Effect of solid solution and aging treatment on the microstructure, mechanical properties, corrosion behavior and antimicrobial properties of Ti-5Mn alloys

In this paper, Ti-5Mn alloy was subjected to different heat treatments to explore the possibility of preparing antimicrobial Ti-Mn alloys and to examine the effect of precipitate on the properties of the alloy. The microstructure, phase composition, hardness, biocorrosion properties and antimicrobial properties of Ti-5Mn alloys after different heat treatments was analyzed by metallurgical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), microhardness tests, electrochemical tests and antimicrobial tests. The results have shown that the phase composition of the solid solution treated Ti-5Mn(T4) was mainly β-Ti phase, and the aged Ti-5Mn was composed of α-Ti phase and Ti17Mn3 phase, while Ti17Mn3 precipitate gradually increased with the extension of the aging time. Ti-5Mn(T4) showed the highest hardness and the best corrosion resistance and the aging process reduced the hardness of Ti-5Mn(T4) alloy. With the precipitation of Ti17Mn3, the corrosion resistance of the alloy became worse and the hardness was reduced, but the corrosion resistance of Ti-5Mn alloy was still better than that of cp-Ti. It was demonstrated that Ti-5Mn(T4) exhibited no antibacterial properties against Staphylococcus aureus, but the aging treatment improved the antibacterial property of Ti-5Mn(T4) alloy, and the antibacterial rate of Ti-5Mn alloy reached 69% after 50 h aging treatment.


Introduction
The challenge in the application of titanium-based biomaterials in humans is bacterial infections during surgery and post-implantation, and complications due to bacterial infections have been reported in both orthopedic and dental implants [1][2][3][4].Despite having a strict sterilization procedure during the surgical procedure, bacterial infections can still occur, which might eventually lead to implantation failure.Therefore, the prevention of bacteria-related infections in titanium-based biomaterials remains an important challenge to be addressed [5].
To reduce bacterial infections, researchers have developed antimicrobial titanium alloys by adding antimicrobial metal elements (e.g., Ag or Cu) to titanium and titanium alloys [3,6,7].The antimicrobial mechanism has also been described in different ways.Some studies have shown that metal ions, including Ag ions, can destroy cell walls, causing cell membrane damage and eventually leading to bacterial death [8][9][10][11].
On the other hand, other researchers have found that the antimicrobial effect of antimicrobial metals does not depend completely on the amount of metal ions dissolved, but is related to the precipitates in the titanium alloy.Fu et al [12] investigated the effect of the second phase on the antibacterial properties and antibacterial mechanism associated with Ti-Au alloys.Their results showed that the aged Ti-Au(T6) alloy and Ti-Au(S) sintered alloy had strong antimicrobial rates due to the formation of Ti 3 Au phase.Cui [13] designed and prepared Ti-15Mo-xAg (x = 10, 12) alloys to obtain low elastic modulus and excellent antimicrobial ability.Their results showed that aging treatment and silver content had a significant effect on the elastic modulus and the antimicrobial ability, and high silver content and precipitation of the Ti 2 Ag phase were beneficial to antimicrobial properties.Wang [14] et al prepared Ti-Cu alloys with different Cu contents and revealed that Ti-Cu alloys with α-Ti+Ti 2 Cu microstructure had excellent antimicrobial properties and corrosion resistance.Therefore, the antimicrobial effect of titanium alloys is not entirely controlled by the metal ions, but the second phase in titanium alloys also plays an important role in the sterilization process [15].
However, it is still not clear how the second phase affects the antimicrobial properties of titanium alloys in the antimicrobial process.It has been shown that surface nanoparticles of antimicrobial materials interfere with the extracellular electron transfer behavior of bacteria [16,17].The effect of electron transfer in alloys on bacteria has been reported by some researchers in Cu-containing stainless steels, where the potential difference between the Cu-rich second phase in the alloy and the matrix promotes the conversion of Cu(II) to Cu(I), leading to a large release of Cu ions, which has a bactericidal effect [18].Wang [19] suggested that the antibacterial mechanism of Ti surface-encrusted AgNPs was that the electron transfer between AgNPs and Ti causes a large amount of ROS production in the bacterial cytosol and medium, which in turn leads to bacterial death.All these results suggest that the electrode potential difference between Ti matrix and the second phase may affect the antimicrobial effect.Also, the study by Fu et al [20] confirmed that the micro area potentials difference (MAPD) on the alloy determined the antibacterial properties.
Mn is a low-cost β-stable alloying element.The main advantages of Mn are its low cost and its low cytotoxicity compared to most β-stable elements [21].Mn is one of the elements necessary for bone growth, which promotes the proliferation of osteoblasts [22].Furthermore, the incorporation of Mn elements into titanium alloys can confer the desired mechanical properties and lower Young's modulus.Santos [23] et al investigated the microstructure, mechanical properties, and biocompatibility of Ti-(6-8)Mn alloys after solid solution treatment.It was reported that Ti-Mn alloys exhibited excellent mechanical properties and cytocompatibility.Kim [24] et al investigated the effect of manganese content on the mechanical properties, oxidation behavior and electrochemical corrosion properties of Ti-Mn alloys and showed that the addition of Mn improved the mechanical properties and corrosion resistance of the alloys, in which Ti-5wt% Mn alloy had the highest hardness (457.2HV) and the lowest modulus (129.9GPa), which makes them a good candidate for dental implant alloy.Asrar [25] et al prepared Ti-5Mn, Ti-10Mn and Ti6Al4V alloys by powder metallurgy and fully characterized their mechanical properties, including hardness and wear resistance.The experimental results demonstrated that Ti-5Mn and Ti-10Mn alloys exhibited higher hardness, lower modulus of elasticity, and superior wear characteristics than Ti6Al4V.Among them, the Ti-5Mn alloy exhibited the most outstanding performance in all the examined parameters.
There is a difference in the electrode potential between Mn and Ti and the aging treatment promotes the precipitation of Ti-Mn intermediate phase.Based on the MAPD mechanism, the precipitation of the Ti-Mn phase might have an antibacterial effect.Therefore, based on previous studies, Ti-5Mn alloy was prepared in this work by casting, and the as-cast alloy was subjected to heat treatment (solution treatment and aging) to adjust the precipitation of Mn-containing second phase.The change in the microstructure, phase composition, hardness, corrosion behavior, and antibacterial effects of Ti-5Mn alloys with the heat treatment was investigated in detail to reveal the possible biomedical application of Ti-Mn alloy.

Materials preparation
High-purity titanium sponge and manganese powder were used as raw materials to prepare Ti-5Mn alloy ingots by a high vacuum arc melting furnace, and the ingots were melted several times to ensure chemical uniformity.Since Mn is volatile during melting, 10% more Mn was added to the ingredients through the Mn compensation amount reported in the literature [26].After casting, the composition of the alloy (shown in table 1) was determined by optical emission spectrometry to be Ti-5.4wt%Mnwith less than 0.01% O, N, and C, named Ti-5Mn(L).Afterward, the ingot was heat treated at 900 °C for 5 h followed by water quenching, named Ti-5Mn (T4), and then aged at 500 °C for 8 h, 20 h, 30 h and 50 h, and named Ti-5Mn(T6-8h), Ti-5Mn(T6-20h), Ti-5Mn (T6-30h) and Ti-5Mn(T6-50h), respectively.

Microstructure characteristics
The phase constitute was analyzed by x-ray diffraction (XRD), with a scanning range of 30°-90°and a scanning speed of 4°min −1 .The samples for microstructure analysis were cut from the ingot, ground with silicon carbide paper up to 2000 mesh, polished, and etched with an etching solution of HF: HNO 3 : H 2 O = 1:2:50.The microstructure of the samples was observed using a metallurgical microscope and the average grain size was calculated by a software.The microstructure was observed under scanning electron microscopy (FE-SEM, JSM 6360LV, JEOL, Japan) with energy-dispersive x-ray spectrometry (EDS).The microscopic crystal structure was observed and analyzed using a field emission transmission electron microscope (JEOL JEM-2100F) equipped with an energy spectrometer.

Hardness
Hardness testing was performed using a micro Vickers hardness tester model MH-500.The test load was 200 g and the loading duration was 15 s.At least 10 points were tested for each sample and the average of 8 points after removing the maximum and minimum values was taken as the mean value with standard deviation.

Electrochemical testing
For electrochemical measurements, a disc sample with 15 mm in diameter and 2 mm in thickness was embedded in cold-curing epoxy resin, exposing only a working surface of Φ15 mm.The sample was ground by 120#, 400#, 600#, 800#, 1200#, and 2000# SiC sandpaper in sequence, and then cleaned with ultrasonic in anhydrous ethanol for 5 min and dried with cold air.The electrochemical testing was conducted on a standard three-electrode system at 37 °C with a platinum counter electrode, a saturated calomel electrode (SCE) as the reference electrode, and the sample as a working electrode.Simulated body fluid (SBF) solution was used as the corrosion medium.The open-circuit potential (OCP) and dynamic potential polarization (Tafel) curves were performed on a VersaSTAT V3-400 electrochemical workstation.Following the electrochemical testing, the samples were dried in a desiccator for 24 h, and then the surface morphology of the samples was observed using a scanning electron microscope (FE-SEM, JSM 6360LV, JEOL, Japan).

Antibacterial test 2.5.1. Plate-count method
The gram-positive strain of Staphylococcus aureus (S.aureus, ATCC25923) was provided by the Dental Laboratory of China Medical University.The antibacterial test was performed by the plate count method with reference to Chinese standard GB/T 2591.The samples were sterilized and placed in 12-well plates.The bacterial solution was diluted to 10 5 CFU ml −1 (called bacterial suspension) with 0.9% NaCl saline and 100 μl of bacterial suspension was dropped on the sample surface.Saline was then filled into the 12-well plate gap.Finally, the samples and culture plates were incubated at 37 °C for 24 h.After the incubation, the samples were washed with 2 ml of physiological saline, and 0.1 ml of the washing solution was spread on solid agar, and incubated at 37 °C for 24 h.Then the colonies number was counted and the antibacterial rate was calculated by equation (1): Where, N control and N experiment denote the average colonies number on the control (cp-Ti) and the samples, respectively.The antimicrobial test was repeated three times to ensure reproducibility.

Morphology of bacteria
100 μl of bacterial suspension with a concentration of 10 5 CFU ml −1 was added dropwise to the surface of each sample.After 24 h incubation at room temperature, the sample surface was washed with saline.Then 2 ml of 2.5% glutaraldehyde solution was added to fix the bacteria at 4 °C for 2 h.The samples were then dehydrated with 30%, 50%, 70%, 90% and 100% ethanol solutions for 10 min.Finally, the samples were dried in a desiccator for 24 h and then sprayed with gold and observed by a scanning electron microscope.

Statistical analysis
Data were presented as mean ± standard deviation.Statistical analysis was conducted using one-way ANOVA.Data were considered significantly different when * p < 0.05.These results suggested that no other phase was formed during the casting and the solid solution treatment or that its content was too low to be detected.The diffraction peaks of Ti 17 Mn 3 and α-Ti were observed after the aging treatment, indicating that Ti 17 Mn 3 phase precipitated during the aging process.Furthermore, the diffraction intensity of Ti 17 Mn 3 increased with increasing aging time, indicating that more Ti 17 Mn 3 phases precipitated with the extension of the aging.
The metallographic micrograph of Ti-5Mn alloy after the solid solution and aging treatment is shown in figure 2. The microstructure of Ti-5Mn(T4) alloy mainly consisted of a single β-Ti phase and the average grain size of Ti-5Mn(T4) alloy was the smallest, about 639 ± 15 μm.
According to the XRD results in figure 1, it can be seen that the Ti-5Mn alloy consisted of α-phase and Ti 17 Mn 3 -phase after the aging treatment.However, as shown in figures 2(b)-(e), only the α-phase appeared in the microstructure of Ti-5Mn(T6) alloy.This may be due to the fact that the size of Ti 17 Mn 3 phase was small to be found under the metallographic microscope.Meanwhile, the grain size of the Ti-5Mn alloy gradually became larger with the extension of the aging time.The average grain size of Ti-5Mn(T6-8h), Ti-5Mn(T6-20h), Ti-5Mn  (T6-30h) and Ti-5Mn(T6-50h) samples were about 676 ± 39 μm, 722 ± 60 μm, 813 ± 90 μm and 850 ± 15 μm, respectively.
Figure 3 shows the SEM microstructure of Ti-5Mn(T4) and Ti-5Mn(T6) samples.There was no second phase in Ti-5Mn(T4), suggesting that the Mn element was solid dissolved into the matrix completely.As shown in figures 3(b)-(e), after the aging treatment, white particles precipitated from the α-Ti matrix, and the number of white particles gradually increased with the extension of the aging time.From XRD patterns, it is clear that the alloy consisted of α-Ti and Ti 17 Mn 3 phases after the aging treatment.Therefore, the white particles are most likely to be Ti 17 Mn 3 phase precipitated from the matrix.To identify the chemical composition of the white precipitates, the Ti-5Mn(T6-50h) alloy, which is rich in the second phase, was analyzed by EDS.
The results are shown in figure 4, where the Ti and Mn elements were uniformly distributed in Ti-5Mn(T6-50h) alloy.It is noteworthy that no elemental enrichment was observed in the Mn elemental distribution map.In order to further determine the microstructure of Ti-5Mn alloy after the aging treatment, Ti-5Mn(T6-50h) with more precipitates was selected for TEM observation.Figure 5(a) shows the bright-field image of Ti-5Mn(T6-50h), accompanied by the energy spectrum analysis and the selected-area electron diffraction analysis of precipitates.The electron micrograph of the rounded area in figure 5(a) clearly shows the morphology of the second phase.The energy spectrum analysis and the selected area diffraction analysis confirm that the precipitated phase is Ti 17 Mn 3 .This indicates that Ti-5Mn alloy underwent a co-precipitation reaction from β-Ti to α-Ti +Ti 17 Mn 3 during the aging treatment.Figure 5(b) shows the distribution of Mn elements.The aggregation of Mn elements on the α-Ti matrix can be clearly seen, which indicates that a large number of fine Mn-rich phases precipitated from the α-Ti matrix during the aging treatment.

Micro-hardness
Figure 6 shows the change in the microhardness of Ti-5Mn alloys with the aging time.The results showed that the hardness of Ti-5Mn alloy reached a maximum of 463 ± 4 HV after the solid solution treatment.Then, the hardness decreased and then increased with the extension of the aging duration.The higher hardness of Ti-5Mn (T4) than that of Ti-5Mn(T6) is due to the solid solution strengthening effect of Mn on the one hand, and the distortion of the lattice structure of the β-phase due to the sufficient solid solution of Mn prevents the dislocation motion.On the other hand, it can be seen from figure 2(a) that the average grain size of Ti-5Mn(T4) alloy was the smallest after the solid solution treatment, and the effect of fine-grain strengthening was the most obvious.
The hardness of Ti-5Mn alloy reached the lowest value, about 396.2 ± 2.3 HV, after aging for 20 h.This phenomenon may be due to the fact that Mn atoms began to aggregate to form precipitate phases, which leads to a weakening of the solid solution strengthening.At the same time, the grain grew gradually and the microstructure of the alloy changed from β-phase to α-phase and Ti 17 Mn 3 phase.This led to a decrease in the dislocation density within the crystal and a relatively weak strengthening effect at the grain boundaries.The combination of the weakened solid solution strengthening effect and grain growth finally led to a decrease in hardness.In addition, Mn element not only has solid solution strengthening after solid solution treatment, but also has precipitation strengthening after the aging treatment.Although precipitated phases were also generated in the Ti-5Mn(T6-20h) alloy, the volume fraction of precipitated phases was probably so small that the precipitation strengthening effect was not significant thus leading to a continuous decrease in hardness.With the further extension of the aging time, the fraction of Ti Mn 3 precipitates increased, and the precipitation-strengthening effect became more and more significant.Although the grain size also increased, the precipitation strengthening compensated for the negative effect of grain growth.As can be seen from figure 3, Ti 17 Mn 3 phase fraction increased with the increasing of the aging time and the hardness of the alloy increased with the increasing of the precipitate phase fraction, as shown in figure 6. Ti-5Mn(T6-50h) alloy exhibited a hardness of 408 ± 1.8 HV, much higher than that of cp-Ti, indicating that the addition of Mn element increases the titanium alloy's hardness [24], even harder than that of Ti-6Al-4V [25].

Corrosion behavior
The open circuit potentials of cp-Ti and Ti-5Mn alloys in the simulated body fluid are shown in figure 7(a).The potential of Ti-5Mn alloys and cp-Ti increased rapidly at the beginning, and then stabilized with the extension of elapsed time, which indicates the formation of passivation film on the surface.The open-circuit potential (OCP, E ocp ) after stabilization was in the order from low to high: cp-Ti < Ti-5Mn(T4) < Ti-5Mn(T6-8h) < Ti-5Mn (T6-20h) < Ti-5Mn(T6-30h) < Ti-5Mn(T6-50h), indicating that the increase in the second phase content drove the OCP of Ti-5Mn alloy toward a more positive direction.This suggests that the addition of Mn and the increase of the second phase can reduce the corrosion tendency of the alloy.Figure 7(b) shows the Tafel curves of the alloys, and table 2 lists E corr and i corr obtained from the Tafel curves.Compared with that of cp-Ti, E corr of Ti-5Mn alloy decreased after the solid solution, and E corr kept moving toward the positive direction with the increase of the aging time.
In terms of i corr , i corr of Ti-5Mn alloy at solid solution condition was smaller than that of cp-Ti.i corr of Ti-5Mn increased with the extension of the aging duration.After 50 h aging, i corr of Ti-5Mn was larger than that of cp-Ti, displaying that Ti-5Mn alloy with single β-Ti microstructure has the most corrosion resistance, and the precipitation of Ti 17 Mn 3 reduces the corrosion resistance.Figure 8 illustrates the surface micromorphology of different samples subjected to electrochemical testing in SBF solution.As shown in figure 8(a-f), no discernible corrosion traces were observed on the surface of cp-Ti and Ti-5Mn alloys following electrochemical testing.Superficial scratches and a few white fine particles were observed.These white fine particles may be attributed to the evaporation of SBF solution remaining on the sample surface.The results of the electrochemical tests as well as the results of the scanning electron microscope micrographs of the alloy surfaces after the electrochemical tests indicate that the heat-treated Ti-5Mn alloy has excellent corrosion resistance.
Ti-5Mn(T4) alloy consisted mainly of β-Ti phase, and the alloy had a homogeneous microstructure, thus exhibiting low electrochemical activity and self-corrosion current density.Following the aging treatment, the microstructure of Ti-5Mn alloy was composed of α-Ti and Ti 17 Mn 3 phases.The potential difference between Ti 17 Mn 3 phase and the α-Ti matrix resulted in the formation of a closed loop in the SBF solution, which initiates the occurrence of galvanic coupling corrosion.The extent of galvanic coupling corrosion was further exacerbated with the increase in the number of Ti 17 Mn 3 phases, which may be responsible for the higher selfcorrosion current density of Ti-5Mn(T6) alloy than that of Ti-5Mn (T4) alloy.However, Ti-5Mn (T6) alloy still exhibits good corrosion resistance compared to cp-Ti.In general, the addition of manganese element makes the alloy more corrosion resistant, which is in agreement with other literature [24], due to its contributes to the formation of passivation films [27].

Antibacterial properties
Figure 9 shows the comparison of the antibacterial properties of cp-Ti and Ti-5Mn alloy after different heat treatments against S. aureus.A wide distribution of colonies was observed on cp-Ti and Ti-5Mn(T4).On the contrary, Ti-5Mn(T6) showed a relative decrease in the number of colonies.Figure 9(g) shows the calculated antibacterial rate of Ti-5Mn alloy.Compared with cp-Ti, the antimicrobial rates of Ti-5Mn(T4) and Ti-5Mn (T6-xh, x = 8 h, 20 h, 30 h, 50 h) were 1.5%, 45.9%, 62.1%, 66.5%, and 69%, respectively.The above results showed that the bacteria grew well on the surface of cp-Ti and Ti-5Mn(T4), and there was no significant difference in the number of colonies between them, indicating that Ti-5Mn(T4) alloy does not have antibacterial properties.The antibacterial rate of Ti-5Mn(T6) increased with the increasing of the aging time.
Figure 10 shows the microscopic images bacteria on different samples after antimicrobial testing.From figures 10(a), (b), it can be seen that the number of Staphylococcus aureus on the surfaces of cp-Ti and Ti-5Mn (T4) alloys was extremely high, and there was no significant difference in the bacteria number between two samples, indicating that bacteria grew well on the surface of cp-Ti and Ti-5Mn(T4) alloys.As can be seen from figures 10(c)-(f), the number of bacteria on the surface of Ti-5Mn alloys decreased significantly after the aging treatment.With the prolongation of aging time, the bacteria number became less and less.
Ti-5Mn alloy was single β-Ti at solid solution condition and composed of α-Ti+ Ti 17 Mn 3 after the aging treatment.Ti-5Mn alloy with β-Ti microstructure does not have antibacterial properties, while Ti-5Mn alloy with α-Ti + Ti 17 Mn 3 microstructure has some degree of antibacterial properties.Therefore, the ability of Ti-Mn alloy to inhibit bacteria should be related to the formation of a second phase.
The results of other studies also demonstrate the key role of the precipitated phase in the antimicrobial process.According to previous studies, the size and content of the precipitate phase has an important influence on antibacterial properties.The more the precipitated phase, the larger the surface area in contact with bacteria and the more significant the antibacterial properties [17,28,29].Therefore, the antibacterial ability of Ti-5Mn alloy increased with the increasing of Ti 17 Mn 3 phase.
However, the antimicrobial rate of the aged Ti-5Mn alloy was not as high as that of Ti-Cu and Ti-Ag alloys after the aging treatment.The antibacterial properties of Ti-Cu [28] alloys and Ti-Ag [30] alloys were greatly improved after the aging treatment, even exceeding 95%, which did not depend entirely on the dissolution of copper and silver ions, but was closely related to the fraction of Ti 2 Cu and Ti 2 Ag phases precipitated during the aging process.
It has been shown [20,31] that the potential difference between the titanium alloy and the second phase on the surface may cause the alloy to develop antimicrobial properties.Through the standard electrode potential table [17], the standard electrode potential difference between Ti and Mn is about 0.445 v or so, while the potential difference is about 1.97 V between Ti and Cu, and 2.42 V between Ti and Ag, respectively.This difference results in the formation of a surface micro area potentials difference (MAPD) between the α-Ti matrix and the precipitate, such as Ti 17 Mn 3 in Ti-Mn, Ti 2 Cu in Ti-Cu, and Ti 2 Ag in Ti-Ag alloy.The larger the MAPD, the better the antibacterial properties of the alloy [12,20].The presence of the MAPD causes electron transfer on the alloy surface, which disrupts the intracellular and extracellular ROS homeostasis and interferes with the normal physiological activities of bacteria, eventually leading to the death of some bacteria.This may be the reason why the aged Ti-5Mn alloy has some degree of bacterial inhibition but the antibacterial ability is not as strong as the aged Ti-Cu and Ti-Ag alloys.

Conclusion
Based on the above analysis, the following conclusions can be drawn: (1) Solid solution treated Ti-5Mn alloy was predominantly composed of β-Ti phase, whereas Ti-5Mn(T6) alloy primarily consisted of the α-Ti and Ti 17 Mn 3 phases.Moreover, the precipitation of Ti 17 Mn 3 phase gradually increased with the extension of the aging time.
(2) Solid solution treated Ti-5Mn alloy demonstrated remarkable hardness and corrosion resistance, but did not have antimicrobial properties.In contrast, the aging-treated Ti-5Mn alloy exhibited a notable enhancement in antimicrobial characteristics, with a slight reduction in hardness and corrosion resistance.

3 .
Results and discussion 3.1.Phase structure and microstructure The XRD patterns of Ti-5Mn alloys under different conditions are shown in figure 1. Ti-5Mn(L) alloy showed diffraction peaks of α-Ti phase and β-Ti phase, while Ti-5Mn(T4) alloy had only diffraction peaks of β-Ti phase.

Figure 1 .
Figure 1.XRD patterns of Ti-5Mn alloys before and after heat treatment.

Figure 6 .
Figure 6.The change of microhardness of Ti-5Mn alloys with the aging time.