Photochemical Surface Modification of Titanium Dioxide Nanotube-Coated Surfaces by Ag-Hydroxyapatite Compositions

Silver-hydroxyapatite coatings prepared from Ag3PO4 microcrystals have been deposited on titanium dioxide nanotubes supported by titanium disks by photodecomposition of predeposited Ag3PO4 microcrystals or their coprecipitate with hydroxyapatite. *e SEM-EDS characterization has confirmed excellent film uniformity and consistent deposition over the surface, which is essential for improving osseointegration of tunable antibacterial bone implants.


Introduction
As invaluable medical devices, titanium (Ti)-based orthopedic and dental implants have become standard as longterm bone replacements capable of supporting biogenic hydroxyapatite (HAp) structures [1,2].Despite their widespread application, early and late implant failures still occur as a result of (i) incomplete osseointegration, (ii) foreign body rejection, or (iii) the formation of pathogenic biofilms on the implant surface [3].Ti and Ti alloys, commonly used in orthopedic and dental implants [4][5][6], are highly corrosion-resistant materials with impressive strength and a low density compared to stainless steels [7][8][9] A compact layer of naturally forming titanium dioxide (TiO 2 ), present on the titanium surface, has shown to enhance chemical bonding of growing bone tissue to the implant [2].A significant research effort is aimed at enhancing bone growth to the Ti surface by tailoring surface roughness and chemical composition [10][11][12], including coating with HAp [13,14] and other calcium phosphate ceramics [15].HAp is a nanocomposite material with a mineral component similar in composition and structure to human bone tissue and has shown to have significant influence over cellular function when applied with specific nanotextures [16][17][18][19][20][21][22].e idea of tailoring nanostructure of a material to influence the overall surface properties has gained major attraction [23,24].For instance, modification of a Ti surface with highly ordered TiO 2 nanotubes (NTs), fabricated via electrochemical anodization, has proven to be an effective method for influencing biological response mechanisms [25][26][27].
To this end, the literature is pervaded with coating methods designed to resolve a combination of the implant failure modes listed above by developing multifunctional implant modification procedures [2,[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42].While it is known that modifying the topography of the implant surface on the nanoscale has shown to be effective in terms of increasing osteogenic phenomena and lessening rejection [43], these coatings do not effectively prevent bacterial colonization.To combat unwanted bacterial growth, imbuing these coatings with antibacterial agents and hierarchical nanostructures, the materials' antibacterial properties are expected to increase dramatically.
Given that implant surfaces are at risk for infection throughout their lifetime, combating implant related infections via controlled, long-term delivery of an antibacterial agent to the local implant environment is critical.
ese preferred properties are often related to the structure and application technique of the antibacterial agent itself.Silver (Ag) is a known antibacterial agent usually deposited on implants by photodecomposition of silver nitrate (AgNO 3 ) or deposition of preprepared Ag nanoparticles (NPs) [16,[44][45][46].However, the resultant Ag coverage is often either nonuniform, likely associated with the low affinity of Ag to TiO 2 , or requires the presence of additional agents (e.g., polyols) [47].In addition, there is little control over the dynamics of Ag release from its deposits exposed to the implant-bone interface [16].erefore, a simple process is needed to evenly distribute Ag onto these nanostructured Ti surfaces while also offering a stable, long-term Ag release profile.Furthermore, studies have shown that a certain concentration of Ag + can have bactericidal capabilities while retaining cytocompatibility, which underscores the importance of controlled (both the quantity and location of Ag on the implant) deposition of Ag [16,48].
Here, we report deposition of silver-hydroxyapatite coatings on titanium dioxide nanotubes supported by titanium disks by phododecomposition of predeposited Ag 3 PO 4 microcrystals or their coprecipitate with hydroxyapatite.First, a Ag 3 PO 4 -containing layer was introduced to the surface driven by the known affinity of Ag 3 PO 4 to TiO 2 [54,55].Next, a uniform Ag coating on the implant's surface was produced by photodecomposition of Ag 3 PO 4 particles which lead to the complete disappearance of the particulate.e capability of HAp to dissolve at high acidity levels creates a perfect opportunity for a controlled release of the antimicrobial silver when it is most needed, while the spatially controlled photodecomposition opens the door for depositing the patterns of antibacterial silver designed for a specific type of the bone implant.
2.2.Nanostructured Ti Disks.Titanium dioxide nanotubes (TiO 2 NTs) were fabricated from titanium (Ti) substrates using a two-electrode electrolytic cell, comprised of Ti anode and platinum cathode.Ti disks with a thickness of 2.0 mm were cut from a 12.7 mm diameter Ti rod, then ground using progressively finer (i.e., 400, 600, 800, and 1200 grit) silicon carbide grinding paper, polished with 1.0 μm alumina powder and finished with a colloidal silica and hydrogen peroxide polishing suspension.Before anodization, disks were consecutively sonicated in deionized water and methanol for 5 mins each.TiO 2 NTs were fabricated using two different electrolytes, denoted as electrolyte 1 and electrolyte 2. Electrolyte 1 consisted of dissolved 0.2 M citric acid, 1 M H 2 SO 4 (sulfuric acid 98.0 w/w.%), and 0.1 M NaF (sodium fluoride ≥ 99%) in continuously mixed deionized water.Electrolyte pH was adjusted to pH 3.5 through addition of anhydrous NaOH (sodium hydroxide) and measured using a pH meter.Electrolyte 2 was made by dissolving 0.3 wt.% NH 4 F (ammonium fluoride ≥99%) and 2 vol.%H 2 O in anhydrous ethylene glycol (EG).Electrolyte 1 was used to fabricate those specimens shown in Figures 1 and 2. All other results were generated using NTs fabricated using Electrolyte 2. It should be noted that the results of this study are not affected by the different electrolytes used and the purpose of this study was not to evaluate the influence of electrolyte composition on Ag deposition.Anodization was carried out using a constant applied potential of 20 V applied using a DC power supply with constant current recording carried out via using KI-Tool software (Tektronix, Beaverton, OR) and multimeter (Keithley 2100 Series: 6(1/2)-Digit USB Multimeter, Tektronix, Beaverton, OR).Anodization continued for 1 h with continuous electrolyte circulation.Following anodization, samples were sonicated in methanol for 3 mins and subsequently annealed for 1 h at 450 °C.Another experiment was performed, where precipitation of Ag 3 PO 4 onto the TiO 2 NT disks surface was achieved using known procedure [55].Following the placement of an untreated TiO 2 NT disk into a beaker filled with 50 mL of water, 2.9 mL of 0.5 M AgNO 3 was added. is solution was slowly agitated at r.t. on a flat-head vortex mixer for 15 mins.Next, 50 μL of 0.1 M Na 2 HPO 4 was added.e NT disk and the supernatant solution were agitated for 30 mins.e NT disk was washed with nanopure water for 1 min and kept at 70 °C overnight.e coated disk was characterized using SEM and EDS and then exposed to UV irradiation for 15 mins with a Xe lamp.UV-treated Ag 3 PO 4 NT disks were characterized using SEM and EDS.

Sonosynthetic Precipitation of HAp onto a TiO 2 NT
Disk.On another untreated TiO 2 NT disks, HAp was precipitated according to a modified known procedure [56].Briefly, a TiO 2 NT disk placed into 25 mL of a 0.1 M calcium precursor solution (4.103 g Ca(NO 3 ) 2 , 31.25 mL 29% NH 4 OH in 250 mL of solution) was ultrasonicated on high power at 25 °C.During ultrasonication, 25 mL of 0.06 M phosphate precursor solution (1.725 g NH 4 H 2 PO 4 , 15.63 mL 29% NH 4 OH in 250 mL of solution) was added at an approximate rate of 13 mL/min.e suspension was ultrasonicated for 1 min after the addition and left to age at r.t.undisturbed for 6 h.Next, the supernatant solution was decanted, and the residue was washed with 25 mL of water.
e TiO 2 NT disk was removed from the beaker, rinsed, washed in water for 15 min, dried at 70 °C overnight.A sample of the residue was added on a TEM grid for SEM, EDS, and TEM characterization.

Coprecipitation of Ag 3 PO 4 and HAp onto a TiO 2 NT Disk.
is procedure was performed in a similar manner to the experimental procedures in Section 2.4.2, except for a further step by adding 1.7 g of AgNO 3 to the calcium precursor solution (1.651 g Ca(NO 3 ) 2 , 1.7 g AgNO 3 , 12.5 mL 29% NH 4 OH in 100 mL of solution).e rest of the experiment was performed the same way.e remaining slurry in the beaker was collected and centrifuged for 8 mins at 3500 rpm.e remaining precipitate after decanting of the supernatant solution was dried in the oven overnight at 70 °C, powdered in a mortar, and characterized as specified in Section 2.4.2.

Ag Deposition on TiO 2 NT Disk by Photolysis of AgNO 3 .
In a control experiment, Ag was deposited by the known procedure of AgNO 3 photolysis [55], and produced a rather patchy (white area) deposition of the TiO 2 NT disk (Figure 3(a)).e EDS results, provided in Table 1, show the presence of Ag element at 1.1 wt.%.

Ag 3 PO 4 Particle Deposition and Subsequent Photolysis.
e Ag 3 PO 4 nano-and microcrystals grown on a TiO 2 NT disk (Figure 3(b)) were retained by the TiO 2 NT surface even after rinsing with water due to the expected affinity of PO 4 3− to the TiO 2 surface.As illustrated in Figure 3(b), the procedure led to a wide particle size distribution.EDS results provided in Table 1, show the presence of Ag (2.2 wt.%) and P (0.7 wt.%).e oxygen content observed is higher than that for the usual untreated TiO 2 NTdisks due to the oxygen of the Ag 3 PO 4 microcrystals.
According to the SEM images provided in Figures 3(c) and 3(d), exposure of the treated surface to UV radiation led to complete breakdown of the Ag 3 PO 4 particles.e EDS results provided in Table 1 illustrates the presence of 0.6 wt.% of Ag and 0.2% of P on the NT disk surface.e ratio of 3 : 1 Ag to P is maintained similar to the preirradiation ratio.
is observation suggests a complete photodecomposition of the Ag 3 PO 4 particles to a subnanometer layer of Ag.
e elemental map presented in Figures 4(c) and 4(d) supports the observation, where Ag was evenly distributed along the surface, as was P after the irradiation of Ag 3 PO 4 microcrystals.1(a) and 1(b)) demonstrate the low affinity of the TiO 2 NT disk toward HAp after its immersion in the HAp aqueous suspension followed by rinsing with water in a control experiment.

Sonosynthetic Precipitation of HAp onto a TiO 2 NT
Disk.
e phosphate ions produced after the reaction (Formula (1)) may provide an opportunity to further deposit HAp on a Ag-coated implant: Formula ( 1) is the expected reaction of Ag 3 PO 4 photodecomposition.e h and ] represent Planck constant and the photon energy frequency, respectively.
Due to a lower surface concentration of Ag and sensitivity of the HAp-based coatings to external stimuli, coprecipitation of HAp with Ag + would provide more control over the release of Ag from the modified implant surface [57].HAp crystals start to dissolve at pH less than 6, which would induce enhanced release of Ag in response to a bacterial infection, which is known to create an acidic microenvironment (pH ≤ 5) [58].

Coprecipitation of Ag 3 PO 4 and HAp onto a TiO 2 NT
Disk.To improve the deposition of HAp, HAp crystals were grown on the TiO 2 NT disks using ultrasonication, both with and without the addition of AgNO 3 (which produces Ag 3 PO 4 resulted from mixing aqueous solutions of AgNO 3 , Ca(NO 3 ) 2 and NH 4 H 2 PO 4 ).Growing HAp particles on the TiO 2 NTdisk using ultrasonication led to the development of a porous array of rod-like HAp crystals (Figures 2(a  anodization process.Figure 5(b) represents an EDS line scan of the same region as in Figure 5(a) that also quantitatively characterizes the element abundance on the surface.Table 2 represents the elemental weight percentage of surface materials obtained using the layered mapping and line scan EDS. is provides another evidence of evenly distributed Ag (0.3% on both) and HAp elements (Ca of 1.9% and 2.1%; P of 1.3% and 1.4%) using the sonosynthetic method.
To access the effect of the TiO 2 NT disk on the coprecipitation of Ag 3 PO 4 and HAp, the precipitates formed in the presence of the TiO 2 NT disk were isolated and characterized.
e supernatant aqueous suspension was drop cast on a TEM grid for EDS analysis to investigate the distribution of Ag in the particulate (Figure 6(a)).e elemental maps of Ag and Ca (Figures 6(b)-6(e)) show the particles, where both elements can be found simultaneously, which suggests either an incorporation of Ag to the HAp phase, or a fine mixture of calcium and silver phosphates.

TEM Analysis of Ag-Free and Ag-Doped HAp.
Figure 7 shows TEM images of the supernatant material obtained through coprecipitation of Ag 3 PO 4 and HAp in the presence of TiO 2 NT disks.e rod-like (HAp) and spherical (Ag 3 PO 4 ) morphology of the nanocrystals are characteristic of HAp and Ag 3 PO 4 .In fact, neither the morphology nor the size of the HAp crystals was affected by the Ag 3 PO 4 coprecipitation technique.is implies that the physical and chemical properties of HAp are unlikely to be affected by their coprecipitation and, therefore, should be retained for further applications.

Conclusions
A highly uniform distribution of Ag over the surface of a TiO 2 NT disk was achieved by the deposition of Ag 3 PO 4 crystals followed by photolysis.Coprecipitation of Ag 3 PO 4 and HAp on a TiO 2 NT disk surface leads to an even distribution of Ag over the surface as well.e availability of evenly distributed Ag and HAp codeposits creates an opportunity for the spatially controlled deposition and environment-controlled release of antibacterial Ag where it is most needed for bacterial infection while maintaining good adhesion of HAp to the TiO 2 surface for potential osteoblast adhesion and bone regeneration.

2. 4 . 1 .
Dip-Coating of HAp onto a TiO 2 NT Disk.An untreated TiO 2 NT disk was immersed in an aqueous 15 mL suspension of 3.31 * 10 −2 mM HAp for 1 hr, rinsed, washed in water for 15 mins, and dried at 70 °C overnight.e sample was characterized with SEM and EDS.

Figure 1 :
Figure 1: SEM micrographs of TiO 2 NT disks mixed with HAp solution and rinsed in water at (a) low and (b) high magnification.

Figure 2 :
Figure 2: e NT disk surface following immersion in a suspension of HAp. e NT disk surface following precipitation of (a, b) HAp and (c, d) Ag-doped HAp layer at two respective magnifications.

Figure 3 :
Figure 3: SEM micrographs of TiO 2 NTdisks (a) treated with AgNO 3 , followed by irradiation using Xe lamp, (b) Ag 3 PO 4 crystals grown on the surface prior to irradiation, and (c, d) two different magnification of TiO 2 NT disks treated with Ag 3 PO 4 followed by irradiation using Xe lamp.

Table 1 :Figure 4 :
Figure 4: EDS elemental maps of TiO 2 NT disk treated with Ag 3 PO 4 followed by irradiation using Xe lamp.e elements represent (a) titanium, (b) oxygen, (c) silver, and (d) phosphorus.

Figure 5 :
Figure 5: (a) Top-down SEM image and overlaid EDS elemental map and (b) EDS line scan obtained from the same region as (a) for coprecipitated Ag and HAp on TiO 2 NT disks.
) and 2(b)).In addition, the HAp surface coverage was significantly improved compared to simple immersion treatment (Section 3.2.1).e affinity remained high when HAp was coprecipitated with Ag 3 PO 4 and produced a Ag-doped HAp layer (Figures2(c) and 2(d)).eEDS elemental map of the disk coated by Ag-doped HAp (Figure5(a)) shows evenly distribution Ag supporting the lack of compact accumulation of Ag.Ca and P were also evenly distributed supporting the presence of HAp. e trace level of F is a contaminant from the electrochemical

Figure 6 :
Figure 6: (a) SEM image of a TEM grid containing the coprecipitated Ag and HAp.EDS elemental maps: (b) Ag, (c) O, (d) P, and (e) Ca of the Ag-HAp NPs.
TiO 2 NTs via Photolysis of Ag Precursors 2.3.1.Ag Deposition on TiO 2 NT Disk by Photolysis of AgNO 3 (Control).A TiO 2 NT disk was treated with 4 mL of 2 M AgNO 3 for 15 mins with shaking, followed by rinsing with DI water (3 × 5 mL), and dried in air at 100 °C for 13 min.e TiO 2 NT disk was irradiated with a Xe lamp for 8 min.e treated disks were characterized using SEM and EDS. 2 Journal of Chemistry 2.3.2.Ag 3 PO 4 Particle Deposition and Subsequent Photolysis.

Table 2 :
Elemental weight percentage of surface materials on the TiO 2 NT disks after deposition of Ag-doped HAp obtained using mapping and line scan EDS.