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Article

Photocatalytic Activity and Antibacterial Properties of Mixed-Phase Oxides on Titanium Implant Alloy Substrates

by
Haden A. Johnson
1,
Darby Donaho
1,
Aya Ali
1,
Amisha Parekh
1,
Randall S. Williamson
1,
Mary E. Marquart
2,
Joel D. Bumgardner
3,
Amol V. Janorkar
1 and
Michael D. Roach
1,*
1
University of Mississippi Medical Center, Department of Biomedical Materials Science, Jackson, MS 39216, USA
2
University of Mississippi Medical Center, Department of Cell and Molecular Biology, School of Medicine, Jackson, MS 39216, USA
3
University of Memphis, Department of Biomedical Engineering, Memphis, TN 38152, USA
*
Author to whom correspondence should be addressed.
Coatings 2024, 14(5), 595; https://doi.org/10.3390/coatings14050595
Submission received: 9 April 2024 / Revised: 25 April 2024 / Accepted: 6 May 2024 / Published: 9 May 2024
(This article belongs to the Special Issue Trends in Coatings and Surface Technology, 2nd Edition)
Browse Figures
Versions Notes

Abstract

:
Titanium alloys are commonly used for implants, but the naturally forming oxides are bioinert and not ideal for bacterial resistance or osseointegration. Anodization processes are a modification technique that can crystallize the oxides, alter oxide surface topography, and introduce beneficial chemistries. Crystalline titanium oxides are known to exhibit photocatalytic activity (PCA) under UVA light. Anodization was used to create mixed-phase oxides on six titanium alloys including commercially pure titanium (CPTi), Ti-6Al-4V (TAV), Ti-6Al-7Nb (TAN), two forms of Ti-15Mo (TiMo-β and TiMo-αβ), and Ti-35Nb-7Zr-5Ta (TNZT). Combined EDS and XPS analyses showed uptake of the electrolyte and substrate alloying elements into the oxides. The relative oxide PCA was measured using methylene blue degradation assays. CPTi and TAN oxides exhibited increased PCA compared to other alloys. Combined XRD and EBSD oxide phase analyses revealed an unfavorable arrangement of anatase and rutile phases near the outermost surfaces, which may have reduced PCA for other oxides. The relative Staphylococcus aureus attachment to each oxide was also assessed. The CPTi and TiMo-αβ oxides showed significantly reduced S. aureus attachment after 1 h of UVA compared to un-anodized CPTi. Cell culture results verified that the UVA irradiation did not negatively influence the MC3T3-E1 attachment or proliferation on the mixed-phase oxides.

1. Introduction

Titanium alloys are commonly used throughout the implant industry due to their desirable properties [1,2,3]. Ti-6Al-4V (TAV) is one of the most commonly used alloys because of the high strength stemming from its duplex microstructure [2]. The duplex microstructure consists of a combination of the hexagonal close-packed alpha-phase (α) and body-centered cubic beta-phase (β) phase microstructures [4]. Due to concerns about vanadium ions being leaked to the body, other duplex alloys, including Ti-6Al-7Nb (TAN) and Ti-15Mo (TiMo αβ), have also recently been introduced [5]. Commercially pure titanium grade 4 (CPTi) exhibits an alpha-phase microstructure and is commonly used for dental or cardiovascular implants due to its higher ductility and formability compared to the duplex alloy counterparts [2]. Although alpha-phase, and duplex alloys are desirable in most circumstances, their higher strength may lead to the stress shielding of bone in certain applications [1,2]. Concerns over potential bone-shielding effects have led to the development of lower-modulus beta-phase alloys such as Ti-15Mo (TiMo β) or Ti-35Nb-7Zr-5Ta (TNZT) [1,6].
One of the key properties that makes titanium a successful implant material is the naturally forming amorphous oxide layer in the presence of oxygen [7,8]. This oxide layer provides excellent corrosion resistance and biocompatibility due to its relative inertness in the body [9,10,11]. However, the naturally forming oxide is bioinert, and provides a less than ideal surface for antibacterial behavior or osseointegration [12]. The surface of titanium can be modified in order to further enhance its ability to interact with cells and promote more optimal bone ingrowth [13]. Anodization processes can crystallize the oxide, alter the oxide surface topography, and introduce beneficial dopant chemistries [14,15,16,17,18]. Previous studies have incorporated phosphorous into the titanium oxide layers and shown increased osteoblast differentiation and better bone-to-implant contact ratios compared to implant surfaces that lacked phosphorous [7,15]. One recent study showed that anatase phase phosphorus-doped anodization oxides could be formed on a wide variety of implant-grade titanium alloy substrates [19].
One of the most unique properties of the anatase and rutile crystalline phases of titanium oxide is their ability to act as a photocatalyst when activated by ultraviolet light of sufficient wavelengths [20,21,22]. Anatase has a bandgap energy of 3.2 eV, and rutile has one that is 3.0 eV [20,23]. Ultraviolet-A (UVA) light possesses the energy necessary to overcome these bandgap energies and causes electron excitation from the valence to the conduction band, resulting in the formation of electron–hole pairs [24,25]. These electron–hole pairs then interact with oxygen and water molecules to form reactive oxygen species (ROS) that are capable of degrading organic dyes and pollutants or killing bacteria [26,27,28,29]. Studies have shown anatase phase titanium oxide to be a better photocatalyst than the rutile phase when comparing singular-phase oxides [30,31]. However, recent studies on mixed-phase titanium oxides have shown a greater photocatalytic effect compared to single-phase oxides when both anatase and rutile phases are activated simultaneously [23,32]. This increased photocatalytic activity is believed to be due to the difference in the bandgap energies of the two phases trapping electron–hole pairs and preventing rapid recombination [33]. Another technique used to increase the photocatalytic activity of titanium oxide has been to use oxide dopants, including transition metals and non-metals [34,35,36,37]. The incorporation of dopant elements can alter the titanium oxide bandgap energy and thus reduce the activation energy needed for the photocatalytic reaction. Some phosphorous-doped titanium oxides have been shown to sufficiently reduce the bandgap energies to enable photocatalytic activation under visible light illumination [34,37].
The primary objectives of this research were to determine the relative photocatalytic activity and the resistance to bacteria attachment of the phosphorus-doped mixed anatase and rutile phase oxides formed on six titanium implant substrate alloys. A secondary objective of the study was to determine if UVA exposure would negatively affect MC3T3-E1 pre-osteoblast cell attachment and proliferation on the oxide surfaces.

2. Materials and Methods

Six titanium implant alloys, including commercially pure titanium grade 4 (CPTi), Ti-6Al-4V (TAV), Ti-6Al-7Nb (TAN), Ti-15Mo duplex alpha + beta phase (TiMo αβ), Ti-15Mo beta phase (TiMo β), and Ti-35Nb-7Zr-5Ta (TNZT), were provided as 12.7 mm diameter bar stock for this study. The nominal compositions for each titanium alloy are compiled in Table 1. Bars were sectioned into 2 mm thick disc specimens using a rotary saw under constant flow of cooling fluid. Following sectioning, disc specimens were ultrasonically cleaned using a laboratory detergent, rinsed with distilled water, and air dried. Immediately prior to anodization, specimens were dipped in a nitric–hydrofluoric acid solution (10:1) for 30 s as an additional cleaning step and to help remove the naturally forming oxide layer.
Anodization was performed in 500 mL of each electrolyte using a DC rectifier (350 V, 10 A, Dynatronix, Amery, WI, USA) and two CPTi counter electrodes. The anodization waveform was applied in potentiostatic 12 V, 10 s steps. The anodization process was initiated at room temperature, but the temperature was allowed to increase throughout the process. Previous anodization trials in our laboratories revealed that three different mixed-acid electrolytes would be needed to produce the mixed-phase oxide layers on the six titanium alloy substrate surfaces. The three anodization electrolytes consisted of mixtures of sulfuric acid (ACS, Fisher Scientific, Waltham, MA, USA), phosphoric acid (ACS, Fisher Scientific, Waltham, MA USA), hydrogen peroxide (30%, Fisher Scientific, Waltham, MA, USA), and oxalic acid (ACS, Alfa Aesar, Haverhill, MA, USA) components. The electrolyte chemistries are compiled in Table 2. The electrolyte and final forming voltage combinations used to form the mixed-phase oxides on each titanium alloy substrate are listed in Table 3.
Thin-film X-ray diffraction (XRD, XDS2000, Scintag, Franklin, MA) was used to determine bulk oxide crystallinity and verify the ratios of anatase and rutile phases within each oxide layer. XRD scans were conducted between two-theta angles of 24° and 30° at a continuous rate of 2°/min. This range was used as it contains the highest intensity diffraction peaks for both anatase and rutile phases at 25.3° and 27.5°, respectively. Peak intensities were extracted from the scans using Jade software (Jade 9, MDI, Livermore, CA, USA). The weight fractions of anatase (XA) and rutile (XR) were then calculated using the following equations: [38,39]
X R = 1 1 + 1.26 I R I A  
X A = 1 − X R  
where IR and IA are the peak intensities of rutile and anatase, respectively [38,39].
Scanning electron microscopy (SEM, Supra 40, Zeiss, Jena, Germany) was used to examine the surface morphology of the anodized oxides. SEM images were acquired using a 8 kV accelerating voltage. Surface porosity was characterized based on multiple (n = 5) images from randomized locations on the oxide surfaces on each titanium alloy substrate. Image analysis software (Vision PE 8.1.690, Clemex, Montreal, QC, Canada) was utilized to examine the surface porosity present on the oxide from each alloy. The total surface area examined for each oxide group was approximately 5000 µm2. Percent surface porosity was calculated as the sum of individual pore areas divided by the total scanned area of each SEM image, and pore density was calculated by dividing the total number of pores from each SEM image by the total scanned area of the image. Welch’s one-way ANOVA (α = 0.05) with a post hoc Games–Howell analyses were utilized to determine significant differences in the total surface pore count, the percent porosity, and the pore density values for the anodized oxides on each titanium alloy substrate. Additionally, size distribution analyses were performed on the anodized layer surface pores found on each titanium alloy substrate. Surface pores found from each oxide were separated into true nanometer pores with diameters less than 100 nm, sub-micron pores with diameters in between 100 nm and 1 µm, and micro-porosity with pore diameters greater than 1 µm groups for comparison purposes.
Atomic force microscopy (AFM, Bioscope Catalyst, Bruker, Santa Barbara, CA) was used to determine the surface roughness of the anodized surfaces on each alloy using 50 μm × 50 μm area scans (n = 5) in ScanAssyst mode (0.25 Hz, 512 samples, line). A first order flattening algorithm was applied to remove tilt from the scans. Ra and Rz were calculated from each flattened scan using Gwyddion software (Version 2.58, Department of Nanometrology, Czech Metrology Institute, Okružní Brno, Czechia). A one-way ANOVA (α = 0.05), followed by a post hoc Tukey analysis, was used to determine statistical differences in Ra and Rz values between anodized oxides on each titanium alloy substrate.
Energy dispersive spectroscopy (EDS, TEAM V4.1.0 Microanalysis System Softwaree Suite, EDAX, Mahwah, NJ, USA) was used at a 15 kV accelerating voltage to determine the bulk oxide chemistry of the anodized surfaces on each alloy. High-resolution X-ray photoelectron spectroscopy (XPS) was also utilized in order to determine the chemical state of the oxide dopants incorporated into the anodized layers for each specimen group. XPS spectra were collected using a K-alpha XPS system (ThermoFisher Scientific Instruments, Waltham, MA, USA) using a monochromatic X-ray source at 1486.6 eV, 12 kV, 6 mA. High-resolution spectra were obtained for Ti 2p, O 1s, P 2p, Al 2p, Nb 3d, Mo 3d, Zr 3d, and Ta 4f at 40 eV pass energy, step size of 0.1 eV and 25 scans. Data analyses were performed using the manufacturer’s software (Advantage 5.9911 Surface Chemical Analysis, ThermoFisher Scientific, Waltham, MA, USA).
Representative specimens from each oxide and substrate alloy combination were also cross-sectioned and mounted in a conductive media (Polyfast, Struers, Cleveland, OH, USA). Cross-sectioned specimens were then polished to a final surface finish of 0.02 μm using a combination of rotary and vibratory polishing methods. SEM was used to measure the oxide thickness values of the polished cross-sectional specimens. A total of 25 oxide thickness measurements were performed for each oxide. A one-way ANOVA (α = 0.05), followed by a post hoc Games–Howell analysis, was used to determine statistical differences in oxide thickness values between anodized oxides on each titanium alloy substrate. EDS (TEAM V4.1.0 Microanalysis System Softwaree Suite, EDAX, Mahwah, NJ, USA) was utilized at a 15 kV accelerating voltage to collect maps of representative cross-sections from each oxide in order to assess the uptake of the alloying and electrolyte chemistries’ anodized layers. Electron backscattered diffraction (EBSD, OIM Analysis 8.0, EDAX, Pleasanton, CA, USA) analyses were also utilized to characterize the spatial distribution and phase fractions of the crystalline oxides present within each oxide cross-section. For this analysis, the polished cross-sectional specimens tilted at 70° were scanned using were using an accelerating voltage of 12 kV and scanned using the manufacture software (APEX Advanced Suite 2.5.1001.0001, EDAX, Pleasanton, CA, USA).
A methylene blue (MB) degradation assay was used to measure the amount of photocatalytic activity generated by the reaction of UVA light with the anodized surfaces [40,41]. Test specimens were placed in a 24-well plate and allowed to soak in 2 mL of 0.001% MB solution (LabChem, Zelienope, PA, USA) overnight. The following morning, MB used for soaking was removed and replaced with 2 mL of fresh MB solution. The specimens were then exposed to an LED lamp (365 nm, 10 W, Chanzon Technology, Shenzhen, China) for 4 h at a working distance of 38 mm, resulting in an UVA intensity of 8 mW/cm2. The 660 nm absorbance of the MB solution was measured at desired timepoints using an ELX800 Universal Microplate Reader (ELX800, BioTek Instruments, Winooski, VT, USA). The photocatalytic activity was calculated as percent degradation using the following equation: [23,41]
P h o t o c a t a l y t i c   a c t i v i t y   % = c 0 − c / c 0 × c 1 / c 0 × 100
where c0is the concentration of the solution before irradiation, c is the concentration of the solution at each timepoint, and c1 is the concentration of the presoak solution [23,41]. Additional details related to the MB protocol used can be found in a previous study [42]. Six anodized specimens of each oxide were tested using the MB degradation protocol. A repeated measures ANOVA (α = 0.05) was used to assess the influence of the oxide on each alloy and the length of UVA exposure on photocatalytic activity for the oxide groups. Post hoc Tukey was used to statistically compare the relative photocatalytic activity at specific timepoints.
Bacterial testing was also performed to compare S. aureus (strain 11–14697 kindly provided by Darlene Miller, Bascom Palmer Eye Institute, Miami, FL, USA) attachment between the oxides on different titanium alloy substrates. A total of 108 colony-forming units per mL (CFU/mL) of logarithmic phase S. aureus were concentrated into a pellet via centrifugation and re-suspended in PBS. Each oxide specimen (n = 6) was placed in one of two 24-well polystyrene cell culture plates and inoculated with 1 mL of the PBS bacterial suspension. One plate, containing half of the test specimens (n = 3 of each oxide), was then exposed to 365 nm UVA irradiation for 1 h using an LED lamp (365 nm, 10 W, Chanzon Technology, Shenzhen, China) aligned at a 38 mm working distance above the cell culture plate. Note this is the same UVA lamp and working distance that was utilized for the photocatalytic activity experiment which resulted in an applied UVA irradiation intensity of 8 mW/cm2. The plate containing the remaining half of the test specimens (n = 3 for each oxide) was covered with foil and kept in the dark for 1 h to serve as controls and a measure of baseline bacterial attachment. Following 1 h of bacterial exposure, test specimens were rinsed with PBS, sonicated for 30 s in fresh PBS, and serially diluted for bacterial CFU counting. Two-sample t-tests were performed between the control and UVA-irradiated oxide groups for each alloy to determine if the photocatalytic reaction significantly reduced bacterial attachment.
MC3T3-E1 mouse pre-osteoblastic cells (Subclone 4; American Type Culture Collection, Manassas, VA, USA) were maintained and expanded at 37 °C and 5% CO2 in alpha-modified Eagle’s minimum essential medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mM sodium pyruvate with pH adjusted to 7.4. Approximately 50,000 cells were seeded on specimens that had been autoclaved and exposed to the 365 nm UVA for 1 h. Empty tissue culture polystyrene (TCPS) wells were used as a control. Cells were supplemented with 1 mL of fresh medium every 48 h.
Cellular morphology on specimen surfaces was assessed on day 7 of culture through Rhodamine–phalloidin (R415, ThermoFisher Scientific, Waltham, MA, USA) and DAPI (ThermoFisher Scientific, Waltham, MA, USA) staining. After rinsing with PBS, the cells were fixed using 4% paraformaldehyde, followed by permeabilization using 0.1% Triton X-100, blocking non-specific binding sites using 1% BSA and finally incubating in stain solutions. Images were captured using an epifluorescence microscope (IX81, Olympus, Hachioji, Tokyo, Japan) with image analysis software (Slidebook 4.2.0.10, Olympus, Center Valley, PA, USA).
The quantity of cells present on multiple specimen surfaces from each oxide (n = 3) was measured using a DNA quantification assay (CyQUANT™, ThermoFisher Scientific, Waltham, MA, USA) after 1 and 7 days of culture, according to the manufacturer’s protocol. After the specified culture period, the cell-seeded specimens were briefly rinsed with PBS, followed by incubation in trypsin–EDTA and neutralization with FBS-supplemented DMEM media. The cell suspension was pelleted and re-suspended in PBS. The cells were then lysed by sonicating for 1 min at 10% amplitude. The fluorescence of DNA bound to DNA-binding Hoechst dye was measured using a fluorescence plate reader (ELX-800, Biotek, Winooski, VT, USA) at an excitation of 480 nm and emission of 520 nm.

3. Results

3.1. Oxide Surface Characterization

Representative XRD scans are shown in Figure 1. Each oxide exhibited a nearly 50:50 ratio of anatase to rutile, except for TiMo β, which was closer to 65% anatase. The crystalline peak positions for TNZT have shifted slightly compared to the other alloys. This shift in the anatase and rutile peak positions was likely due to the large number of alloying elements present in the TNZT substrate causing differences in the oxide lattice spacings.
Representative SEM surface images for each oxide are shown to the left side of Figure 2. Table 4 provides a summary of the surface porosity analyses performed on each oxide. Each oxide revealed a relatively homogeneous distribution of surface pores. The TAV oxide exhibited the highest number of surface pores, followed by a group containing the TiMo αβ and TiMo β oxides, then a group containing the TAN and CPTi oxides, and finally, the TNZT oxide with the least surface pores. The pore-density analysis revealed the same ranking, as the same total surface area was examined for each oxide group. However, the percent porosity analysis revealed substantially different oxide rankings. The TiMo αβ oxide exhibited a significantly higher percent porosity compared to the TAV, TAN, and TNZT oxides. Additionally, the CPTi and TiMo β oxides also revealed significantly higher percent porosities compared to the TAN oxide. A secondary porosity analysis divided the surface pores into bins of nano-sized pores with diameters less than 100 nm, sub-micron pores with diameters between 100 nm and 1 μm, and micro-porosity with pore diameters greater than 1 μm. The CPTi oxide revealed 40.1% nanopores, 59.7% sub-micron pores, and 0.2% micropores. The TAN oxide exhibited 48.4% nanopores and 51.6% sub-micron pores, while the TAV oxide was shown to have 54.0% nanopores and 46.0% sub-micron pores. The TiMo αβ oxide was shown to have 44.1% nanopores and 55.9% sub-micron pores, while its TiMo β oxide counterpart was shown to have 27.3% nanopores with 72.7% sub-micron pores. Finally, the TNZT oxide exhibited 60.8% nanopores, 30.3% sub-micron pores, and 9.8% micropores.
Representative AFM scans for each oxide are shown to the right side of Figure 2. Ra and Rz measurements for each oxide are included alongside the 3-D scans. The average Ra and Rz values of the oxide formed on TNZT were significantly higher than those for all other titanium alloys except TiMo αβ.
EDS bulk surface chemistry results from the anodized alloy surfaces are provided in Table 5. Each titanium substrate alloy incorporated some amount of phosphorous from the phosphoric acid within the electrolyte into its oxide layer. Additionally, each of the major alloying elements within each alloy substrate were revealed in the surface EDS datasets.
Representative high-resolution XPS spectra for each oxide are compiled in Figure 3. A strong doublet for the Ti 2p region at approximately 459 eV and 465 eV was observed for all specimens. This indicates that titanium is present in the Ti 4+ state and binding with oxygen to form the oxide layer [42,43]. The shift in the Ti 2p 459 eV peak from 458.7 eV to 459.2 eV shown in the range of titanium alloys was again attributed to differences in lattice strains for some of the complex alloys. The O 1s peak at approximately 530 eV for all groups corresponds to oxide groups such as titanium oxide. A range from 530.0 and 530.6 eV is also shown for the 530 eV O 1s peak position within the different titanium alloy oxides. The additional O 1s peak around 532 eV indicates the presence of hydroxyl groups and phosphates. A P 2p peak at approximately 134 eV indicates that phosphorus was primarily incorporated into the anodized layers as a phosphate species such as TiPO4 [7,42,43,44]. The position of the 134 eV P 2P peaks was shown to vary between 133.5 eV and 134.0 eV in the different titanium alloy oxides. The Al 2p peaks near 74 eV shown for the TAN and TAV alloy oxides indicate the presence of Al2O3 [5,45,46,47]. The Nb 3d doublet at 207 and 210 eV indicates the presence of Nb2O5 in the TAN and TNZT surfaces [5,45,46,47,48]. The peak intensity is much more pronounced in the TNZT oxide scans due to the higher concentration of Nb in the substrate alloy. The Mo 3d doublet near 233 and 236 eV for both TiMo oxides indicates the presence of MoO3 [49]. The Zr 3d doublet near 133 and 135 eV indicates the presence of ZrO2 in the TNZT oxide; additionally, the Ta 4f doublet near 26 and 28 eV confirms the presence of Ta2O5 [48]. The V 2p scans of the TAV oxide are not presented due to negligible vanadium oxide peaks being detected. Although this result was surprising, it is consistent with the XPS analysis of anodized TAV alloys in a previous study [5]. This result is likely attributed to vanadium’s weakened affinity to oxygen in comparison to titanium and aluminum [50].

3.2. Characterization of Oxide Cross-Sections

Figure 4 compiles representative SEM and EBSD phase images of cross-sectional views of each oxide. The cross-sectional SEM images to the left side of Figure 4 show that the oxide porosity extended throughout the thickness of each oxide layer down to the substrate alloy interface. The cross-sectional EBSD phase images to the right side of Figure 4 provide the spatial distribution of the anatase and rutile phases within the oxide layers to complement the bulk XRD oxide phase distributions shown in Figure 1. A color legend for the alpha titanium, beta titanium, anatase titanium oxide, and rutile titanium oxide phases is provided to the far right of Figure 4. The CPTi and TAN oxide EBSD phase images show a mixture of anatase (green) and rutile (yellow) throughout the oxide. Most importantly, both oxide phases are present on, or near, the outermost surface to increase photocatalytic activity. The TAV EBSD phase image, on the other hand, reveals the anatase phase to primarily be on the outermost surface of the oxide layer and on top of the rutile phase oxide. The TiMo αβ oxide shows a primarily rutile phase oxide with small portions of anatase close to the substrate–metal interface. The TiMo β oxide shows alternating portions of anatase and rutile in the thin oxide layer. Finally, the TNZT oxide is very thick and contains a mostly anatase phase with a smaller rutile phase present near the outmost surface.
Figure 5 compiles the EDS cross-sectional chemistry maps for representative oxides on each titanium alloy substrate. All oxides showed Ti, O, and P within the oxide layers. Additionally, the individual alloys were shown to contain other major alloying elements in the oxide cross-sections. The TAV and TAN oxides were shown to contain Al within the oxide layers as well. The map also shows that, in the TAV oxide, V is present within the oxide layer cross-section. Nb was shown to be present in the oxide layer cross-sections of the TAN and TNZT oxides, though the Nb was much more prominent in the TNZT oxide, which agrees with the EDS surface data in Table 5. The TNZT oxide also showed evidence of Zr and Ta in the oxide cross-section. Finally, Mo was shown to be present in both oxide cross-sections the TiMo αβ and TiMo β oxides. A close examination of Figure 5 also shows that the distributions of P and alloying element uptake into the anodization oxides were relatively uniform throughout the oxide cross-sections.
Figure 6 provides relative the oxide thickness values measured from the cross-sectional SEM images. The TNZT oxides were significantly thicker compared to those on all other alloy substrates. The CPTi and TAN alloy oxides exhibited the next thickest oxide group, followed by the TAV alloy oxides, then the TiMo αβ alloy oxides, and finally the TiMo β oxides that represented the thinnest oxide group.

3.3. Photocatalytic Activity of the Oxides

Photocatalytic activity results determined by MB degradation for the anodized layers on each alloy are shown in Figure 7. A general upward trend in photocatalytic activity was observed for most of the surfaces over the four-hour test period. The least amount of photocatalytic activity was observed for the oxides on the TAV alloy. The CPTi oxide exhibited a significant increase in MB degradation compared to all other alloys beginning after only 60 min of UVA exposure. This trend continued to the 240 min timepoint. The TAN oxide also showed a significantly increased photocatalytic effect compared to the TAV and TNZT oxide surfaces at 240 min.

3.4. Bacteria Attachment to the Oxides

S. aureus attachment results for each oxide surface are shown o the left side of Figure 8. All oxide surfaces exhibited a reduction in attached bacteria after 1 h of UVA exposure, except for those formed on TAN substrates. Two-sample t-tests revealed the reduction in bacteria attachment on the CPTi (p = 0.019) and TiMo αβ (p = 0.001) surfaces to be statistically significant. The TAN oxides actually showed an increase in the number of attached bacteria, although the increase was not shown to be statistically significant. To help illustrate the changes in bacterial attachment that occurred due to UVA exposure, a percentage reduction or efficacy value was calculated comparing the number of bacteria that were attached to foil-wrapped oxides under dark conditions to oxides that were exposed to 1 h of UVA irradiation. This efficacy value was calculated using the following equation [51]:
B a c t e r i a l   a t t a c h m e n t   r e d u c t i o n   e f f i c a c y   % = n 0 − n i n 0 × 100  
where n0 represents the mean bacterial attachment for the foil-wrapped condition, and ni represents the mean bacterial attachment for the UVA-treated condition for the same specimen group [51]. A composite comparison of the bacterial attachment reduction efficacy for each of the anodized alloys is provided on the right side of Figure 8. CPTi and TiMo αβ, which showed significant reductions in bacterial attachment, had reduction efficacies of 88% and 64%, respectively. Although the increased number of bacteria that attached to UVA-irradiated TAN surfaces was not found to be significantly higher, it did represent an overall increase of 74% more bacteria on the surface.

3.5. Pre-Osteoblast Response to the Oxides

DNA quantification assay results on all oxide surfaces and TCPS control surfaces are shown in Figure 9. All oxide surfaces had a similar number of MC3T3-E1 cells present after 1 day of incubation. The number of cells significantly increased on all surfaces between day 1 and day 7, except on the CPTi. Additionally, TAV and TiMo β oxide surfaces observed significantly more pre-osteoblast proliferation than CPTi and TAN at day 7. Although the number of cells attached to the oxide surfaces did increase over the tested timeframe, TCPS control surfaces still maintained a significantly higher number of cells compared to the different titanium alloy substrates. Rhodamine/DAPI staining and imaging was performed to verify DNA assay results and assess cellular morphology. Images of cells on the oxide surfaces after 7 days of incubation are shown in Figure 10. Cells were found to be confluent across and exhibited similar cytoplasmic actin filament spreading on each oxide surface.

4. Discussion

Phosphoric acid is known to retard the anatase-to-rutile phase transition in the oxide when it is present in the anodization electrolyte [14,16,52]. Anodization electrolytes containing phosphoric acid previously developed in our laboratory formed either anatase phase or anatase and rutile mixed-phase oxides on CPTi substrates [14]. In the present study, mixed-phase oxides were produced on five additional titanium alloy substrates. However, the amount of anatase and rutile formed within each oxide varied due to the electrolyte chemistry, the forming voltage applied, and the substrate alloy chemistry. For example, the oxides formed on the TAN and CPTi alloys, used the same electrolyte and final forming voltage, but the TAN alloy oxide was shown to exhibit less anatase compared to the CPTi alloy oxide. This result was attributed to the additional presence of niobium in the TAN substrate alloy. Niobium has been shown to retard the anatase-to-rutile phase transition and likely also affects the amorphous-to-anatase phase transition [53]. The TNZT substrate alloy contains approximately five times as much niobium as the TAN alloy but was still able to form substantial amounts of anatase using a different electrolyte that contained less phosphoric acid and a higher final forming voltage.
Another interesting observation was the shift in the anatase and rutile peak locations in the XRD data in Figure 1 for the TNZT alloy oxide. The XPS dataset also showed substantial peak shifts in the position of the Ti 2p peak for some alloys. The TNZT and TAV alloys exhibited the largest shifts in the Ti 2p peak positions. The XRD and XPS peak shifts were attributed to the large number of alloying elements in some of the Titanium substrate alloys that also incorporated in the oxide layers through the anodization processes.
The XRD dataset in Figure 1 revealed the bulk distributions of a nearly 50:50 ratio of anatase and rutile phases. However, the EBSD phase images of the oxide cross-sections in Figure 4 show that the anatase and rutile phases were often evenly distributed throughout the oxides. As examples, the TAV oxide showed an anatase layer on top of a rutile layer in the oxide; the TiMo β oxide showed alternating blocks of anatase or rutile oxide. However, recent studies have shown that intermixed anatase and rutile phases near the outermost surface of the oxide layers generally produce the highest photocatalytic effects [23,33,42]. The results of this study also showed how the EBSD datasets are complementary to XRD datasets in that they provide valuable additional information on the localized spatial distributions of the oxide phases.
In addition to controlling oxide crystallinity, phosphorous doping of titanium oxide has beneficial effects such as improving osteoblast attachment and proliferation and enhancing the photocatalytic effect. Previous studies with anodized titanium oxides indicate that the incorporation of phosphorous to the oxide may help to elucidate biochemical bonding between bone cells and titanium implant surfaces compared to those that are anodized solely with sulfuric acid electrolytes [7]. Phosphorous atoms alter the titanium oxide lattice structure, which can reduce the amount of energy necessary to overcome anatase and rutile’s bandgaps of 3.2 and 3.0 eV, respectively [54,55]. This in turn results in oxide surfaces that produce more photocatalytic activity and may even be activated by interactions with visible light [55]. A quick comparison of the EDS dataset in Table 5 for the anodization processes on each alloy reveals substantial differences in the phosphorus dopant uptake levels in the oxides. The variations in phosphorus uptake for the anodization processes on each alloy were confirmed by the differences in the XPS P 2p peak intensities shown for the different oxides. Moreover, a comparison with the oxide thickness dataset in Figure 6 shows that increased phosphorus uptake did not directly correlate with increased oxide thickness. Additionally, the EDS map data, as shown in Figure 5, revealed that phosphorus uptake into the oxides was distributed throughout the oxide cross-sections.
In a previous study, we formed phosphorus-doped anatase phase oxides on the same six titanium alloy substrates [19]. The TAN and TNZT Nb-containing anatase phase oxides in that study revealed inhomogeneous distributions of surface porosity [19]. In contrast, each of the mixed-phase oxides in the present study exhibited a relatively homogenous distribution of surface porosity. It was also interesting to note that each mixed-phase oxide except for TNZT showed a higher pore-density value compared to its anatase phase oxide counterpart [19]. This finding indicated that most of the mixed-phase oxides revealed a higher number of surface pores per unit area than the comparative anatase phase oxides. However, the percent porosity values noting the surface pore areas versus the total scanned area revealed similar orders of magnitude between the anatase and mixed-phase oxides [19]. This combination most likely indicates that a higher number of smaller surface pores were shown on the mixed-phase oxides in this study compared to the anatase phase oxides in the previous study.
Based on the SEM images, the oxide thicknesses of the alloys generally increased with the final forming voltage that was used to create the oxide. This corroborates well with our previous studies using similar anodization techniques [14]. Surprisingly, most of the oxides produced on each substrate alloy were shown to exhibit similar surface roughness values. Some of the largest Ra and Rz values were measured for the oxides formed on TNZT. This was likely due to the large variation in pore sizes formed on the TNZT alloy surfaces.
MB degradation has often been used as a screening technique to determine the relative photocatalytic activity of titanium oxides. Previous studies have shown that anatase phase titanium oxide exhibits upwards of 80% MB degradation after extended exposure to UVA light [56,57]. Other studies have shown that combination oxides containing both anatase and rutile crystalline phases become even more photocatalytic due to the synergistic effect the two phases have on the photocatalytic reaction [23,30,32,42]. Chemical dopants have also been shown to affect the photocatalytic activity of titanium oxides [53,58,59,60]. The oxide formed on CPTi degraded MB at an accelerated rate compared to the oxides formed on the other substrate alloys. This result corroborated well with the XRD results, which showed that the anatase and rutile peak intensities formed on CPTi were generally higher than those formed on the other substrate alloys. One exception to this trend was the oxide formed on TNZT. Even though substantial oxide growth and an increase in both anatase and rutile XRD peak intensities was observed, the oxide on TNZT greatly underperformed compared to most of the oxides on the other alloys when evaluating MB degradation. The large number of alloying elements in TNZT are likely causing shifts in the anatase and rutile phase unit cell structures resulting in reduced photocatalytic activation when the surfaces are exposed to UVA light. Additionally, the limited amounts of rutile phase oxide present near the outermost oxide surface, as shown in the cross-section EBSD dataset in Figure 4, may also be contributing to the reductions shown in photocatalytic activity.
Promising MB degradation results were also observed by the oxides formed on TAN. The oxide on TAN exhibited significantly more MB degradation than the oxides on both TAV and TNZT. This was an interesting result since TAN had lower anatase and rutile XRD peak intensities than TNZT, but a better mixture of the two oxide phases near the TAN oxide outermost surface, as shown in the cross-sectional EBSD dataset. The oxide formed on TAV exhibited the least amount of MB degradation. The XRD peak intensities for both crystalline phases are the lowest for TAV, indicating that the anodization technique used for this substrate alloy in the present study may require further optimization. Furthermore, the cross-sectional EBSD data for TAV showed anatase phase oxide formed on top of the rutile phase instead of forming a blended mixture near the outermost surface. Nonetheless, the TAV photocatalytic activity results were in direct contrast to the results of another study that showed TAV anodized in orthophosphoric acid to form anatase phase oxides that significantly degraded MB compared to similar anatase phase oxides formed on commercially pure titanium—grade 2 [61].
Based on photocatalytic activity results from MB degradation testing, CPTi and TAN oxides were predicted to perform the best against bacteria. Oxides on CPTi and TiMo αβ substrate alloys did exhibit a significant reduction in S. aureus attachment after 60 min of UVA exposure. However, TAN actually showed an increase in bacterial attachment after UVA exposure, but the increase was not determined to be statistically significant. S. aureus was the focus of the bacterial testing in this study due to its role as an early colonizer in the formation of biofilms and its prevalence in implant-related infections [62,63]. Specifically, Staphylococci species are a cause of up to four out of every five orthopedic implant infections [62]. Additionally, S. aureus possess several cell-surface adhesion molecules that help to facilitate attachment to both bone matrix and implant surfaces [62]. Further testing against other bacterial strains that are typically associated with the formation of biofilms may yield different results.
The main goal of the cell culture experiments with MC3T3-E1 pre-osteoblasts was to ensure that exposing the anodized surfaces with UVA for 1 h prior to seeding with cells would not negatively affect their ability to attach to, and proliferate, across the surfaces. The UVA exposure was not anticipated to harm the osteoblasts due to the rapid recombination of the electron–hole pairs [64]. From the results of the DNA assay, it was observed that a similar number of cells initially attached to each of the alloys. After 7 days, all surfaces, besides CPTi, saw a significant increase in the number of cells present. However, CPTi was still statistically similar to TAN. At any rate, the cells were confluent across each of the alloys, as evidenced by the Rhodamine and DAPI images.

5. Conclusions

Anodization electrolyte chemistry and final forming voltage were varied to form optimized oxides on a range of titanium implant alloys. All substrate alloys formed nano- and microporous phosphorous-doped oxide layers. The most photocatalytic oxides were determined to be those formed on CPTi and TAN. A significant reduction in S. aureus attachment was shown for the oxides on CPTi and TiMo αβ. Oxides on all substrate alloys except TAN reduced the number of bacteria attached after 365 nm UVA irradiation. The results of the short-term cell culture experiment did not reveal any negative impact of UVA irradiation pretreatments on osteoblast attachment and proliferation on the oxides. Although photocatalytic activity was observed in both MB and bacteria testing, the results of the two experiments did not directly correlate with one another. These initial findings are encouraging and warrant further research to determine if these oxides may be more effective against other strains of bacteria that are associated with implant-related infections.

Author Contributions

Conceptualization, H.A.J. and M.D.R.; methodology, H.A.J. and M.D.R.; investigation, H.A.J.; data collection, H.A.J., D.D., A.A. and A.P.; data analysis, H.A.J.; writing—original draft preparation, H.A.J.; writing—review and editing, A.V.J., M.E.M., R.S.W., J.D.B. and M.D.R.; supervision, M.D.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors would like to acknowledge Fort Wayne Metals for donating the CPTi and TAV alloys, Depuy Synthes for donating the TiMo αβ and TAN alloys, and ATI Specialty Materials for donating the TiMo β and TNZT alloys for the study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Coatings 14 00595 g001
Figure 1. XRD results from representative scans of the oxides on each titanium alloy substrate. The analysis of each oxide revealed both anatase and rutile crystalline titanium oxide phases to be present. Peak intensities are provided in units of counts per second (cps).
Figure 1. XRD results from representative scans of the oxides on each titanium alloy substrate. The analysis of each oxide revealed both anatase and rutile crystalline titanium oxide phases to be present. Peak intensities are provided in units of counts per second (cps).
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Figure 2. (Left side)—Representative surface SEM images showing the oxide surface topography and porosity for the oxides formed on each titanium alloy substrate. (Right side)—Representative 50 µm × 50 µm 3D AFM scans for the oxides on each titanium alloy substrate. Ra values are represented as mean ± SD nm, and Rz values are represented as mean ± SD µm. The superscript letters provided with the Ra and Rz values give the statistical groupings from the ANOVA analysis of the surface roughness parameters. The roughness values for oxide groups showing the same superscript letters were not significantly different.
Figure 2. (Left side)—Representative surface SEM images showing the oxide surface topography and porosity for the oxides formed on each titanium alloy substrate. (Right side)—Representative 50 µm × 50 µm 3D AFM scans for the oxides on each titanium alloy substrate. Ra values are represented as mean ± SD nm, and Rz values are represented as mean ± SD µm. The superscript letters provided with the Ra and Rz values give the statistical groupings from the ANOVA analysis of the surface roughness parameters. The roughness values for oxide groups showing the same superscript letters were not significantly different.
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Figure 3. High-resolution XPS scans for Ti 2p, O 1s, and P 2p for each oxide surface. The peak positions for Ti 2p, O 1s, and P 2p in each alloy were shown to shift due to lattice distortions attributed to the complexity of the alloy microstructures. High-resolution regions are also included for the Al, Zr, and Ta alloying elements found in some of the respective titanium alloy substrates.
Figure 3. High-resolution XPS scans for Ti 2p, O 1s, and P 2p for each oxide surface. The peak positions for Ti 2p, O 1s, and P 2p in each alloy were shown to shift due to lattice distortions attributed to the complexity of the alloy microstructures. High-resolution regions are also included for the Al, Zr, and Ta alloying elements found in some of the respective titanium alloy substrates.
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Figure 4. (Left side)—Cross-sectional SEM images showing the representative porosity distributions for the oxides formed on each titanium alloy substrate. (Right side)—Representative EBSD phase images for the oxide cross-sections showing the spatial distributions of anatase and rutile phases within the anodized layers. A color-coded legend for the phases is provided to the far right.
Figure 4. (Left side)—Cross-sectional SEM images showing the representative porosity distributions for the oxides formed on each titanium alloy substrate. (Right side)—Representative EBSD phase images for the oxide cross-sections showing the spatial distributions of anatase and rutile phases within the anodized layers. A color-coded legend for the phases is provided to the far right.
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Figure 5. Cross-sectional EDS maps of mixed-phase oxides on each titanium alloy substrate. The far left image contains the SEM image of the selected region. To the right of the oxide SEM image, individual element maps are shown for Ti, O, P, Al, V, Nb, Mo, Zr, and Ta. Each oxide exhibited P uptake from the anodization electrolyte and some uptake of the elements in the respective titanium alloy substrate.
Figure 5. Cross-sectional EDS maps of mixed-phase oxides on each titanium alloy substrate. The far left image contains the SEM image of the selected region. To the right of the oxide SEM image, individual element maps are shown for Ti, O, P, Al, V, Nb, Mo, Zr, and Ta. Each oxide exhibited P uptake from the anodization electrolyte and some uptake of the elements in the respective titanium alloy substrate.
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Figure 6. Cross-sectional oxide thickness measurements (n = 25) are shown for each oxide. Data are represented as mean ± SD µm. Oxides showing the same letter grouping did not exhibit significantly different anodized layer thickness values.
Figure 6. Cross-sectional oxide thickness measurements (n = 25) are shown for each oxide. Data are represented as mean ± SD µm. Oxides showing the same letter grouping did not exhibit significantly different anodized layer thickness values.
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Figure 7. (Top)—The relative oxide photocatalytic activity versus time is shown for the oxides formed on each titanium alloy substrate. (Bottom left)—The relative photocatalytic activity for each oxide is shown at the 60 min timepoint. (Bottom right)—The relative photocatalytic activity for each oxide is shown at the 240 min timepoint. Data are represented as mean ± SD% photocatalytic activity. The relative photocatalytic activities of oxides showing the same letter grouping were not significantly different.
Figure 7. (Top)—The relative oxide photocatalytic activity versus time is shown for the oxides formed on each titanium alloy substrate. (Bottom left)—The relative photocatalytic activity for each oxide is shown at the 60 min timepoint. (Bottom right)—The relative photocatalytic activity for each oxide is shown at the 240 min timepoint. Data are represented as mean ± SD% photocatalytic activity. The relative photocatalytic activities of oxides showing the same letter grouping were not significantly different.
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Figure 8. (Left side)—The relative oxide S. aureus attachment results for each oxide after 1 h under dark (n = 3) and UVA-irradiated (n = 3) conditions are provided. Two sample t-tests (α = 0.05) were performed on the bacterial attachment results between the dark and UVA-irradiated conditions for each oxide. Statistical p-values are shown for the t-tests for oxides that exhibited significant reductions in bacterial attachment between the dark and UVA-irradiated conditions. (Right side)—the relative percentage of bacterial attachment reduction or bacteria reduction efficacy between the UVA-irradiated and dark conditions is shown for each oxide.
Figure 8. (Left side)—The relative oxide S. aureus attachment results for each oxide after 1 h under dark (n = 3) and UVA-irradiated (n = 3) conditions are provided. Two sample t-tests (α = 0.05) were performed on the bacterial attachment results between the dark and UVA-irradiated conditions for each oxide. Statistical p-values are shown for the t-tests for oxides that exhibited significant reductions in bacterial attachment between the dark and UVA-irradiated conditions. (Right side)—the relative percentage of bacterial attachment reduction or bacteria reduction efficacy between the UVA-irradiated and dark conditions is shown for each oxide.
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Figure 9. The relative DNA assay quantitative results for each oxide (n = 3) and TCPS plate control surfaces after 1 and 7 days of incubation. DNA data are represented as mean ± SD ng/mL. Titanium alloy oxides showing the same letter indicate that no statistically significant differences were shown in DNA values.
Figure 9. The relative DNA assay quantitative results for each oxide (n = 3) and TCPS plate control surfaces after 1 and 7 days of incubation. DNA data are represented as mean ± SD ng/mL. Titanium alloy oxides showing the same letter indicate that no statistically significant differences were shown in DNA values.
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Figure 10. Representative rhodamine/DAPI images of cells after 7 days of incubation on: (A) CPTi; (B) TAV; (C) TAN; (D) TiMo αβ; (E) TiMo β; and (F) TNZT oxide surfaces. The rhodamine/DAPI evaluation revealed confluency of the cells and distinct uniform cytoplasmic actin filament spreading with elongated cellular morphology on day 7 of culture for all oxide groups.
Figure 10. Representative rhodamine/DAPI images of cells after 7 days of incubation on: (A) CPTi; (B) TAV; (C) TAN; (D) TiMo αβ; (E) TiMo β; and (F) TNZT oxide surfaces. The rhodamine/DAPI evaluation revealed confluency of the cells and distinct uniform cytoplasmic actin filament spreading with elongated cellular morphology on day 7 of culture for all oxide groups.
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Table 1. Nominal composition of titanium substrate alloys.
Table 1. Nominal composition of titanium substrate alloys.
Titanium AlloyElemental Composition (wt %)
Ti 1FeOAlVNbMoZrTa
CPTibal.0.030.26------
TAVbal.0.150.095.904.12----
TANbal.0.140.135.88-6.56---
TiMo βbal.0.030.09---14.64--
TiMo αβbal.0.030.09---15.16--
TNZTbal.0.050.19--34.03-7.535.97
1 Balance with alloying elements shown.
Table 2. Mixed-acid anodization electrolyte chemistries.
Table 2. Mixed-acid anodization electrolyte chemistries.
ElectrolyteSulfuric Acid
(M)		Phosphoric Acid
(M)		Hydrogen Peroxide
(M)		Oxalic Acid
(M)
A3.50.09380.750.25
B3.50.04690.750.25
C1.40.030.75-
Table 3. Anodization processes to form mixed-phase oxides.
Table 3. Anodization processes to form mixed-phase oxides.
AlloyElectrolyteFinal Forming Voltage (V)
CPTiC180
TAVB108
TANC180
TiMo αβA108
TiMo βA108
TNZTC228
Table 4. Surface porosity analysis of the oxides.
Table 4. Surface porosity analysis of the oxides.
Titanium
Alloy		Total Pore
Count 1		Pore
Density 1		Percent
Porosity 1
(%)		Pore Size Distribution (%)
<100 nm100 nm–1 µm>1 µm 2
CPTi2354 ± 232 C2.4 ± 0.2 C17.6 ± 3.9 AB40.1 ± 5.259.7 ± 5.30.2 ± 0.2
TAN2349 ± 276 C2.4 ± 0.3 C9.0 ± 1.5 C48.4 ± 2.651.6 ± 2.5--
TAV7010 ± 893 A7.2 ± 0.9 A9.8 ± 3.1 BC54.0 ± 3.046.0 ± 3.0--
TiMo αβ3805 ± 525 B3.9 ± 0.5 B18.1 ± 2.3 A44.1 ± 2.755.9 ± 2.7--
TiMo β3655 ± 131 B3.7 ± 0.1 B17.2 ± 3.8 AB27.3 ± 1.772.7 ± 1.7--
TNZT599 ± 42 D0.6 ± 0.1 D10.7 ± 1.1 BC60.8 ± 3.230.3 ± 3.28.9 ± 0.6
1 Oxides with the same letter superscripts were not statistically different. 2 Values for micro-porosity less than 0.1% are not shown.
Table 5. Bulk surface EDS results from the oxide surfaces on each titanium alloy.
Table 5. Bulk surface EDS results from the oxide surfaces on each titanium alloy.
Element
Composition (wt %)		Titanium Alloy
CPTiTAVTANTiMo αβTiMo βTNZT
Ti56.4 ± 2.058.6 ± 0.649.8 ± 0.855.0 ± 0.555.9 ± 2.034.6 ± 1.4
O41.8 ± 2.038.2 ± 0.542.4 ± 0.941.4 ± 0.539.4 ± 1.636.9 ± 1.6
P1.7 ± 0.10.4 ± 0.01.6 ± 0.11.0 ± 0.21.3 ± 0.40.5 ± 0.1
Al--1.9 ± 0.12.7 ± 0.1------
V--1.0 ± 0.1--------
Nb----3.3 ± 0.1----20.1 ± 0.5
Mo------2.4 ± 0.23.3 ± 1.9--
Zr----------5.2 ± 0.1
Ta----------2.4 ± 0.1
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Johnson, H.A.; Donaho, D.; Ali, A.; Parekh, A.; Williamson, R.S.; Marquart, M.E.; Bumgardner, J.D.; Janorkar, A.V.; Roach, M.D. Photocatalytic Activity and Antibacterial Properties of Mixed-Phase Oxides on Titanium Implant Alloy Substrates. Coatings 2024, 14, 595. https://doi.org/10.3390/coatings14050595

AMA Style

Johnson HA, Donaho D, Ali A, Parekh A, Williamson RS, Marquart ME, Bumgardner JD, Janorkar AV, Roach MD. Photocatalytic Activity and Antibacterial Properties of Mixed-Phase Oxides on Titanium Implant Alloy Substrates. Coatings. 2024; 14(5):595. https://doi.org/10.3390/coatings14050595

Chicago/Turabian Style

Johnson, Haden A., Darby Donaho, Aya Ali, Amisha Parekh, Randall S. Williamson, Mary E. Marquart, Joel D. Bumgardner, Amol V. Janorkar, and Michael D. Roach. 2024. "Photocatalytic Activity and Antibacterial Properties of Mixed-Phase Oxides on Titanium Implant Alloy Substrates" Coatings 14, no. 5: 595. https://doi.org/10.3390/coatings14050595

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