompatibility, easy manipulation, high corrosion resistance, high modulus of elasticity, accessible acquisition and affordability (Gimmel'farb, &Abrarov 1980, Thelen et al.,2004Simdabe, 2014). Ti implants should remain in the human body for a long time and bear the same loads as the surrounding bone, so it is important that these implants not only allow osseointegration, but also present mechanical characteristics similar to bone tissue. Although the widespread use of Ti for the implant area, intense investigation efforts have been carried out in order to optimize the properties of the implanted surfaces for a rapid and adequate biofixation (Kohavi et al.,2013). When Ti is exposed to atmospheric air, a thin passivating layer of native Ti oxide is formed, which facilitates biocompatibility (Yamagami et al.,2014). However, it can be rapidly destroyed by relative movements and friction between the implant and the tissue (Mandl et al., 2001). Because of these frictions, Ti particles can be locally deposited and further transported to gingiva, lung and spleen, possibly causing sequelae (Mandl et al., 2001, Guglielmotti et al., 2015, Heringa et al., 2018. Long-term effects may cause distinct issues, such as loss of the implant itself, often resulting in prosthetic corrective surgeries. In this context, it is essential to evaluate interactions between osteoblastic cells and the biomaterial. Among several surface treatment proposed for Ti implants (Munoz-Castro et al.,2009, Valencia-Alvarado et al., 2010, Xiao et al., 2012 oxygen ion implantation represents an effective tool, capable of changing morphology, composition and crystalline phase, as well as mechanical and tribological properties , Savonov et al., 2011Da Silva et al., 2006, Peláez-Abellán et al., 2012. In addition, it has been previously demonstrated that O-PIII treatment improves osseointegration in rat femurs after 3 months of incubation when compared to untreated Ti implants (Mandl et al., 2001;Mandl et al., 2002). Yang et al. (2015) demonstrated that O-PIII treated Ti provides better protein adsorption, adhesion, migration, proliferation, mineralization and differentiation of hMSCs (mesenchymal stem cells), indicating that an appropriate treatment with O-PIII may improve the biocompatibility and functionality of Ti surface.
Several characteristics of the implants, including surface composition, topography, hydrophobicity, load, microstructure and flexibility influence bacterial adhesion and biofilm formation (Morais et al., 2013, Do Prado et al., 2013. Implant-related infections are considered the most serious complication common to the risk of surgical infection, which increases when foreign material is implanted. Biomaterial-associated infections are challenging to treat, since bacteria in the biofilm are protected by the host immune system. Moreover, ineffective antibiotics represent another consistent obstacle (Zhao et al., 2015). Highly resistant bacterial strains, contamination of the surgical area and the surrounding tissue endanger the health of the patients, affecting the osseointegration (Zhao et al., 2015, Zaatreh et al., 2016. The objectives of this study were the characterization of O-PIII-treated Ti surfaces, the association between the materials and osteogenesis (in vitro) and the formation of monotypic microbial biofilms on the surface of the materials.

Samples preparation and characterization
A Grade-4 Ti ingot was machined into disks (12.7 mm in diameter and 3 mm thick) and used as the substrate. Prior to oxygen PIII treatment, the specimens were ultrasonically cleaned with acetone for 20 min, and then dried in air.
Samples were treated in a high temperature PIII reactor (Oliveira et al., 2010) by performing the heating of the substrates at controlled temperatures simultaneously with the ion implantation. Thus, three set of samples were immersed in oxygen plasma for 1 h, being individually treated under the same operational conditions: at 3x10-3 Torr, with pulses of 7.7 kV / 30 µs / 420 Hz.
Substrate temperature was the distinct parameter between the three groups (Group 1: Ti O-PIII at 400°C; Group 2: Ti O-PIII at 500°C; Group 3: Ti O-PIII at 600°C). Group 4 corresponds to untreated titanium.
Quantitative analysis of oxygen content present on the surface of the samples was performed by EDS (Energy dispersive X-ray spectrometry, Bruker, model XFlash Detector 410-M). The formation of new phases was confirmed by X-ray diffraction (XRD) with a diffractometer (Panalytical, model X'PERT POWER), CuK α radiation source (λ = 1,5406 Å), angular range at 2θ varied from 35º to 90º. Measurements were performed at room temperature, in continuous scan mode, with a speed of 1.2º per minute and an angular step of 0.02º. Voltage and electric current used were 45 kV and 40 mA, respectively. The identification of the diffracted peaks was done with the help of X'Pert Highscore software, provided by PANalytical BV, taking into account the JCPDS (Joint Committee on Powder Diffraction Standards) database.
The topography of the samples was evaluated by Atomic Force Microscopy using a Veeco equipment, model MULTIMODE V. An area of 2 x 2 μm of each sample was probed, using the Flex-Axiom MFA system.
Corrosion resistance of the samples and the effects of O-PIII treatments, in relation to the chemical treatment, was evaluated at chemical level, through the EIS (Electrochemical Impedance Spectroscopy) test.
For electrochemical analysis a three electrode cell was used, with Ag/AgCl as the reference electrode, a platinum wire as the counter electrode and the Ti sample as the working electrode. Tests were performed in simulated body fluid (SBF) solution.
EIS analyzes were performed with a 10 mV disturbance signal amplitude, in the range between 105 and 10-2 Hz. Measurements were calculated after 0, 2, 4, 8, 12 h of submersion. Autolab (Metrohm, 302N) was used to evaluate the samples. In order to verify possible microstructural changes resulting from the corrosion tests, the corroded surfaces were examined by scanning electron microscopy (SEM, FEI Inspect S50 model).
Cell interaction with the materials was evaluated by SEM after seven days of incubation (n = 02). Cells were fixed with paraformaldehyde (4%) at room temperature, dehydrated through an ascending series of ethanol (70%, 90% and 100%) and, prior to the analysis, all samples were coated with a thin layer of gold, using a sputter-coating system (Emitech, model SC7620).
The equipment used was the Inspect S50 model of the FEI brand (Thermo Fisher Scientific, Massachusetts, USA).
Cells were incubated in contact with the materials for 3 days, thereafter cell viability was quantified by exposing the cells to the toxic agent by incubation with the MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Saint Louis, USA) at a concentration of 0.5 mg / mL. After 1 h of incubation, at 37 ºC and 5% CO2, the organic solvent DMSO (Dimethylsulfoxide) (Sigma-Aldrich, Saint Louis, USA) was added to solubilize the formazan crystals. For each experimental group, n = 05 was determined. Colorimetric microplate reader was used at wavelength 570 nm (Biotek, model ELx808cse).
After being platted and cultivated for 10 days (n = 05 per group), total protein content was calculated, according to the modified method of Lowry et al. (1951). For protein extraction, sodium surfactant lauryl sulfate (Sigma-Aldrich, Saint Louis, USA) at 0.1% was used. The solution was mixed with Lowry reagent (Sigma-Aldrich, Saint Louis, USA), at room temperature.
Then, Folin-Ciocalteau reagent (Sigma-Aldrich, Saint Louis, USA) was added to the mixture. A spectrophotometer (Micronal, model AJX 1900) at 680 nm was used to calculate the absorbance and the total protein content was measured from a standard curve from bovine albumin pre-determined and expressed as µg / ml unit.
After ten days of cell culture, alkaline phosphatase activity was analyzed and, for each experimental group, n = 05 was considered. Thymolphthalein monophosphate and diethanolamine buffer (0.3 M and pH 10.1) substrates were added in glass tubes. To this solution, an aliquot of the lysates from each well was added, samples were maintained at 37 °C. Then, Na2CO3 (0.09 M) and NaOH (0.25 M) solutions were added to the tubes. Absorbance was calculated on a spectrophotometer (Micronal, model AJX 1900), at 590 nm.
After a period of fourteen days of incubation (n = 05 for each experimental group), mineralized nodules were quantified.
Hank's solution (H6136 -Sigma-Aldrich, Saint Louis, USA) was used for cell culture nutrition, in addition to Alizarin S red dye (Sigma-Aldrich, Saint Louis, USA), at a concentration of 2 mg /ml, responsible for staining calcified areas. After one day, acetic acid (10%) solvent was added, contents were transferred to centrifuge micro tubes (Sigma-Aldrich, Saint Louis, USA) and vortexed (Vortex, model QL 901). Samples were then centrifuged for 20 minutes and the supernatants were transferred to 96well plate (Thermo Fisher Scientific, Rochester, NY, USA). Reading was performed using a microplate reader (Biotek, model ELx808 IU), under the wavelength of 405 nm.
After a one-day incubation period, MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Saint Louis, USA) dye was added at a concentration of 0.5 mg / ml. After incubation under light protection, the solution was removed, DMSO (Sigma-Aldrich, Saint Louis, USA was added and kept under stirring, in an orbital table. aliquots of 100 μl of each well were transferred To 96-well plate (Thermo Fisher Scientific, Rochester, NY, USA). Absorbance reading was performed by a microplate reader (Biotek, model ELx808cse), at a wavelength of 570 nm.
These procedures were performed as described by Mello et al. (2019).

Statistical analysis
Quantitative data of the results were statistically analyzed by GraphPad Prism 6 software, using one-way ANOVA and Tukey's multiple comparison tests. The level of significance adopted was the conventional value of 5%, 0.1% or 0.001%.

Surface characterization
According to Figure 1, Ti group (pretreatment) presented, comparatively, low amount of O on their surface (6.94% O), while O-PIII-treated Ti groups at 400ºC, 500ºC and 600ºC presented the mean values of 26.31%, 45.66% and 56.61% of O, respectively, demonstrating that the higher the temperature reached in the PIII treatment, the greater the atomic percentage of O on the surface. In fact, the reactivity of the metal for oxygen uptake is increased with the temperature. The mechanism involves the dissolution of oxygen in the metal and its absorption. In addition, the oxygen ions implanted on Ti surface can reach deeper layers at higher temperatures due to diffusion process. The oxidation at high temperatures also promotes the development of crystalline phases. and Ti O-PIII at 600ºC. Dispersive energy spectroscopy. ** p <0.05, *** p <0.001 and **** p <0.0001 compared to Ti * group (ANOVA and Tukey test). Asterisks indicates a statistical difference.
Source:Authors. Figure 2 shows the presence of TiO2 (rutile phase) peaks in the O-PIII-treated groups at 500ºC and 600ºC, characterized mainly at the angle of 27.5º, considering the most intense peak of this phase. The presence of this peak was not identified in Ti (pretreatment) and Ti O-PIII groups at 400°C. In fact, a consequence of the increase of the substrate temperature during PIII is the facilitation for the formation of the rutile phase, as observed in figure 2, due to the increase of the ratio of O to Ti. The presence of Ti-α had also been observed in all groups.  Fig. 3 (b, c and d). According to our results, the higher the temperature reached in the O-PIII-treatment, a more numerous of such structures is observed. Figure 4 shows the roughness (RMS -Root mean square) and surface area profile values, confirming that increasing the O-PIII temperature, higher the RMS and the surface area. Figure 5 shows that Ti (pretreatment) sample, compared to the other O-PIII-treated Ti samples, had the lowest impedance in all evaluated frequencies, of almost one order of magnitude lower. This is an indication that the oxygen enriched modified surface layer produced via O-PIII protects the metal surface from corrosion in SBF medium. O-PIII-treated Ti sample at 600ºC was the one with highest impedance at medium frequency. At low frequencies, impedance was not differentiated between samples.  Additional investigation about the corrosion of the surfaces was performed by examining the topography of the surfaces. Figure 6 (a) demonstrates a well-defined corrosion site on the surface of Ti sample (pretreatment) as a consequence of SBF exposure. In Figure 6 (b, c and d), no specific corrosive sites were identified, only specific areas were observed, due to the production process of the samples. Source:Authors.

In vitro cell assays
The cell morphology can be seen in Figure 7 (a) for untreated Ti sample and in Figure 7 (b, c, d) for O-PIII samples treated at 400°C, 500°C and 600°C, respectively. In general, all samples allowed cell spreading. We suggest that samples submitted to O-PIII treatment had more evident cell adhesion, as cell prolongations were better observed in these treated groups. Concerning the cellular viability, no statistical difference is observed in Figure 8 Concerning the total protein content, Figure 8 (b) demonstrates that groups submitted to O-PIII treatment at 400ºC and 500ºC had no alterations in protein expression when compared with the Ti group (pretreatment). However, O-PIII-treated Ti group at 600ºC had protein expression statistically increased (p <0.0001), when compared to all groups.
After evaluating the surface characterization and MG-63 assays (Figure 1 to 8) carried out so far, O-PIII-treated group at 400ºC was not considered for the following assays as it did not present significant results in the current study.
Concerning the alkaline phosphatase activity shown in Figure 8   Viability. b) Total protein content. c) Alkaline phosphatase activity. d) Quantification of mineralization nodules. ** p <0.05 and **** p <0.0001 compared to the * (ANOVA and Tukey test). Asterisks indicates a statistical difference. Source:Authors.

Formation of Microbial Biofilms
As observed in Figure 9 (a), in Ti (pretreatment) and O-PIII-treated Ti samples at 500ºC and 600ºC, the monotypic biofilm formation of P. aeruginosa was statistically decreased (p <0.001, p <0.001 and p<0.00001, respectively) compared to the control group. However, the group treated with O-PIII at 600°C statistically decreased the amount of P. aeruginosa when compared to the O-PIII-treated group at 500°C and Ti (pretreatment).
In Figure 9 (b), only the group treated with O-PIII at 600ºC presented a statistical decrease (p <0.001) compared to the control group, regarding monotypic biofilm formation of S. aureus. However, Ti (pretreatment) and O-PIII-treated Ti groups at 500ºC and 600ºC did not show significant changes in the amount of S. aureus when compared between themselves.
In Figure 9 (c), groups submitted to O-PIII treatment at 500ºC and 600ºC showed a statistical decrease (p <0.001) in the quantification of S. mutans, compared to Ti (pretreatment) and control groups, however, these groups had no alterations when compared to each other.
In Figure 9 (d), O-PIII-treated at 500ºC and 600ºC groups presented statistical decrease (p <0.001) in the amount of C. albicans compared to Ti (pretreatment) and control groups, however, these groups had no significant differences when compared to each other.

Discussion
Treatment with plasma immersion ion implantation (PIII) is responsible for modifying complex surfaces not altering sample size, moreover, it is possible to work with PIII in a wide temperature range, allowing the formation of structures and phases without balancing (Rossi et al., 2004, Gupta, 2011 .The advantages of PIII implantation system consist in the fact that the period of treatment do not depend on sample size and does not require special sample manipulation, even when surfaces are irregular (Ueda et al., 2007).
Atmospheric oxygen has strong affinity for Ti, forming a passive layer of oxide on its surface, which serves as a protective layer against corrosion. The thickness of this oxide layer may undergo some changes according to environmental conditions and treatment (Hansen et al., 2015). Recently, Mohan et al. (2017) evaluated chemical compositions of surface composed by Ni, Ti and O on NiTi alloy substrates subjected to O-PIII technique. They observed an increase in oxygen percentage in the substrates of the studied alloys, and their spectra showed the presence of nickel, Ti and oxygen on these surfaces, indicating that enriching the surface with the oxygen treatment is essential for the formation of stoichiometric TiO2.
These results corroborate our study, in which the formation of a greater amount of oxygen in the samples of Ti treated with O-PIII at 400ºC, 500ºC and 600ºC was observed. Moreover, we demonstrated that the higher the temperature used in the treatment, the higher the presence of oxygen on the surface.
The formation of rutile phase TiO2 after Ti treatment has recently been reported in the literature by several studies (Rafieian et al., 2015, Guan, & Lou 2018, Sasahara et al., 2018. Yang et al. (2015) performed studies with the O-PIII treatment to create dense and thin layer of TiO2 on the surface of Ti for dental implants, they observed the formation of an oxide layer, mainly consisted of ruthenium TiO2. In our study, TiO2 (rutile phase) peaks observed in the groups treated with O-PIII at 500ºC and 600ºC, attested the efficacy of the treatment under these conditions, a consequence of the higher oxygen content. In contrast, Tóth et al. (2004) published that no crystalline oxide phase could be detected in TiAlV (titanium aluminum vanadium) alloy after oxidation. These authors suggested that treatment under low temperature and the presence of aluminum oxide may have prevented crystallization.
In this study, we observed that O-PIII-treated samples exhibited nanoporous surface structure, demonstrating a nanostructured TiO2 layer in the form of round grains, which is increased as the temperature increases in the treatment. The increase of the nanoscale roughness caused by O-PIII treatment had been previously reported (Hung et al., 2016, Wu et al., 2018. Hung et al. (2016) analyzed the morphology of Ti surfaces pre-and post-treated with O-PIII and, as in our results, Ti had relatively flat surfaces only with parallel polishing traces, however O-PIII-treated Ti showed nanoporous surface structure, exhibiting a more uniform, denser, nanostructured TiO2 layer, with higher round grain magnification compared to Ti (pretreatment) samples.
EIE is used for different purposes, ranging from electronic transport in semiconductor devices to the electrochemical kinetic processes of the most different natures, ie processes that occur in photovoltaic cells and corrosion systems (Bisquert et al., 2000, Gratzel, 2001. The measurement of corrosion resistance in this study showed that increasing surface treatment temperature resulted in greater inertia against corrosion in SBF medium. Consequently, the higher the temperature used in O-PIII treatment, the higher the sample impedance. Pan et al. (1994) observed a dark pigmentation in Ti exposed for several weeks to phosphate buffered saline (PBS) with H2O2 additions, representing a suggestive point of corrosion caused by the treatment.
Recently, a range of studies involving cell analysis on Ti surfaces have been reported in the literature (Gehrke et al., 2018, Urenã et al., 2018, Kasnak et al., 2019 all of them designating Ti as a successful research tool, as cell interactions with the surface of the biomaterial can be evaluated in more detail. In our study, it was observed that all groups permitted cellular adhesion, moreover, O-PIII-treated samples had more evident cell adhered with more cellular projections. Soares et al. (2018) corroborate our results, describing that MG-63 cells also presented prolonged formats, surrounded by abundant cytoplasm, welldefined and intact when in contact with Ti. Kiran et al. (2018) after evaluating the viability of MG-63 cells on different Ti surfaces, observed that all samples stimulated cell growth over a period, increasing viable cells; when samples were submitted to treatments with higher degree of morphological changes in the surfaces, a greater increase of cellular viability was described. Our results corroborate this previous study, showing a statistical increase in cell viability in Ti group submitted to O-PIII treatment at 600ºC, compared to the other groups.
Total protein content is an important parameter in osteogenesis. In this study, it was observed that the group submitted to the highest treatment temperature (Ti O-PIII at 600ºC) had a higher amount of proteins when compared to the other groups. Cheg et al. (2018) identified that surfaces treated with hydrothermal oxidation of low temperature microwaves promoted positive influence on MG-63 DNA content, but did not alter alkaline phosphatase and osteoprotegerin. However, in the present study, Ti submitted to treatment with O-PIII at 600ºC increased alkaline phosphatase values.
A study conducted by Baranowski et al. (2016) allowed us to consider that the assay performed with Alizarin S Red dye revealed an increase in calcium deposition in osteoblast populations cultured with bone sialoprotein with morphogenetic bone protein additive 7 (BMP-7) compared to the control group with no additives (proliferation medium). Our results, which were also performed with Alizarin S Red dye, showed that Ti group treated with O-PIII at 600ºC statistically increased the amount of mineralized matrix nodules compared to the other groups. Ren et al. (2014) stated that cell culture assays clearly show that micro/nanotexturide surface of TiAlNb (titanium aluminum niobium) stimulates fixation, dissemination, proliferation and increased cell alkaline phosphatase activity better than polished surfaces. In our study, it was observed that the surface changes of O-PIII treated Ti samples at 600ºC improved MG-63properties, regarding adhesion, viability, protein expression, alkaline phosphatase activity and matrix mineralization.
Decontaminating microorganisms and their toxins on implant surfaces consists in a prerequisite for preventing periimplantitis, in addition to achieving therapeutic success of implants. It has been shown that Gram-positive S. aureus bacteria have high affinity to titanium substrates, particularly to those with a rough surface finish (Aguayo et al., 2015, Izquierdo-Barba et al., 2015. Giannelli et al. (2016) observed the effects of laser diode on S. aureus biofilm adherent to Ti oxide surface of dental implants. They observed that this treatment in pulsed and continuous modes induced statistical reduction of viable cell compared to untreated biofilm. In the present study, only Ti group submitted to O-PIII treatment at 600ºC statistically decreased cell viability compared to the control group, while the other groups did not demonstrate significant alterations in the quantification of S. aureus when compared between themselves. Although no statistical differences were observed, Ti and O-PIII-treated Ti at 500ºC groups showed a trend to reduce cell viability. Fatani et al. (2017) after evaluating orthodontic stainless-steel brackets coated with TiO2 as anti-adherent and antibacterial tool for Streptococcus mutans, observed a decrease in biofilm formation in brackets coated with Ag + TiO2. Our results demonstrated that O-PIII treatment of Ti samples at 500ºC and 600ºC statistically reduced the amount of S. mutans compared to Ti (pretreatment) and control groups.
Furthermore, we observed that, after one day of cultivation, the groups Ti and O-PIII-treated Ti at 500ºC and 600ºC showed a significant reduction of the quantification of P. aeruginosa in comparison with the control group. The group Ti O-PIII at 600ºC was the one with higher significant decrease compared to the other groups. Studies carried out by Nunes Filho et al.
(2018) stated that the chemical composition of Ti surface interferes with the biofilm formation of Pseudomonas aeruginosa.
After quantifying the adhesion of bacteria in Ti and TiN samples, the authors observed that TiN showed higher performance against bacterial adhesion and aggregates formation, and that Ti (pretreatment), after the first 3 h of incubation, led to less than 20% of bacteria adhesion, while after 6 h this percentage increased. In TiN samples, after 3 h, about 80% of the bacteria were adhered and, after a period of 6 h of exposure to nitride surfaces, bacteria were unable to bind. These results indicate a correlation between the chemical state of Ti and the interference observed in the first steps of P. aeruginosa biofilm formation. Jeyachandran et al. (2007) suggested that chemical status and roughness represent the main influences on the interaction of bacteria with the surface of an implant.
Previous studies have shown that Candida biofilms exhibit increased resistance to antifungal agents and, although several hypotheses have been suggested, none of them clearly elucidates the phenomenon of increased resistance. However, surface roughness has been shown to directly influence the adhesion of microorganisms to medical devices such as catheters and dental implants (Ramasay, & Lee 2016, Vargas Blanco et al., 2017. In a study carried out by Tsang et al. (2007) about biofilms of Candida albicans on Ti plates with different roughness, it was observed that, from sandblasting with 99.6% aluminum oxide of different grain sizes, there was no statistically significant difference in fungal adherence between groups. In contrast, herein, we observed that Ti groups submitted to O-PIII treatment at 500ºC and 600ºC showed a statistical decrease in the quantification of C. albicans compared to Ti (pretreatment) and control groups.

Conclusion
O-PIII treatment in Ti samples at different temperatures (400ºC, 500ºC and 600ºC) promoted an increase in surface roughness, corrosion resistance, oxygen presence and formation of TiO2-rutile, as the temperature used in the treatment increased.
These modifications in surface properties resulted in MG-63 cells activity improvements when these cells are cultivated in contact with the materials, especially for O-PIII treatment at 600°C. O-PIII treatment also reduced viability of monotypic microbial biofilms (P. aeruginosa, S. aureus, S. mutans and C. albicans), regardless of the temperature used in Ti treatment.
From these data obtained, it is suggested to use the O-PIII treatment at 600ºC in Ti grade IV, due to the increase in the substrate temperature during this technique, it is to facilitate the formation of the rutile phase, improving the physical-chemical properties of the samples treated. In addition, it presented good results in cell analysis, as well as a decrease in the quantification of monotypic microbial biofilms, becoming a promising material for clinical application. Thus, for future works is recommended in vivo studies to analyze the influence of Ti O-PIII treatment at 600°C in commercial implants both in bone neoformation, as in the biomechanical test, in order to evaluate the force fixation the osseointegration.