Anodization of implant materials

Abstract

Disclosed are titanium compositions and methods of manufacturing titanium compositions that comprise an oxide coating formed via an anodization process. An anodization electrolyte used in the anodization process comprises (1) a chelating acid component, (2) a phosphate component, and (3) a phosphate binder component. The resulting oxide coating comprises α-tricalcium phosphate and / or hydroxyapatite crystalline compounds with a calcium to phosphate (Ca / P) ratio within a range of human bone.

Description

ANODIZATION OF IMPLANT MATERIALSBACKGROUND

[0001] The present invention is related to improved titanium implants. More specifically, the present invention is related to anodization processes to form either calcium titanate or hydroxyapatite-containing coatings on implant materials.

[0002] As humans live longer, the need for improving the performance and effective lifetimes of implants is increasing. Osseointegration, a direct structural and functional connection between ordered, living bone and the surface of implants, is considered as the physiological basis of successful endosseous implantation. Insufficient bone-implant contact can lead to fibrous scar tissue formation which may result in loosening of the boneimplant interface. Implant failures are often associated with insufficient bone-implant contact and / or aseptic loosening (impaired implant fixation). Accordingly, there is an ongoing need for implants and implant manufacturing processes that promote improved osseointegration.SUMMARY

[0003] The disclosed embodiments of the present invention are directed to an anodization process to form calcium titanate and / or hydroxyapatite oxides on titanium or titanium alloy implant surfaces (e.g., commercially pure titanium). The anodization process can comprise the use of an electrolyte solution (also referred to herein as an anodization electrolyte) and an anodization waveform applied in that electrolyte solution for a predetermined process duration. The anodization electrolytes used in the disclosed embodiments can include (1) a chelating acid component comprising multiple carboxylic acid groups, such as a citric acid solution, (2) a phosphate component, and (3) a phosphate binder component. Certain embodiments of this invention include using juiced fruit, or commercially available fruit juice, as a component of the anodization electrolyte. Other embodiments utilize anodization electrolytes comprising (e.g., completely comprising) synthetic laboratory chemicals. The anodization electrolyte components can comprise commercially available edible substances and / or calcium and phosphate chemistries that already have a history of use for clinical applications.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Features of this disclosure will be apparent from the following description, taken in conjunction with the accompanying drawings and the appended claims, all of which form a part of this specification. In the Drawings, like reference numerals may be utilizedto designate corresponding or similar parts in the various Figures, and the various elements depicted are not necessarily drawn to scale.

[0005] Figure 1 illustrates effective and poor osseointegration around a titanium dental implant.

[0006] Figure 2 illustrates some example applications of anodized titanium alloys in the human body.

[0007] Figure 3 illustrates example citrus fruit-based anodization electrolytes for Embodiment 1.

[0008] Figure 4 illustrates the final forming voltages recorded from galvanostatic anodization in each citrus fruit-based electrolyte. All three citrus fruit-based oxides formed in each electrolyte revealed similar final forming voltage ranges of 230 ± 9 V (CFOJ), 233 ± 10 V (OJ), and 234 ± 2 V (MJ), respectively.

[0009] Figure 5 illustrates Optical Microscopy and SEM images of citrus fruit-based oxide groups showing complex micro and nanoscale surface topographies (left column - optical micro-topography at 100X, middle column - SEM micro-topography at 5,000X, right column - SEM nano-topography. Optical microscopy images reveal a greyish-white surface appearance for each oxide group. The nanoscale SEM surface topography for each oxide group exhibited a unique cauliflower-like surface morphology.

[0010] Figure 6 illustrates AFM images of citrus fruit-based anodized oxide groups showing micro and nanoscale roughness profiles, (left column 50 x 50 pm2 scan areas, right column - 1 x 1 pm2 scan areas). Multiscale micro and nano-scale surface roughness profiles were observed for each oxide.

[0011] Figure 7 illustrates surface roughness values for each citrus-based oxide group. A) Average surface roughness (Ra) values, B) surface roughness values. 50 x 50 pm2 scan areas showed Sa values less than 500 nm, while 1 x 1 pm2 scan areas showed true nano-roughness Sa values below 90 nm. Additionally, the unique cauliflower-like nanoscale surface topographies were confirmed within the 1 x 1 pm2 AFM roughness profiles for each group.

[0012] Figure 8 illustrates representative EDS spectra from each citrus fruit-based oxide group. All three oxide spectra revealed prominent dopant uptake peaks for Ca, and P besides the Ti and O peaks.

[0013] Figure 9 illustrates representative XPS spectra for each citrus fruit-based oxide group. A) survey full spectra, B) Ti2p, C) Ols, D) Ca2p, E) P2p, and F) Cis.

[0014] Figure 10 shows EDS and XPS Ca and P dopant uptake and Ca / P uptake ratios for each citrus fruit-based oxide group. A) EDS Ca and P dopant uptake levels, B) XPS Ca and P dopant uptake levels, C) EDS Ca / P uptake ratios, and D) XPS Ca / P uptake ratios. The EDS and XPS oxide surface Ca / P ratios for all oxide groups were shown to fall within the 1.5 to 1.7 Ca / P ratio range commonly reported for human bone.

[0015] Figure 11 shows representative X-ray diffraction scans of the citric fruit-based oxides. Full two-theta scan range of 20° to 80° are demonstrated on the left side. A zoomed-in two-theta angle region 24° to 36° is shown to the right side to emphasize the formation of crystalline titanate phases. All the three oxides showed formation of anatase and calcium titanate crystalline compounds.

[0016] Figure 12 shows cross-sectional thickness values for each citrus fruit-based oxide group. The average oxide thickness value for each of the oxide groups was found to be less than 500 nm.

[0017] Figure 13 shows Ca2+ion cumulative release profile for the CFOJ oxide group over a 30-day period. An initial burst release was observed, followed by a sustained release reaching an average cumulative amount of 76 ppm by day 30.

[0018] Figure 14 shows example citrus fruit juice-based electrolytes for Embodiment 2.

[0019] Figure 15 shows representative X-ray diffraction scans of the CPTi4 substrate material and each oxide. Full range of 20° to 80° two-theta scans are shown on the left side. A zoomed-in 24° to 36° two-theta angle region is shown to the right side to emphasize the formation of crystalline calcium oxide phases. The anatase phase was formed in oxide A. Calcium titanate and calcium diphosphate phases were formed in all the oxides. Oxides C and D showed a combination of a-tricalcium phosphate and hydroxyapatite phases, with oxide D having predominantly hydroxyapatite.

[0020] Figure 16 - Optical microscopy and SEM images (left column - 100X optical images, middle column - SEM 5,000X micro-scale topography, right column - SEM 75,000X nano-scale topography) for CPTi4 substrate material and each anodized oxide. Oxide A exhibits a uniform greyish-white appearance with nanoscale porosity and small white deposits. Oxide B shows increased white deposits with micro and nano-scale porosity. Oxides C and D exhibit micro and nano-scale petal-like structures.

[0021] Figure 17 - Representative 3D optical profilometer oxide surface roughness values. Oxide C was found to have significantly higher A) Oxide average surface roughness (Sa) values (p < 0.05) and B) oxide peak-to-valley roughness (Sz) values (p <0.001) compared to the other oxides. Oxide D also had significantly higher Szvalues compared to oxide A (p < 0.01) and oxide B (p < 0.05).

[0022] Figure 18 - Representative oxide surface Ca / P ratios. The EDS-derived surface Ca / P ratios were shown to increase with the changing anodization electrolytes.

[0023] Figure 19 - Representative FTIR spectra. The CPTi4 substrate and Oxide A lack definitive peaks, while oxides B, C, and D exhibit a strong PO43peak at 1050 cm The oxide D exhibits a more pronounced peak. Broad O-H bands (3000-3600 cm ') are observed in oxide D and attributed to adsorbed water molecules. Oxides C and D show CO32substitution peaks at wavelengths of 875, 1450, and 1570 cm1confirming the formation of bone-like carbonated apatite.Figure 20 - Representative SEM cross-sectional images of tested oxides.

[0024] Figure 21 - Representative oxide cross-sectional thickness values: A) Inner layer oxide, and B) Outer layer oxide. C) Total oxide. Oxides C and D show significantly thicker inner oxide layers (1.5 pm) compared to oxide A (p<0.0001) and oxide B (p<0.01). Among the bi-layered oxides, oxide C has the thickest outer layer (26 pm), significantly greater than oxide D (14 pm) (p<0.001) and oxide B (4 pm) (p<0.0001). Oxides C (27.6 pm) and D (15 pm) have the highest total oxide thickness, significantly greater than oxide B (5.5 pm) (pO.OOOl) and oxide A (1 pm) (pO.OOOl).

[0025] Figure 22 - Representative EDS line scans of oxide dopant distributions across the layer cross-sections. The Ti and O element distributions are provided in the left column, and distributions of the Ca, P, and Mg dopant elements incorporated from the anodization electrolyte are provided in the right column. The substrate interface is located on the left side of each window, followed by the inner and outer oxides moving left to right. Oxide A revealed a single layer oxide structure consisting of Ti, O, Ca, P, and Mg. Oxides B, C, and D revealed a bi-layered oxide structure, with the concentration of Ti decreasing from the substrate toward the outer oxide. Ca, P, and Mg-dopant uptake begins in the inner oxide layer and continues to increase before reaching a relatively stable level in the outer oxide for oxides B, C, and D. X-axis scales are adjusted to best represent the oxide thickness.

[0026] Figure 23 - A) EDS-derived Ca, P, and Mg-dopant uptake levels from the outermost 0.5 microns for oxide A and the outermost 2 microns of oxides B, C and D. B) Corresponding EDS Ca / P ratios within the outermost oxide surfaces. Ca / P surface ratios range from 1.1 to 1.7 for the oxides.

[0027] Figure 24 - Oxide layer adhesion quality results for each oxide. The oxide B revealed some microcracking and delamination. The oxides A, C, and D did not exhibit any delamination while oxides C and D exhibited minor microcracking.

[0028] Figure 25 - Oxide D microhardness evaluation.

[0029] Figure 26 - Representative XPS spectra for oxide D. A) survey full spectra, B) Ti2p, C) Ols, D) Cis E) Ca2p, F) P2p, and G) Mg Is.

[0030] Figure 27 - Oxide D A) Ca ion release profile, B) Mg ion release profile, C) Ca ion cumulative release profile, D) Mg ion cumulative release profile. Ca and Mg ion release profiles showed an initial burst release followed by a gradual release, indicating a sustained cumulative release over a period of 30 days. The Ca ion release reached a cumulative amount of approximately 120 ppm whereas the Mg release was much lower at approximately 4 ppm over a period of 30 days.

[0031] Figure 28 shows representative XRD oxide crystallinity results for test anodization processes shown in Table 4.

[0032] Figure 29 shows the representative oxide crystallinity results for anodization in electrolytes containing three different concentrations of citric acid with equivalent amount of calcium additions as were utilized in the E2 group D anodization process as listed in Table 5.

[0033] Figure 30 isolates the 0. IM citric acid E3 anodization process oxide which showed substantial hydroxyapatite and tricalcium phosphate formation.

[0034] Figure 31 shows optical microscopy and SEM images of the 0.1M citric acid E3 anodization process oxide showing complex topographies (left column - optical micro topography at 100X, middle column - SEM micro topography at 5,000X, right column - SEM nano topography at 75,000X).

[0035] Figure 32 shows a representative surface EDS spectrum from the 0. IM citric acid E3 anodization process oxide.

[0036] Figure 33 shows A) EDS dopant uptake levels for Ca and P, B) Average EDS Ca / P ratios for the 0. IM citric acid E3 anodization process oxide.

[0037] Figure 34 shows a Representative FTIR spectrum for the 0.1M citric acid E3 anodization process oxide.

[0038] Figure 35 shows oxide thickness values. A) Individual layer oxide thickness values. B) Stacked average oxide thickness values in each layer in the 0. IM citric acid E3 anodization oxide.

[0039] Figure 36 shows the representative XRD oxide crystallinity results for testing of the function of calcium acetate electrolyte component used in the E3 anodization process.

[0040] Figure 37 shows the representative oxide crystallinity results for the calcium acetate (CA-Citric) and calcium carbonate (CaCCh-Citric) containing phosphate binder electrolytes for the E3 and E4 anodization processes.

[0041] Figure 38 isolates the calcium carbonate binder E4 anodization process oxide which showed substantial hydroxyapatite and tricalcium phosphate formation.

[0042] Figure 39 shows optical microscopy and SEM images of the calcium carbonate binder E4 anodization process oxide showing complex topographies (left column - optical micro topography at 100X, middle column - SEM micro topography at 5,000X, right column - SEM nano topography at 75,000X).

[0043] Figure 40 shows a representative surface EDS spectrum from the calcium carbonate binder E4 anodization process oxide.

[0044] Figure 41 shows A) EDS dopant uptake levels for Ca and P, B) Average EDS Ca / P ratios for the calcium carbonate binder E4 anodization process oxide.

[0045] Figure 42 shows a Representative FTIR spectrum for the calcium carbonate binder E4 anodization process oxide.

[0046] Figure 43 shows oxide thickness values. A) Individual layer oxide thickness values. B) Stacked average oxide thickness values in each layer in the E4 anodization oxide.

[0047] Figure 44 shows the representative oxide crystallinity results for from each oxide group from the E5 anodization electrolyte trials.

[0048] Figure 45 shows optical microscopy and SEM images of the E5 anodization process oxides showing complex topographies (left column - optical micro topography at 100X, middle column - SEM micro topography at 5,000X, right column - SEM nano topography at 75,000X).

[0049] Figure 46 shows a representative surface EDS spectrum from the E5 anodization process oxides.

[0050] Figure 47 shows A) EDS dopant uptake levels for Ca and P, B) Average EDS Ca / P ratios for the E5 anodization process oxides.

[0051] Figure 48 shows A) EDS dopant uptake levels for Mg in the E5 anodization process oxides.

[0052] Figure 49 - Representative XPS spectra for oxide D. A) survey full spectra, B) Ti2p, C) Ois, D) CisE) Ca2p, F) P2p, and G) Mgis.

[0053] Figure 50 shows a Representative FTIR spectrum for the E5 anodization process oxides.

[0054] Figure 51 shows oxide thickness values. A) Individual layer oxide thickness values. B) Stacked average oxide thickness values in each layer in the E5 anodization oxides.

[0055] Figure 52 - Anodization oxides from E3, E4, and E5 can be formed using any multiple carboxylic chelating acids in the anodization electrolyte, such as citric acid, EDTA, or malic acid in combination with a phosphate component and a phosphate-binder component.

[0056] Figure 53 - XRD of anodized oxide surfaces formed using MP-CA formula (citric acid-MPCA) by replacing citric acid with other multiple carboxylic chelating acids such as EDTA and malic acid.

[0057] Figure 54 - FTIR of anodized oxide surfaces formed using MPCA formula (citric acid-MPCA) by replacing citric acid with other multiple carboxylic chelating acids such as EDTA and malic acid.

[0058] Figure 55 - Representative X-ray diffraction scans from each titanium alloy oxide group. The full range of 20° to 90° two-theta scans are shown on the left side to show all diffraction peaks formed on each titanium alloy surface. A zoomed-in 24° to 36° two- theta angle region is also provided to the right side to emphasize the formation of crystalline calcium phases within each oxide. Hydroxyapatite, a-tricalcium phosphate, and calcium titanate were shown to be formed in the oxides on each titanium alloy. Evidence of calcium diphosphate formation was also shown in the TiMo a + P and TAV duplex alloy oxides.

[0059] Figure 56 - Optical microscopy and SEM images of each oxide group showing complex surface topographies (left column - optical images at 100X, middle column - SEM micro-scale topography at 5,000X, right column - SEM nano-scale topography at 75,000X). CPTi, TiMo P, and TAV oxides reveal uniform distributions of dark and light areas, while TiMo a + P oxide displays localized white patches within darker regions in optical images. Micro-scale SEM images revealed a similar rough surface appearance on all oxides, while nano-scale SEM images revealed white deposits and surrounding nanopores.

[0060] Figure 57 - A) Representative LCM images for each oxide group. B) Oxide average surface roughness (Ra) values and C) oxide peak-to-valley roughness (Rz) values revealed statistically similar roughness in CPTi, TiMo a + P, and TAV oxides. TiMo Poxide, exhibited significantly lower Raand Rzvalues compared to the CPTi oxide. (p=0.03).

[0061] Figure 58 - Representative surface Ca / P ratios for each oxide group. The EDS derived surface Ca / P ratios for each oxide group were shown to be approximately 2.

[0062] Figure 59 - Representative FTIR spectrum for each oxide group. Each oxide surface shows a strong PO43absorption peak at 1050 cm indicative of hydroxyapatite and a-tricalcium phosphate formation. Additionally, weak O-H bands between 3000- 3600 cm1represent the bending mode of adsorbed water, with a poorly defined characteristic hydroxyapatite OH' peak at 3570 cm1. High-intensity CO32substitution peaks at 875, 1450, and 1570 cm1confirmed the presence of bone-like carbonated apatite on all oxide surfaces.

[0063] Figure 60 - Representative cross-sectional oxide thickness values: A) Inner oxide layer, and B) Outer oxide layer. TiMo P and TiMo a+P oxides exhibit significantly thicker inner oxide layers compared to CPTi and TAV (p < 0.0001). Conversely, CPTi shows significantly lower outer oxide thickness than other alloys (p < 0.0001), resulting in the lowest total oxide thickness

[0064] Figure 61 - Representative EDS line scans of dopant element distributions across the cross-sections of each oxide group. The Ti, O, and substrate alloying element distributions are shown in the leftmost column, while distributions of elements incorporated from the anodization electrolyte (Ca, P, and Mg) are shown in the right-most column. The alloy substrate is located on the left side of each image, followed by the inner oxide layer and then the outer oxide layer. The presence of a bi-layered oxide structure is clarified for each oxide, with the concentration of substrate (Ti) and alloying elements (Mo, Al, V) decreasing across the inner oxide layer and being absent in the outer oxide layer. The uptake of Ca, P, and Mg begins in the inner oxide layer and continues through each outer oxide layer

[0065] Figure 62 - A) EDS Ca, P, and Mg-dopant uptake levels in the outermost two microns of each oxide group. B) Corresponding EDS Ca / P ratios within the outermost two microns of each oxide group. These Ca / P surface ratios range from 1.3 to 1.7, which are within the range of bone tissue.

[0066] Figure 63 - Relative oxide layer adhesion quality results for each alloy group. A) CPTi, B) TiMo P, C) TiMo a+P and D) TAV. The CPTi, TiMo a+P, and TAV oxide groups exhibited minimal microcracking and no delamination, indicative of acceptable adhesion strengths. The TiMo P oxide showed significant delamination andmicrocracking, which was presumably caused by the combination of the omega phase and alpha case formed during the anodization processes on this alloy substrate.

[0067] Figure 64 - Micro-CT images of an anodized bone screw with seamless boneimplant interface and high BIC after 4 weeks in a rat femur.

[0068] Figure 65 provides a comparison of solid and 3D printed porous gyroid titanium lattice substrates, for clarity.

[0069] Figure 66 shows the representative oxide crystallinity results for from each oxide group from the E7 anodization electrolyte trials on 3D printed titanium substrates.

[0070] Figure 67 shows optical microscopy and SEM images of the E7 anodization process oxides showing complex topographies (left column - optical micro topography at 100X, middle column - SEM micro topography at 5,000X, right column - SEM nano topography at 75,000X).

[0071] Figure 68 shows A) EDS dopant uptake levels for Ca, P, and Mg, B) Average EDS Ca / P ratios for the E7 anodization process oxides.

[0072] Figure 69 shows representative FTIR spectra from the E7 anodization process oxides.DETAILED DESCRIPTIONOverview of Conventional Implant Anodization

[0073] Global Demand and Need for Improving Implant Longevity. With the rising number of implant procedures worldwide, the demand for dental and orthopedic implants with improved durability and reliability continues to increase. The global dental and orthopedic implant markets have experienced significant expansion. [1-3] While dental implants have a high success rate (90-97%), the American Dental Association (ADA) estimates that between 0.15 and 0.5 million patients in the United States require revision surgeries annually. [4-6] Aseptic loosening, caused by impaired implant fixation, remains the leading cause of prosthetic failure, accounting for approximately 76% of prosthetic reoperations (50% in hip prostheses and 30% in knee prostheses). [7,8] Osseointegration is a structural, functional, and chemical connection between the bone and the implant and is a key determinant of implant success. [9,10] Osseointegration plays a significant role in implant stability by determining the speed of bone-implant sealing and the extent of bone remodeling.

[0011] Inadequate osseointegration can cause fibrous scar tissue formation at the bone-implant interface, leading to loosening and, eventually, implant failure.

[0012] Thus, osseointegration is vital for the long-term stability of the implants and for reducing the risk of failure.

[0074] Titanium and Its Alloys as Implant Materials'. Titanium and its alloys are commonly used in orthopedic and dental implants due to their advantageous properties, high strength-to-weight ratios, excellent corrosion resistance, and biocompatibility. [13- 15] Titanium primarily exists in two crystalline lattice structures: the hexagonal close- packed (HCP) alpha-phase, which is stable at lower temperatures, and the body-centered cubic (BCC) beta-phase, which is stable at higher temperatures.

[0016] The alpha-phase alloys are characterized by good ductility and formability, making them suitable for applications such as dental implants, cardiovascular stents, spinal devices, and trauma fracture fixation plates and screws. [17,18] On the other hand, Beta-phase titanium alloys have comparatively higher ductility and lower elastic modulus, thereby reducing the stress-shielding effects on bone growth, making these alloys effective for spinal implants and trauma fracture fixation devices. [16,19-21] Titanium alloys are often processed into duplex microstructures that combine alpha and beta phases, which can result in enhanced strength compared to single-phase alloys.

[0018] These duplex alloys are commonly used in demanding applications like total joint replacement components and intramedullary nails. [17,22]

[0075] Challenges of Bio-Inert Implant Surfaces with Osseointegration'. Titanium implants can face challenges relating to their bio-inert surfaces. Upon exposure to air, titanium rapidly forms a less than 5 nm thick, amorphous oxide layer, which is bio-inert in nature and lacks osteoinductive properties, causing hindrance to effective integration with surrounding bone tissue. [23-26] Various surface modification techniques have been developed to address this limitation and enhance the bioactivity of titanium implants. Commonly employed methods include sol-gel processing, chemical vapor deposition, plasma immersion ion implantation and deposition, plasma spraying, and anodization. These techniques have demonstrated improvements in bone tissue response and have contributed to the increased success and longevity of titanium-based implants in vivo. [27-30] As discussed above, however, limitations remain.

[0076] Hydroxyapatite is a vital bone mineral and amounts to about 65-70% of bone weight. [31,32] It has wide applications as a biomaterial in dental and orthopedic devices and has been suggested to be an effective intermediate in the osseointegration of titanium implants. [10,33] It has a hexagonal crystal structure that can be verified by its stoichiometric Ca / P chemistry ratio of 1.67 or by its characteristic X-ray diffraction (XRD) pattern.

[0034] Plasma spraying has been utilized to deposit hydroxyapatite coatings with various levels of crystallinity on titanium substrate and found that the rate of cellproliferation on the surface was directly proportional to the crystallinity of the hydroxyapatite in vitro and in vivo.

[0035] Moreover, plasma-sprayed hydroxyapatite coatings with 100% crystallinity have been reported not to show resorption like their amorphous counterparts.

[0036] Since resorption of the hydroxyapatite coating appears primarily in contact with bone of lower density, high crystallinity coatings have been suggested for implants placed in trabecular or cancellous bone.

[0037] Hydroxyapatite binds chemically to bone, forming direct contact between the tissue and the implant surface.

[0038] Thus, hydroxyapatite coatings have commonly been used on the surface of metallic implants to improve their bioactivity and to increase the bone-implant contact (BIC) area. [39-42] Several methods, including plasma spraying, sputtering, pulsed laser deposition, and sol-gel techniques, have been employed to deposit hydroxyapatite coatings on titanium and improve osseointegration. [38,39,43,44] Plasma-sprayed coatings are made by spraying hydroxyapatite particles on the implant surface at high temperatures and cooling rapidly. They are commonly used in clinical practice.

[0045] These plasma-sprayed hydroxyapatite coatings bind easily to bone tissue but have shown low bonding strengths to metallic implant alloy surfaces. Primary factors influencing the bonding strength of these coatings include differences in the coefficient of thermal expansion (CTE) between hydroxyapatite and titanium alloy substrate and structural changes or degradation of hydroxyapatite at high spraying temperatures. [45,46] Poor adhesion or delamination between the coating and the substrate may eventually cause early implant failure due to the coating's cracking and peeling or delamination. [45,46] Moreover, the rapid temperature changes during the plasma spraying process allow the formation of amorphous and / or secondary phases such as tetra calcium phosphates (TTCP), which are highly bioresorbable. Figure 1 illustrates adhesion between hydroxyapatite spray coatings and a titanium implant substrate.

[0077] Anodization of Titanium Implants'. Anodization is an electrochemical surface modification technique popularly used to modify the surface topography, chemistry, and crystallinity of titanium implants in a single processing step, making it an effective approach for improving implant performance. [47-49] Anodization as a surface modification technique is increasingly employed to address the drawbacks of the plasmaspraying technique.

[0050] Figure 2 represents some example applications of anodized titanium alloys in the human body.

[0078] Anodization for Surface Modification'. Anodization can achieve complex micro and nano-scale surface topography on titanium surfaces. Anodization studies that useforming voltages exceeding the dielectric limit can form micro or nanoscale surface porosity, which significantly contributes to the overall surface topography of the oxide layer. Oxide surfaces with micro-scale roughness have shown improved mechanical bone interlocking [10,51,52], In contrast, surfaces with nano-scale roughness have shown increased protein absorption and osteoblast cell attachment and differentiation, promoting faster osseointegration. [10,48,49] Biomaterials devoid of surface roughness in the micro- and nanoscale range have been shown to hinder cell osteogenic differentiation.

[0010] Rougher surfaces (mean average roughness Ra> 0.5 pm) were correlated to increased bone-to-implant contact (BIC) and described to be preferred by bone cells compared to smooth surfaces. [10,53] Some anodization processes have been shown to produce oxide surfaces exhibiting complex surface topographies integrating micro and nano-scale surface features to promote cell proliferation, differentiation, and extracellular matrix formation. [54-57] Thus, anodized surfaces can improve macroscopic and microscopic mechanical retention and alter surface chemistry, stimulating biological processes that can promote bone growth.

[0058] Two distinctive types of surface topography can be obtained through anodization, namely, microporous anodized titanium (MAT) and titanium dioxide nanotubes (TNTs).

[0059] Anodization also enables incorporating various bioactive elements into the titanium oxide layer individually or in combination. Some of the commonly added dopants include calcium (Ca), phosphorus (P), and magnesium (Mg), all of which are important elements found in the bone and can help to promote osseointegration. [60,61] Mg is found to be 0.72 wt% in bone.

[0062] Anodization processes can also thicken and crystallize the titanium dioxide layer into anatase and rutile phases. Anatase and rutile are generally shown to be more bioactive than their amorphous titanium dioxide counterparts. [52,63,64] Additionally, the atomic lattice structure of anatase has been reported to be a better match to the apatite lattice and thus promotes apatite formation on the oxide surfaces. [65,66] Recent advancements have focused on developing crystalline calcium-phosphate (CaP) compounds on titanium implant surfaces to mimic a natural bone composition for improving implant integration and performance. [23,28,67] The following sections discuss anodization studies that have incorporated Ca, P, or Mg-containing chemical compounds into the anodization electrolyte for the purpose of creating bio-chemically-doped MAT surfaces designed to improve titanium osseointegration.

[0079] Multifunctional Anodization Coatings'. Approximately 80-90% of the mineral content of bone is made up of Ca and P.

[0068] Ca and P are also the key components ofhydroxyapatite, the primary mineral phase of bone. Anodization processes enable the incorporation of essential chemical elements into the oxide layer. Anodization studies have explored the addition of Ca and P into the titanium oxide layer, individually or in combination, as CaP oxides. [23,28,69]

[0080] Ca-doping oxide studies'. Blood proteins adsorb onto the implant surface upon implant placement, and clot formation occurs. Ca2+ions help activate thrombin and fibrin formation and stabilize the clot, which facilitates the recruitment of bone-forming cells.

[0070] Ca2+ions have been known to play a critical role in the osseointegration of implants by regulating key proteins involved in osteoblast differentiation and bone regeneration, as evident from the results of a recent study showing that Ca-modified titanium surfaces showed early bone regenerative matrix formation, leading to faster osseointegration compared to unmodified surfaces. [70-72]

[0081] Anodization of titanium using three distinct electrolyte groups: sulphuric acid, phosphoric acid, and calcium hydroxide in an applied voltage range of 20 to 130 V in galvanostatic current densities ranging between 5 to 40 mA / cm2resulted in S-doped, P- doped and Ca-doped implants. The Ca-doped implants exhibited porous surface morphology characterized by crater-like structures (~1.3 pm in diameter) and calcium titanate (CaTiCh) formation at the outermost surface. The oxide revealed high calcium incorporation (Ca 11 at%) and a crystalline anatase phase. In vivo testing in rabbit tibia revealed that Ca implants demonstrated the highest removal torque (RTQ) levels and bone-to-implant contact (BIC), while analysis in rabbit femur revealed that Ca implants exhibited the most mature bone structure, with increased osteocyte activity and enhanced mineralization compared to the P-doped, S-doped, and unanodized control groups.

[0052] It was hypothesized that electrostatic interactions between the CaTiCh (perovskite) layer and bone matrix proteins could enhance bone mineralization and implant stability. Anodization of alumina-blasted titanium implant surfaces in an electrolyte containing sodium glycerophosphate (GP) hydrate and calcium acetate (CA) at an applied voltage range of 20 to 130 V in galvanostatic current densities ranging between 5 to 40 mA / cm2resulted in oxides having a porous surface topography with pore sizes ranging from 0.2 to 0.5 pm, containing 1.8 at% Ca, and demonstrated the highest RTQ value and interfacial shear strength compared to alumina-blasted controls in a rabbit tibia study.

[0073]

[0082] P-doping oxide studies'. Approximately 85% of the phosphorus found in the body is in the bone.

[0068] Phosphorus-containing compounds have essential roles in maintaining cell structure and cellular metabolism and regulating subcellular processes likemaintenance of cell membrane integrity, cellular metabolism, acid-base homeostasis, and bone mineralization.

[0074] Adequate phosphorus levels are essential to maintain the regular osteoblast and osteocyte activity required in matrix mineralization.

[0074]

[0083] Initially, researchers exploring P-doped anodization processes focused on understanding its effect on surface topography optimization, osteoblast behavior, and thermal treatments to enhance apatite deposition. Anodization of titanium in a 0.2M phosphoric acid-containing electrolyte at 70 A / cm2current density resulted in P-doped oxides having a smooth surface topography at lower voltages, transitioning to micro- porous structures (~0.5 pm pores) at higher voltages, with oxides containing -10 at% P.

[0075] These P-doped oxides demonstrated improved osteoblast attachment and proliferation compared to the unanodized controls.

[0084] Titanium anodized at 20 V in 1 M phosphoric acid electrolyte resulted in oxides having a flower-like surface topography with 1.4 at% P incorporation and exhibited a combination of anatase and rutile crystalline phases.

[0076] Subsequent immersion of these oxides in SBF facilitated apatite deposition in 21 days, with significant improvement in deposition following heat treatments at 400 °C and 600 °C, indicating enhanced bioactivity through thermal processing. Biologically, these heat-treated oxides demonstrated superior osteoblast attachment, proliferation, extracellular matrix (ECM) formation, and differentiation potential compared to the sulphuric acid-anodized and unanodized controls.

[0085] Anodization in a 1.4 M phosphoric acid electrolyte at a high voltage of 200 V revealed oxide surfaces having a distinct porous structure with crater formations and an anatase crystalline phase.

[0077] Anodization in potassium pyrophosphate (K4P2O7) and potassium triphosphate (K3PO4) containing electrolytes at a current density of 200 mA / cm2for 300 s revealed K4P2O7 oxides to have a more complex and rough surface with crater-like micropores, whereas K3PO4 oxides had comparatively smoother surfaces with pit-like pores.

[0078] Additionally, K4P2O7 oxides exhibited higher amounts of anatase phase, while K3PO4 oxides contained more rutile phase due to thermal effects experienced during anodization. Upon immersion in SBF, the anatase-rich K4P2O7 oxide surfaces achieved full apatite coverage by day 14, while the rutile-dominated K3PO4 oxides showed only partial apatite formation.

[0086] Later on, researchers exploring P-doping of anodized oxides focused on using multi-acid combination electrolytes to achieve improved surface topography and crystallinity for better osteoblast activity outcomes. Anodization of titanium in a sulfuric-phosphoric acid combination electrolyte with a 200 V forming voltage produced a rough, porous oxide layer (~5 pm pore size) having a distinct crater wall (thickness ~3 mm) with predominant anatase crystallinity and enhanced corrosion resistance.

[0079]

[0087] Biological assessments of the P-doped oxides demonstrated superior osteoblast adhesion, proliferation, differentiation, and mineralization, compared to unanodized controls. Anodization in a complex electrolyte composed of phosphoric acid, sulfuric acid, oxalic acid, and hydrogen peroxide at 180 V forming voltage resulted in anatasephase oxides exhibiting uniform surface porosity, which showed enhanced osteoblast differentiation and maturation compared to the sulphuric acid anodized controls. [47,80]

[0088] Ca andP combination-doping oxide studies'. Numerous anodization studies have incorporated Ca and P oxide dopants simultaneously to create surfaces with calcium-to- phosphorus (Ca / P) ratios in the range of human bones. [23,81-83] Ishizawa et al., the first research group to form CaP combination oxides on titanium, initially tried combinations of phosphoric acid and various calcium compounds, forming oxides with less than desirable surface Ca / P ratios below one. [82,84] The same research group switched to a P-glycerophosphate (P-GP) disodium salt and calcium acetate (CA) electrolyte and anodized at 50 mA / cm2. It successfully generated oxides with porous surface topography having Ca / P ratios within the range of hydroxyapatite (1.67) and human bone (1.50 - 1.70). [82,84] When followed by subsequent hydrothermal treatments, the oxides formed hydroxyapatite on the surfaces. Most subsequent CaP oxide anodization studies have utilized similar aqueous electrolytes consisting of GP and CA to explore the oxide in vitro and in vivo. Anodization in a similar electrolyte containing P-GP disodium salt pentahydrate and CA monohydrate at 350V for 10 minutes resulted in surfaces having a porous topography with donut-shaped pores ranging from 1 to 2.7 pm in diameter, accompanied by visible surface cracks. Crystallinity analysis confirmed the formation of both anatase and rutile phases.

[0085] The biological response was evaluated by immersing the anodized samples in simulated body fluid (SBF) for 7 days under additional UVC irradiation energy (254 nm), which successfully formed the crystalline hydroxyapatite phase. The study proposed a mechanism suggesting that UV irradiation-induced the generation of hydroxyl radicals (»OH), which modified the TiCh surface chemistry by forming Ti-OH groups. These negatively charged Ti-OH groups facilitated the adsorption of Ca2+ions from SBF, forming an amorphous calcium titanate layer, which subsequently reacted with phosphate (POT ) to nucleate and grow hydroxyapatite. Anodization in a P-GP and sodium acetate electrolyte at 50 mA / cm2, followed byhydrothermal treatment to induce hydroxyapatite formation on the titanium surface, formed microporous oxides with hydroxyapatite crystals that exhibited high BIC values in rat femur model compared to non-hydrothermally treated anodized surfaces and unanodized controls.

[0086] Anodization of titanium in similar aqueous electrolytes containing Ca-GP and CA under varying processing conditions consistently resulted in the formation of CaP-enriched rough and porous oxides with variations in crystallinity, mostly anatase, sometimes rutile and even a combination of the two. Some of these oxides demonstrated improvement in osteoblast attachment, proliferation, and differentiation in vitro, while some demonstrated enhanced bone-implant bonding strength in vivo compared to unanodized controls. Additionally, some oxides also demonstrated the highest Ca / P precipitation in simulated body fluid (SBF). [87-91]

[0089] Anodization in an electrolyte composition containing calcium phosphate, CA, and sodium ethylenediamine tetra-acetic acid (EDTA) produced oxides exhibiting a high surface atomic Ca / P ratio (~1.3).

[0092] These anatase phase oxides also revealed a porous and rough surface topography. n vitro dissolution experiments on these oxides demonstrated that the CaP phases in the coatings were partially soluble, releasing calcium and phosphate ions over 14 days.

[0090] Anodization in a potassium phosphate (KH2PO4), calcium hydroxide (Ca(OH)2), calcium formate (Ca(HCOO)2), and EDTA-containing electrolyte at 100-150 mA / cm2resulted in oxides having microporous surface topographies with pore sizes from 3-4 pm to -100 nm.

[0093] These CaP oxides were found to have a low Ca / P ratio (<0.26 by XPS), but XRD analysis revealed the formation of anatase at lower current densities and rutile phase at higher current density. Despite the low Ca / P surface ratios, the coatings successfully promoted apatite deposition in SBF and exhibited favorable protein adsorption, enhancing osteoblast adhesion and proliferation.

[0091] Anodization to form titanate crystalline compounds'. Calcium titanate (CaTiCE), known as perovskite, is a well-known bio-ceramic used as an anodization and electrospinning coating material for implants. [94,95] It has good biocompatibility, an intermediate coefficient of thermal expansion between bone and hydroxyapatite, and the ability to increase cell attachment and proliferation. [95,96] Additionally, CaTiCE has been shown to enhance the adhesion between the titanium substrate and hydroxyapatite coatings and reduce the dissolution of hydroxyapatite in acidic environments in previous electrospinning and sol-gel studies. [95,97] Ion implantation techniques have also been utilized to form calcium titanate coatings on titanium implants by integrating calcium ionsinto the titanium dioxide layer. The ion-implanted coatings exhibited enhanced biocompatibility when soaked in SBF.

[0098] Radiofrequency sputtering has also been used to form an approximately 50 nm layer of CaTiCh on a titanium substrate that showed the formation of bone-like apatite after subsequent soaking in Hank’s solution.

[0099] Thus, it has been suggested that CaTiCh surfaces would readily form bone-like apatite and promote faster bonding to bone tissues. [96,100]

[0092] While most previous CaP-doped studies could only form anatase and rutile crystalline phases, some could also produce crystalline titanate phases within the oxides through anodization using similar [3-GP and CA-based electrolyte recipes. Some Ca-only doped, and recent CaP-doped oxide studies have shown that the formation of crystalline titanate compounds during anodization improves the oxide apatite forming ability during subsequent bioactivity testing [23,94,101-105],

[0093] Anodization in a sodium [3-GP pentahydrate and CA monohydrate electrolyte at high forming voltages of 450 V produced oxides exhibiting porous and rough surface topography having Ca / P ratios ranging between 1.3 and 1.8. [94,106] A crystallinity analysis on these oxides confirmed the presence of CaTiCh, [3-dicalcium phosphate (P- Ca2P2O?), and a-tricalcium phosphate (a-Cas(PO4)2) along with anatase and rutile. Subsequent immersion in SBF revealed apatite formation on the oxide surfaces as early as 14 days and 28 days. Furthermore, FT-IR analysis on the SBF-soaked oxides confirmed the presence of CCh2bands, indicating the apatite formed was carbonated hydroxyapatite. The apatite layer was continuous and covered the entire surface after 56 days of immersion. The following mechanism was hypothesized for apatite formation: The negatively charged TiCE surface facilitates the adsorption of Ca2+ions, initiating apatite nucleation, after which CaTiCE hydrolyzes in simulated body fluid (SBF), releasing Ca2+, OH , and TiO(OH)2 species. This, in turn, increases the local supersaturation of calcium and phosphate, enhancing apatite precipitation. Subsequently, Ca- and P-containing phases further contribute to apatite nucleation and growth, leading to the formation of a carbonated hydroxyapatite layer that resembles natural bone apatite. Once apatite nuclei form, they grow spontaneously by consuming Ca2+and phosphate ions from the surrounding solution, ultimately forming a dense apatite layer with prolonged immersion.

[0094] Anodization in phosphoric acid and CA electrolyte formed oxides with ~0.3 pm sub-micron pores with a -1 pm spacing across the surfaces.

[0107] , EDS analysis of the oxides revealed P (-11 at%) and Ca (9-15 at%) incorporation, while XRD analysesconfirmed the presence of anatase and CaTiOs. Similarly, anodization in an aqueous electrolyte composed of Ca-GP and CA at a current density of 0.212 A / cm2resulted in a porous and rough surface topography with a Ca / P ratio of 1.67.

[0108] XRD analysis confirmed the presence of anatase, rutile, and CaTiOs phases in the oxide. Subsequent hydrothermal treatment in a sodium hydroxide (NaOH) solution formed needle-like hydroxyapatite crystals. Cross-sectional SEM analysis revealed a 15 pm thick oxide, with a noticeable depletion of Ca and P beneath the surface, suggesting the diffusion of ions from the inner layer to the outer surface, followed by their dissolution in the solution and subsequent precipitation as hydroxyapatite crystals. The study proposed a mechanism where Ca2+and PO43ions migrated outward, while Ti-OH groups, formed in the alkaline environment, acted as nucleation sites facilitating the crystallization of needle-like hydroxyapatite structures on the surface. A subsequent study added additions of NaOH to the Ca-GP and CA electrolytes and performed anodization using a current density of 70 mA / cm2for 10 minutes, resulting in the deposition of a nanoflower-like structure on the surfaces.

[0102] Crystallinity analysis confirmed the formation of sodium titanate and calcium titanate within the anodized layer. The biological response was evaluated through 7-day immersion in SBF, which led to complete surface coverage with bone-like apatite. The study further proposed a mechanism suggesting that sodium titanate functioned as a bioactive phase, where Na+ions were exchanged for H3O in SBF, generating Ti-OH groups. These hydroxyl groups subsequently facilitated the adsorption of Ca2+and PO43ions, leading to the formation of amorphous calcium phosphate, which later crystallized into bone-like apatite.

[0095] Anodization to form tricalcium phosphate and hydroxyapatite'. Hydroxyapatite and Tricalcium phosphate are widely studied calcium phosphates and are commonly used in bone cement and bone substitution applications.

[0109] Tricalcium phosphate has a Ca / P ratio of 1.5 and has two phases, a-phase and P-phase. [110, 111] Both phases are common components of CaP bone cement.

[0112] Anodization in an electrolyte containing Ca-GP and CA monohydrate at 500 V, produced a porous TiCE layer composed of rutile, anatase, and a-tricalcium phosphate (a-TCP). Upon subsequent immersion of the anodized oxides in Hank's solution, the a-TCP and amorphous Ca-P compounds showed gradual transformation into hydroxyapatite.

[0113] Previously, a-TCP has shown increased boneimplant contact and peri-implant bone volume in the femur of rabbits.

[0112] Biological apatite is bone-like apatite, often called carbonated hydroxyapatite. It has characteristics similar to that of hydroxyapatite in natural bone and can be created by chemical reactionsusing solutions that mimic the composition of bodily fluids.

[0114] It has been reported previously that bone-like apatite is more beneficial for osseointegration as compared to other bioactive ceramics. [115,116] Thus, it has been suggested that the deposition of a bone-like apatite coating on the titanium implant would enhance osseointegration. Hydroxyapatite that is carbonated apatite consists of carbonate substitutions.

[0117] In literature, two types of carbonate substitutions are explained: (a) A-type - direct substitution of OH- with CO32' and (b) B-type - tetrahedral group of PO43' substituted by CO32’. These substitutions may cause changes in the lattice parameters, crystal symmetry, thermal stability, morphology, solubility, and physical, chemical, and biological characteristics of apatite.

[0096] Anodization of titanium in an electrolyte containing sodium biphosphate dihydrate and CA monohydrate under a current density of 0.5 A / cm2for 60 minutes resulted in oxides having a porous and uneven network structure surface topography, with pores ranging from 1 to 5 pm in diameter and a total of 9% porosity.

[0118] Cross-sectional analysis revealed a 20 pm thick coating without a distinct interface with the titanium substrate. Surface chemistry analysis using EDS confirmed the presence of Ca and P with a Ca / P ratio of 1.63 on the surface. Crystallinity analysis using XRD identified the presence of anatase and rutile phases of TiCE along with broad hydroxyapatite peaks. The mechanism for the formation of hydroxyapatite involved hydroxyl (Ti-OH) group formation on the surface, which acted as nucleation sites for phosphate ion adsorption, followed by calcium ion attraction and calcium phosphate precipitation, leading to hydroxyapatite formation.

[0097] Anodization at 450V in an electrolyte containing [3-GP disodium salt pentahydrate and CA monohydrate for 5 minutes with a pulse frequency of 100 Hz and a duty cycle of 30% resulted in oxides exhibiting a porous and uneven surface topography with interconnected pores (2 to 10 pm pore size).

[0119] Cross-sectional analysis revealed a 20 pm thick coating with no distinct interface between the oxide layer and the titanium substrate. EDS confirmed the presence of Ca and P, with a Ca / P ratio of 2.6, while XRD identified the presence of anatase and rutile phases, along with highly crystalline hydroxyapatite. Biological evaluation using MC3T3-E1 osteoblast cells showed excellent cell attachment, spreading, and proliferation on the oxide surfaces.

[0098] A two-step anodization process using an electrolyte containing sodium phosphate monobasic dihydrate and CA monohydrate, initially at a current density of 10-16 A / dm2for 10 minutes, followed by a second anodization at an increased electrolyte concentrationand 450V for 40 minutes resulted in a dense oxide layer with dispersed surface pores ranging from 1 to 5 pm in diameter.

[0120] Cross-sectional SEM analysis showed an oxide thickness of approximately 27 pm, with a seamless transition between the oxide and the substrate, suggesting strong adhesion. XRD analysis revealed the presence of anatase and rutile phases, along with broad hydroxyapatite peaks after the first anodization. After the second anodization, the hydroxyapatite phase became more pronounced, along with residual anatase and rutile phases.

[0099] Anodization in an electrolyte containing [3-GP disodium salt pentahydrate and CA at 480 V for 20 minutes with a pulse frequency of 100 Hz and a 6% duty cycle resulted in oxides composed of hydroxyapatite, a-TCP, CaCCh, CaTiCh, and rutile phases, forming a bi-layered hydroxyapatite / titanium dioxide structure.

[0121] The mechanism of hydroxyapatite involved the breakdown of the TiCE dielectric layer, followed by the incorporation of Ca2+, HPOE , and OH ions, and subsequent thermal decomposition of Ca-deficient hydroxyapatite into hydroxyapatite and a-TCP, with CaCOs formed due to the electrochemical oxidation of acetate ions at the anode. Tensile strength tests showed a cohesive failure within the oxide coating (not at the interface), indicating strong adhesion between the coating and the substrate, and revealed a strength of 24 MPa.

[0100] Anodization in an electrolyte containing sodium GP salts and CA at 350V for 3 minutes resulted in a porous and rough surface topography with high pore density (pore sizes ranging from 70 to 650 nm) and a surface roughness of ~9.8 nm.

[0122] EDS and XPS analyses confirmed a Ca / P ratio of -1.60, which is close to the stoichiometric ratio of hydroxyapatite. XRD analysis identified the presence of hydroxyapatite, anatase, a- TCP, and CaTiCE in the oxide, with hydroxyapatite being the predominant phase. The crystalline size of the hydroxyapatite phase was -42 nm, indicating a nanocrystalline structure.

[0101] Anodization in an electrolyte containing sodium dihydrogen phosphate and CA dissolved in deionized water at 390 V with a pulse frequency of 900 Hz, a 50% duty cycle, and a treatment time of 15 minutes.

[0123] The electrolyte temperature was maintained at 10°C with a cooling system. The surface morphology of the oxides revealed a porous structure covered with petal-like apatite crystals. The coating exhibited a double-layered structure, with an outer porous apatite layer (-16 pm thick) and an inner dense TiCE layer (-6 pm thick). XRD analysis showed that the coating contained hydroxyapatite as a major phase, along with anatase and rutile. FTIR analysis confirmed the presence of phosphate (PCE3) groups at 1100 cm ' / 1030 cm1(vs P-0 stretching) and 602 cm ' / 56 I cm1(v4 P-O bending). The carbonate (CCE2) absorption band was detected at 1450 cm indicating the incorporation of carbonate into the apatite structure. The OH bands characteristic of hydroxyapatite at 3560 cm1were not clearly visible, suggesting the presence of carb onate- substituted apatite. XPS analysis confirmed the presence of Ca and P in the oxides. The study further confirmed that the hydroxyapatite formation mechanism involved the role of higher thermal energy from anodization, which enhanced ion migration and diffusion. This led to the nucleation and growth of apatite crystals on the surface. Initially, the hydroxyapatite particles sintered onto the surface, and over time, their deposition increased, and the particles grew into a continuous petal-like structure.

[0102] Anodization in an electrolyte containing CA and monosodium orthophosphate at 390 V with a pulse frequency of 700 Hz and a duty cycle of 20% for a duration of 3 minutes resulted in oxides having a porous surface topography covered by a flower-like apatite layer arising from the self-assembly of plate-like calcium phosphate precipitates. [124,125] XRD revealed hydroxyapatite to be the dominant phase in addition to anatase and rutile. The surface chemistry analysis conducted using EDS and XPS confirmed the presence of Ca2+and P5+ions, with a Ca / P atomic ratio of 1.25 on the flower-like surface but 1.65 on the inner porous surface, indicating that the diffusion of Ca2+was less in the plate-like structures leading to the formation of calcium -deficient apatite. FTIR analysis identified phosphate groups (PCE3) at 1100 cm ' / 1030 cm1(vs P-0 stretching) and 602 cm ' / 56 I cm1(v4 P-0 bending). The carbonate absorption band (CCE2) was detected at 1450 cm1while the characteristic OH band of hydroxyapatite at 3560 cm1was absent, suggesting the presence of carbonate-substituted apatite. The mechanical properties of the coating were evaluated using scratch testing. The adhesive force between the apatite and inner porous TiO? layers was 26.5 N, while the adhesive force between the TiO? layer and the titanium substrate was 48.5 N. The improved adhesion was attributed to the TiO? layer acting as a transitional interface, reducing interfacial stress, and mechanically interlocking with the hydroxyapatite layer. Further optimization of the anodization parameters in the same electrolyte by applying 360V, 0.7A current, a pulse frequency of 100Hz, and a duty cycle of 50% for a treatment duration of 3 minutes resulted in similar bi-layered oxides consisting of an inner porous TiCE layer and an outer petaling hydroxyapatite layer.

[0125] The porous TiCE layer had an average thickness of 8 pm, while the petaling layer added an additional 5 pm. EDS analysis revealed a Ca / P ratio of approximately 1.6 on the petal surfaces. XRD analysis identified hydroxyapatite as the dominant phase, while FTIR analysis detected phosphate (PO43) absorption peaks at1100 cm1and 1030 cm1(vs P-0 stretching), as well as 602 cm1and 561 cm1(v4 P-0 bending). Additionally, a carbonate (CCE2) absorption band at 1450 cm1indicated carbonate substitution within the apatite structure, and the absence of the characteristic OH band at 3560 cm1suggested the formation of carb onate- substituted hydroxyapatite. The biological evaluation demonstrated that these anodized oxides exhibited the highest MC3T3-E1 cell adhesion, spreading, and proliferation compared to polished titanium controls. Cell proliferation, assessed using the CCK-8 assay, showed the highest optical density (OD) values at 1, 3, and 5 days of culture. Furthermore, alkaline phosphatase (ALP) expression was significantly elevated, confirming enhanced osteogenic differentiation.

[0103] Anodization in an electrolyte containing sodium biphosphate dehydrate, CA monohydrate, and strontium (Sr) acetate at various concentrations conducted at 450V for 10 minutes resulted in the formation of strontium-containing hydroxyapatite oxides (Sr- HAp) while the hydroxyapatite oxides (HAp-TiCE) served as controls.

[0126] The surface topography analysis of the oxides, as revealed by SEM, showed a porous structure with fine porosity on the Sr-HAp coating, while the HAp-TiCE coating exhibited a crater-like morphology. The thickness of the coatings varied, with Sr-HAp at 65 pm and HAp-TiCE at 75 pm. XRD analysis confirmed the crystallinity of the oxides with hydroxyapatite as the predominant phase in both groups, along with CaTiCE. Additionally, HaP-TiCE had a rutile phase in the coating. The biological effects of the coatings were assessed through in vitro and in vivo studies. Cell adhesion and proliferation tests showed that both groups significantly improved osteoblast attachment compared to uncoated titanium controls. Among these, HAp-TiCE revealed the highest osteoblast differentiation. Further in vivo tests conducted on rabbit femurs for 12 weeks demonstrated that the HAp-TiCE coating had the best bone-implant integration, with cortical bone growing closely along and adhering to the implant surface. Mechanical testing using push-out bonding strength measurements revealed that the HAp-TiCE oxides had the highest bonding strength (5.37 MPa) after 12 weeks. The presence of TiCE interlayer in the HAp-TiCE coating contributed to its superior mechanical stability, preventing delamination.

[0104] Anodization in a different electrolyte composition consisting of calcium chloride (CaCE) and potassium phosphate monobasic (KH2PO4) conducted at a voltage of 320- 340 V and a current of 34-35 A for 4 minutes at an electrolyte temperature of 50°C resulted in oxides having a rosette-like porous surface topography, with nano-sized hydroxyapatite crystals (50-100 nm) densely covering the surface.

[0127] The oxides werefound to be 10-25 m in thickness and demonstrated a seamless transition between the oxide and the titanium substrate, suggesting a strong adhesion, which was attributed to the formation of an amorphous CaTiCh interlayer (3-5 pm thick). EDS analysis revealed a Ca / P ratio of 1.66, and XRD confirmed the formation of a purely crystalline hydroxyapatite phase. A unique mechanism for the formation of highly crystalline hydroxyapatite was demonstrated wherein the formation of an amorphous CaTiCh interlayer was initiated, which, upon exposure to H+ions from potassium phosphate, transformed into TiO(OH)2, generating a high density of Ti-OH groups. These hydroxyl groups then acted as nucleation sites, attracting Ca2+and PO43ions from the electrolyte, leading to the crystallization and growth of hydroxyapatite. The thermal energy from anodization further promoted ion diffusion and uniform crystal growth, resulting in a dense, nanocrystalline hydroxyapatite layer.

[0105] Anodization in an electrolyte containing CA and P Ca-GP for 120 minutes resulted in a thick (63.4 pm), highly porous oxide layer with interconnected pores with an average pore size of 20 pm. [128-130] XRD analysis showed that hydroxyapatite was the dominant crystalline phase, along with CaTiCh, TCP, TiP?, and rutile. XPS analysis confirmed the presence of Ca, P, Ti, and O within the oxides. FTIR analysis identified vibrational modes characteristic of hydroxyapatite and calcium apatite-based phases. The phosphate (PO43) groups were detected at 1025-1030 cm1and 1182-1185while hydroxyl (OFF) groups were observed at 650-653 cm1and 3570-3853 cm confirming the presence of hydroxyapatite. Additionally, carbonate (CO32) vibrational modes were detected at 760-7671414-1420 cm and 1508-1518indicating potential carbonate substitution in the hydroxyapatite structure. Anodization in the same electrolyte using the same parameters for a shorter duration of 60 minutes resulted in oxides exhibiting a dense inner layer and a porous outer layer with an average pore size of approximately 8 pm and a total thickness of 47 pm. EDS mapping confirmed a uniform distribution of Ca and P with a Ca / P ratio of 1.95. XRD analysis identified anatase, rutile, TiP?, TCP, Cas(PO4)2, CaTiCh, and hydroxyapatite phases, with hydroxyapatite and CaTiCh being the dominant phases. The oxides exhibited strong adhesion to the titanium substrate as tested using a micro scratch test conducted using a 200 mm radius Rockwell C diamond tip, attributed to the presence of CaTiCh in the oxide. Additionally, the oxides also demonstrated significantly enhanced wear resistance, with a wear rate of 0.48 x 104mmVNm, compared to uncoated titanium controls. Furthermore, using these oxides as controls, anodization performed at 90 minutes in the same conditions, and electrolyterevealed oxides having higher porosity and more pronounced cracks due to thermal expansion. XRD analysis confirmed oxides to have anatase and rutile, TCP, perovskite- CaTiCh, and hydroxyapatite. The control 60-minute duration oxides had a higher proportion of rutile, while the 90-minute duration anodized oxides showed increased amounts of perovskite-CaTiCf and hydroxyapatite. Secondary apatite formation was observed on both oxides after 14 days of SBF immersion, with higher apatite crystals found in the 90-minute oxides, as seen in SEM images. FTIR analysis, conducted after 14 days of immersion in simulated body fluid (SBF), identified phosphate groups at 950- 960 cm1and 1030-1050 cm Carbonate groups were observed at 1460-1465 cm1(A- type), 1420-1425 cm1(B-type), and 870-875 cm1(bending mode in carbonated hydroxyapatite). Additionally, a band in the range of 710-767 cm1confirmed carbonate substitutions. The mechanical properties evaluation revealed that the 60-minute oxides had the lowest wear rate (0.42 x 104mm3 / N m), whereas the 90-minute oxides exhibited a slightly higher wear rate (0.84 x 104mm3 / N m). Finally, it was found that the 90- minute oxides exhibited the highest corrosion resistance.

[0106] Anodization in an electrolyte containing disodium EDTA (Na2H2EDTA 5.5H2O) as a chelating agent, along with CaO as the calcium source and sodium hexametaphosphate (NaePeOis) as the phosphate source with pH of the electrolyte was maintained at 8.9 while the temperature was maintained at 35°C throughout conducted at an applied voltage up to 300V, and an average current density of 0.4 A / cm2for a duration of 10 minutes resulted in oxides having a thickness of 32 pm, with the surfaces having a porous topography containing densely packed round pores and craters of uniform size (6- 8 pm for large pores and 0.5-2 pm for small pores).

[0131] The EDS analysis revealed a Ca / P atomic ratio of 1.18 in surface layers and 1.24 in cross-sections. The coatings primarily consisted of P-TCP and hydroxyapatite, as identified by XRD analysis, along with anatase and rutile phases. After thermal treatment at 800°C, the crystallinity improved, and the hydroxyapatite peaks became more defined. Anodization in a 400 mL electrolyte solution containing sulphuric acid (H2SO4), P-GP, and CA conducted at 350V and 70 mA / cm2for 10 minutes at room temperature (~25°C) resulted in oxides exhibiting needle-like hydroxyapatite structures as confirmed by SEM and XRD analysis.

[0132] The contact angle measurement was unmeasurable, as the water droplet was rapidly absorbed, indicating high hydrophilicity. Anodization using a more concentrated electrolyte volume of 200 mL instead of 400 mL, without the addition of sulfuric acid, while maintaining the same anodization waveform parameters resulted in oxides exhibiting well-defined surfacetopography consisting of microporous, donut-shaped structures (1-6 pm) embedded within a TiCE matrix.

[0059] During the anodization process, the pH decreased from approximately 7.8 to 6.25 due to hydrolysis and ionization reactions, indicating significant electrolyte transformation and enhanced ion incorporation. EDS analysis revealed a Ca / P ratio of 2.3, with Ca and P incorporation of 7.46 at% and 3.23 at%, respectively. XRD confirmed the presence of hydroxyapatite, a-TCP, and CaCCE, along with anatase and rutile. Biological evaluations indicated that the oxides had significantly enhanced osteoblast viability and mineralization compared to untreated titanium controls. These surfaces showed sustained osteoblast proliferation over 14 days, while Alizarin red staining confirmed early-phase calcium deposition and osteogenic differentiation without the need for additional growth factors. These oxides, with a thickness of 18.6 pm, were evaluated through microhardness indentation testing (HV 0.3), revealing an average hardness of 350 MPa. Their adhesion to the titanium substrate was examined using microscratch testing and demonstrated strong bonding.

[0107] Magnesium Doped Coatings'. Magnesium (Mg) is the fourth most abundant element in the body and is the most abundant in cells in its ionic form. [133,134] Approximately 50 % of Mg in the human body is stored in bones.

[0135] Mg ions (Mg2+) play a vital role in bone metabolism and osseointegration by regulating osteoblast activity, mineralization, and cellular adhesion. [136,137] Mg2+regulates cellular functions like growth and proliferation, signaling, and metabolism.

[0138] Mg2+also plays a crucial role in integrin-mediated cell adhesion, wherein it facilitates integrin-ligand interactions, particularly with RGD motifs, thereby strengthening cellular adhesion and supporting early bone formation. [136,137] The presence of Mg2+in bone tissue is associated with improved structural integrity, as it modulates bone turnover and prevents osteoclast differentiation and bone resorption. [133,139] Mg-dopant additions incorporated onto titanium surfaces have been shown to enhance osseointegration by promoting osteogenesis, cell adhesion, and angiogenesis through pathways such as PI3K, ERK, and BMP-4.

[0140] Anodized coatings on titanium implant surfaces, followed by subsequent Mg incorporation by hydrothermal treatment, have been shown to enhance osteogenic differentiation in bone.

[0141] Previously, Mg -hydroxyapatite combination sol-gel dip coatings on titanium surfaces showed improved interfacial coating adhesion strengths and push-out forces compared to the hydroxyapatite-only counterparts.

[0142] Furthermore, micro-CT analyses of these Mg-hydroxyapatite combination coatings showed improved trabecular bone formation and osseointegration abilities.

[0108] Anodization of titanium in a Mg2+ions containing mixed electrolyte system at high forming voltages and current densities in galvanostatic mode resulted in 3.4 gm thick oxides exhibiting a porous structure with numerous craters, with an average pore size of 1.5 pm, while machined titanium controls had a smooth, non-porous surface with an oxide thickness of 17.4 ± 6 nm.

[0143] XPS analysis of the oxides confirmed the presence of TiCh and Mg (9.3 at%). XRD revealed a mixture of anatase and rutile phases. The biological in vivo evaluation in a rabbit tibia model demonstrated significantly enhanced osseointegration speed and mechanical fixation. RTQ values were 15.3 N.cm at 3 weeks and 20.1 N.cm at 6 weeks, compared to machined implant controls with RTQ values of9.4 N.cm at 3 weeks and 12.7 N.cm at 6 weeks. Bonding failure analysis revealed that implants with Mg oxides fractured within the bone tissue while the machined control implants failed at the bone-implant interface.

[0109] Anodization in an electrolyte composed of 1.5 M phosphoric acid (H3PO4) and1.5 M magnesium sulphate (MgSOQ performed at 150 V and 0.5 A for 10 minutes at room temperature (25 °C) resulted in oxides having a porous surface topography, with pores 5-10 times larger than standard anodic porous titania (100-200 nm).

[0144] Additionally, EDS mapping demonstrated a uniform distribution of Ti, Mg, and P across the oxide surfaces, confirming a consistent and homogeneous distribution of Mg (~1.3 at%) and P (-10 at%). Raman spectroscopy revealed peaks for anatase-phase in the oxides.

[0110] Anodization in an electrolyte solution containing calcium gluconate (6 g / L), sodium hexametaphosphate (6 g / L), sodium hydroxide (8 g / L), ethylenediaminetetraacetic acid (EDTA, 2 g / L), and magnesium gluconate (15 g / L) conducted using a unipolar square pulse at a constant voltage of 430 V, a frequency of 1000 Hz, a duty cycle of 30%, and a treatment duration of 5 minutes resulted in oxides containing a microporous surface topography with evenly distributed pores measuring 2- 6 pm in diameter and nanograins ranging from 30 to 60 nm.

[0145] EDS analysis confirmed a high uptake of Mg (15.1 wt%), along with Ti (12.3 wt%), O (43.2 wt%), P (13.5 wt%), and Ca (9.2 wt%). Additionally, XPS identified the presence of MgO, with a homogeneous distribution of Mg throughout the oxide surface. XRD analysis confirmed the presence of anatase and rutile phases. Mg2+ion release from the oxides was assessed using inductively coupled plasma mass spectrometry (ICP-MS), revealing a sustained release over 12 days, with cumulative Mg2+ion concentrations reaching approximately 50 ppm in the surrounding medium. Biological in vitro evaluations demonstratedenhanced protein adsorption, along with significantly improved MC3T3-E1 osteoblast adhesion, spreading, and proliferation. Gene expression analysis indicated upregulation of osteogenic markers, including ALP, BMP-2, RUNX2, OCN, and COL-I, confirming enhanced osteogenic differentiation. Furthermore, Western blot analysis showed a significant increase in ERK phosphorylation and c-Fos expression, suggesting that Mg- enriched oxides activated the ERK / c-Fos signalling pathway. In vivo osseointegration studies using a rabbit femoral implantation model demonstrated significantly greater bone volume and trabecular thickness around the Mg-enriched implants compared to non-Mg- containing control groups. Micro-computed tomography (micro-CT) and histological analysis confirmed increased new bone formation, with well -organized trabeculae and a higher BIC. Toluidine blue and Van Gieson staining revealed extensive mineralized tissue formation around the implants. By 16 weeks, continued bone remodelling and mineralization was observed, with no signs of inflammation, fibrous encapsulation, or implant loosening.[OHl] Anodization in an electrolyte composed of disodium ethylenediamine-tetra- acetate (Na2H2EDTA 5.5H2O) as a chelating agent, with CaO serving as the Ca source, sodium hexametaphosphate (NaePeOis) as the P source, and a Mg additive conducted at an applied voltage of up to 300V, an average current density of 0.4 A / cm2for a duration of 10 minutes resulted in 13 pm thick oxides having a porous surface topography characterized by a uniform distribution of round pores and craters.

[0131] EDS analysis confirmed the complete substitution of Ca with Mg, with an atomic Mg / P ratio of 0.80 in the surface layers and 0.47 in the cross-sections. XRD analysis identified the presence of anatase and rutile phases, along with magnesium carbonate (MgCCh) and dolomite (CaMg(COs)2). Following thermal treatment at 800°C, the crystallinity of the oxides improved, and Mg-rich phases, such as Whitlockite (Cag.sMgo.sPvCEs), dominated the surfaces.

[0112] Anodization in 85% phosphoric acid (EEPO^-based electrolyte containing 166.7 g / L calcium nitrate tetrahydrate (Ca(NOs)2 4H2O), 166.7 g / L magnesium nitrate hexahydrate (Mg / NCff-OILO), and 166.7 g / L of either copper(II) nitrate trihydrate (Cu(NOs)2 3ILO) or zinc nitrate hexahydrate (Zn(NOs)2 6H2O) conducted at 650 V for 3 minutes resulted in oxides having a highly porous and rough surface topography.

[0146] EDS analysis determined a Ca / P atomic ratio of 0.071, while XRD analysis confirmed the presence of Ca(EL>PO4)2 EL>0 (monocalcium phosphate monohydrate) andTi(HP04)2-H20 (titanium hydrogen phosphate hydrate) in both the zinc- and copper- containing oxide groups.

[0113] Anodization in an electrolyte composed of 0.35 M CA monohydrate, 0.02 M P- GP pentahydrate, and 0.1 M magnesium acetate tetrahydrate conducted at 300 V and 2.5 A for 1 minute resulted in 7.7 pm thick oxides with a highly porous surface topography.

[0147] The Rockwell C. indentation adhesion test revealed the oxides to have better adhesion compared to those formed with longer treatment durations, as fewer cracks propagated around the indentation site. EDS analysis confirmed the successful incorporation of Ca (-4.5 at.%), P (~1.8 at.%), and Mg (-1.7 at.%), with a Ca / P ratio of approximately 2.5, while XRD analysis revealed the presence of both anatase and rutile phases, with anatase being the predominant phase.

[0114] Chelating acids including multiple carboxylic acid groups '. The structure of citric acid consists of multiple carboxylic acids, and citric acid is a chelating acid that can chelate metal ions.

[0148] Additional or alternative chelating acids that include multiple carboxylic acid groups include ethylenediaminetetraacetic acid (EDTA), malic acid, tartaric acid, ascorbic acid, oxalic acid, and / or succinic acid. [148-153] The concept of using chelating acids that include multiple carboxylic acid groups, such as citric acid and / or malic acid, for anodization processes to form hydroxyapatite on titanium implant surfaces has never been explored before.

[0115] Phosphate binders: Almost all the above anodization studies that created hydroxyapatite on their surfaces have made use of electrolyte compositions similar to calcium acetate and P-glycerophosphate CaP electrolytes. An early CaP study started using calcium acetate as a potential source of calcium in their electrolytes due to its high solubility and the ability to produce high Ca / P ratios on the surface of titanium.

[0154] Thereafter, almost all the studies that have formed hydroxyapatite on the surface using anodization have used calcium acetate. [132,155-159] Calcium acetate is known to be a strong phosphate binder.

[0160] Besides calcium acetate, calcium carbonate, and calcium citrate are also well-established phosphate binders. Calcium acetate and calcium carbonate have been used clinically as treatment for hyperphosphatemia. Calcium citrate is a weak phosphate binder and is less commonly clinically. [160-162] In patients with Chronic Kidney Diseases (CKD), these phosphate binders work by exchanging the phosphate anion with an active cation (acetate, carbonate, or citrate) to form a nonabsorbable compound that can be excreted from the body.

[0163] The concept of usingother calcium-based phosphate binders for anodization processes to form hydroxyapatite on titanium implant surfaces has never been explored before.

[0116] The following disclosure describes several embodiments of anodization methods, titanium compositions formed thereby, and implantable devices that include such titanium compositions. Any of the methods, concepts, and / or features described in the foregoing overview can be used in conjunction with the embodiments described below, except where indicated otherwise or where clearly incompatible.Selected Terms & Definitions

[0117] Modifications to embodiments described in this document and other embodiments will be evident to those of ordinary skill in the art after a study of the information provided in this disclosure. The information provided in this disclosure, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding, and no unnecessary limitations are to be understood therefrom. Further, while the terms used herein are believed to be well-understood by one of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0118] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

[0119] Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “an implantable device” includes a plurality of such devices.

[0120] When the term “about” or its synonyms is used in conjunction with a stated amount, value, or condition, it may be taken to mean an amount, value or condition that deviates by less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the stated amount, value, or condition.

[0121] Unless indicated otherwise, percentages expressing proportional amounts are made on a weight basis.

[0122] The term “exemplary” is used herein synonymously with the term “example.” An “exemplary embodiment” is therefore understood as an example embodiment that canillustrate features and / or concepts of the general invention and does not necessarily indicate that it is the most desired or preferred embodiment.Embodiment 1: Anodization Process Using Citrus Fruit Juice-Based Electrolyte to Form an Oxide Coating that Includes Calcium Titanate Crystalline Compounds

[0123] Embodiment 1 (El) of the present invention is a fruit juice-based anodization process for modifying titanium implant surfaces to form an oxide layer containing calcium titanate crystalline compounds and hierarchical micro and nanoscale surface roughness profiles.El Materials and methods

[0124] Specimen preparation: Commercially pure titanium grade 4 (CPTi4) disc specimens were made by cutting a 12.7 mm bar stock to 3 mm thickness. The cut specimens were cleaned ultrasonically using laboratory detergent (Alconox®, White Plains, NY, USA), followed by rinsing with distilled water. The discs were then dipped in a 10: 1 ratio solution of nitric acid-hydrofluoric acid (TURCO NITRADD, Henkel Corporation, Madison Heights, MI, USA) for a period of 30 seconds to activate the surface for anodization.

[0125] Anodization'. Three citrus fruit-based electrolytes were developed for this study, as shown in Table 1 and Figure 3. The first electrolyte (CFOJ) consisted of a mixture of commercially available calcium-fortified orange juice (Kroger, Cincinnati, OH, US) and 0.15M calcium acetate (99% Spectrum chemical, New Brunswick, NJ, US). The other two electrolytes required juicing navel oranges (Kroger, Cincinnati, OH, US) and mandarins (Kroger, Cincinnati, OH, US) using a commercially available fruit juicer (Eurolux ELCJ-1800S, 300 W, China). The second electrolyte (OJ) consisted of a mixture of juice from juicing 5 navel oranges, monobasic calcium phosphate (90%, Thermo Fisher Scientific, Waltham, MA, US), and calcium acetate. The third electrolyte (MJ) consisted of a mixture of juice from juicing 7 mandarins, monobasic calcium phosphate, and calcium acetate. The pH of all three electrolytes was recorded prior to anodization. The anodization reaction cell consisted of a 500 ml beaker of each electrolyte, the CPTi4 disc anode, and two CPTi4 strip cathodes. Anodization of activated disc specimens was performed using a DC rectifier (350 V, 10 A, Dynatronix, Amery, WI) using a galvanostatic direct current density waveform. Current densities of 700 mA / cm2 were applied for 20 seconds in the CFOJ electrolyte, and current densities of 1000 mA / cm2 were applied for 20 seconds in the OJ and MJ electrolytes. Multiple specimens (n=4) wereanodized in each electrolyte, and the final forming voltage was recorded for each specimen.Table 1. Composition of anodization electrolytes for forming CaP oxides.Oxide Citrus fruit-based electrolyte Calcium Phosphate CalciumGroup (M) AcetateCFOJ Calcium-fortified orange juice - 0.15(500 mL)OJ Orange juice (500 mL) 0.07 0.15MJ Mandarin juice (500 mL) 0.07 0.15

[0126] Oxide surface characterization: Optical microscopy imaging (Keyence, Osaka, Japan, VHX-1000) and Scanning electron microscopy (SEM, Zeiss, Jena, Germany, Supra 40) were used to document the surface morphology of the anodized surfaces on micro-nano scale dimensions. SEM imaging was conducted using a 12 kV accelerating voltage. Atomic force microscopy (AFM, Bruker, Santa Barbara, CA, Bioscope Catalyst) was also used in ScanAssyst mode (0.25 Hz, and 512 samples / line) to capture the micro and nano-scale surface roughness topographies of the anodized surfaces. 50 x 50 pm2and 1 x 1 pm2scan areas were collected on multiple specimens (n=4) from each anodized group. Gwyddion software was used to calculate average roughness (Sa) and peak-to- valley roughness (Sz) values for each specimen. Energy dispersive X-ray spectroscopy (EDS, EDAX, Mahwah, NJ, TEAM EDS Software Suite) was used to collect bulk chemistry spectra from the anodized oxide surfaces using an SEM accelerating voltage of 12 kV, magnification of 5000X, and a 30 pm SEM aperture to identify the bulk oxide chemistry. Spectra were collected from triplicate areas from each specimen on each oxide group (n=4). Average atomic percentage values for each element present in each oxide group were calculated from the EDS spectra datasets. This methodology allowed the calculation of the Ca and P dopant uptake levels into the anodized oxide layers and, thus, the calculation of the surface oxide Ca / P ratio for each oxide group. Since the below- surface interaction volumes for EDS of our oxides penetrated to depths that extended through the entire oxide layer thickness and into the surface of the CPTi substrate material, it was of interest to examine the oxide-only surface chemistry using a different analytical technique. The oxide-only surface chemistry of the anodized surfaces was measured using X-ray photoelectron spectroscopy (XPS, Thermo Scientific, Waltman, MA, K-Alpha XPS System Avantage v5.9911 Software Suite) for comparison to the EDS datasets. The XPS system was equipped with a monochromatic X-ray source at 1486.6eV, corresponding to the Al Kaline. The X-ray power of 75 W at 12 kV was used for all experiments with a spot size of 400 pm2. The base pressure of the K-Alpha instrument was at 9.0 x IO'10mbar. The instrument was calibrated to give a binding energy of 84.0 eV for Au 4f? / 2 and 284.8 for the Cis line of adventitious (aliphatic) carbon present on the non-sputtered samples. Photoelectrons were collected from a take-off angle of 90° relative to the sample surface. A series of XPS spectra were done in the Constant Analyzer Energy mode. Triplicate survey spectra were collected from different areas of a representative specimen (n=l) from each oxide group at a pass energy of 200 eV and an energy step size of 1.0 eV. Triplicate high resolution (HR) core level spectra of C Is, O Is, P 2p, Ca 2p, and Ti 2p were also taken from different areas of a representative specimen (n=l) from each oxide group at a 40 eV pass energy, an energy step size of 0.1 eV, and using an average of 50 scans. Thin-film X-ray diffraction (XRD, XDS 2000, Scintag, Franklin, MA) was utilized to check for the presence of crystalline anatase or rutile titanium oxide phases and titanate compound formation within the anodized oxide layers. Triplicate representative specimens from each group were rotated 1° away from the copper X-ray source (0.154 nm Cu-Ka) to enhance the X-ray interaction volume within the thin anodized layers. XRD scans were conducted at two-theta angles ranging from 20° to 80° since this range contains the highest intensity diffraction peaks for anatase, rutile, and titanate compounds.

[0127] Oxide cross-sectional characterization: Representative specimens from each oxide group were sectioned, mounted in conductive epoxy, and polished in order to view the cross-sectional thicknesses of the citrus-based oxide layers in the SEM. Five oxide cross-sectional images were collected per specimen, and five thickness measurements were performed on oxide each image, resulting in a total of 25 thickness measurements per oxide group.

[0128] Ion release rates: Inductively coupled plasma optical emission spectrometry (ICP-OES, SPECTRO AMETEK, SPECTROGREEN, Kleve, Germany, SPECTRO ICP Analyzer Pro Software) was used for measuring the release profiles and cumulative release profiles of Ca2+ions from the CFOJ group. Representative anodized disc specimens (n=3) were immersed in 10 mL of calcium-free phosphate-buffered saline (PBS) solution (MP Biomedicals, Santa Ana, CA, USA). The specimens were transferred to fresh solution at specific time points: 2, 4, 6, 8, 10, 12, 14, 18, 22, and 30 days. The large particulate matter in each collected solution was dissolved by adding 300 pL of nitric acid and 50 pL of hydrochloric acid, followed by filtration using 0.4 pm syringefilters. The filtered solutions were analyzed for Ca2+ion release concentrations in parts per million (ppm).

[0129] El Results The CFOJ, OJ, and MJ citrus fruit-based electrolyte mixtures shown in Table 1 and Figure 3 revealed pH values of 4.96, 5.14, and 5.10, respectively. The final forming voltage ranges recorded for all the specimens anodized using galvanostatic waveforms in the CFOJ, OJ, and MJ electrolytes are compiled in Figure 4. Interestingly, the citrus fruit-based oxides formed in each electrolyte revealed similar final forming voltage ranges of 230 ± 9 V, 233 ± 10 V, and 234 ± 2 V, respectively.

[0130] Oxide surface morphology and roughness: Optical microscopy and SEM images for each citrus fruit-based oxide group are shown in Figure 5. Low-magnification optical microscopy images are shown in the left column of Figure 5 and reveal a greyish-white surface appearance for each oxide group. SEM images at higher magnifications for each group revealed a complex micro and nanoscale surface topography, as shown in the middle and right columns of Figure 5. The nanoscale surface topography for each oxide group exhibited a unique cauliflower-like surface morphology. Representative AFM 50 x 50 pm2 and 1 x 1 pm2 scan areas from each citrus-based oxide group are shown in Figure 6 and confirmed the multiscale micro and nano-scale surface roughness profiles for each oxide. The average surface roughness, Ra, and peak-to-valley roughness, Rz, values for each citrus-based oxide group are compiled in Figure 7. 50 x 50 pm2 scan areas showed Ra values less than 500 nm, while 1 x 1 pm2 scan areas showed true nanoroughness Ra values below 90 nm. Additionally, the unique cauliflower-like nanoscale surface topographies were confirmed within the 1 x 1 pm2 AFM roughness profiles for each group.

[0131] Oxide surface chemistry: Representative EDS and XPS spectra for each citrus fruit-based oxide group are shown in Figures 8 and 9. EDS and XPS dopant uptake levels for Ca and P within each citrus fruit-based oxide group are compiled in Figure 10. EDS spectra revealed significant dopant uptake of Ca and P in all citrus fruit-based oxide groups, as shown in Figures 8 and 10. Approximately 6 at % of Ca and 4 at % of P were incorporated into each oxide group as shown in Figure 10A. The resulting average EDS Ca / P ratios for CFOJ, OJ, and MJ, as shown in Figure 10C, were 1.44 ± 0.18, 1.69 ± 0.1, and 1.7 ± 0.2, respectively. Thus, the EDS oxide surface Ca / P ratios were all shown to fall within the 1.5 to 1.7 Ca / P ratio range commonly reported for human bone. [49,84,91] XPS survey spectra collected from the outermost surface of the citrus fruit-based oxides exhibited Ti, O, Ca, P, N, and C peaks, as shown in Figure 9A. The high-resolution spectrafor the Ti2Ppeaks are shown in Figure 9B. The formation of TiCh is represented by the Ti2P3 / 2 peaks having a binding energy of 459 eV. [48,92] Figure 9C shows the Ois peaks having a binding energy of 531.05 and represents the formation of calcium phosphate- containing oxides. [75,92] The Ca2P3 / 2 peaks for each oxide group revealed binding energies of approximately 347.6 eV, as shown in Figure 9D, representing the formation of CaTiCh and calcium phosphates. [48,52,75,92,128,164] The ?2Ppeaks for each oxide group exhibited binding energies of 133.6 eV, as shown in Figure 9E, and represent the formation of phosphorus-containing titanium oxides and phosphates. [48,75,128] Finally, the Cis peaks for each citrus fruit-based CaP oxide group exhibited binding energies of approximately 285 eV and 289 eV providing evidence of C-C bonds and carbonates, as shown in Figure 9F. [48,75] The 285 eV binding energy peaks for Cis are generally attributed to carbon surface contamination. [51,165] The Ca and P dopant uptake shown in the XPS spectra for the outermost oxide surfaces of each citrus fruit-based oxide group is compiled in Figure 10B. Comparing Figures 10A and 10B, the XPS Ca and P dopant uptake levels were very similar to those shown by the EDS spectra, which likely represented the entire oxide cross-sectional thickness as well as the surface contribution from the CPTi substrate. However, the XPS spectra showed slightly higher Ca and P uptake levels when directly compared to the counterpart EDS spectra values. Nonetheless, the resulting XPS spectra Ca / P ratios from the outermost oxide surfaces were still all shown to fall within the ranges commonly reported for human bone. [49,84,91]

[0132] Oxide crystallinity: Representative XRD scans of the citric fruit-based oxides are shown in Figure 11. Full two-theta scan range of 20° to 80° is demonstrated on the left side. A zoomed-in two-theta angle region of 24° to 36° is shown on the right side to emphasize the formation of crystalline titanate phases. CaTiOs was formed in each of the citrus-based oxides which agrees well with the XPS high-resolution Ca2P spectra results that also suggested CaTiCE formation in the outermost layers of the citrus fruit-based oxides. Additionally, all three oxides also showed diffraction peaks for the anatase titanium dioxide phase and hexagonal a-phase substrate CPTi material.

[0133] Oxide thickness: All three citrus fruit-based oxides showed similar thickness values, as shown in Figure 12. The average oxide thickness value for each of the oxide groups was found to be less than 500 nm. Specifically, the CFOJ electrolyte oxide revealed thickness values of 490 ± 59 nm. The juiced navel orange electrolyte OJ oxide showed thickness values of 447± 38 nm. Finally, juiced mandarin MJ oxides revealed oxide thickness values of 417 ± 54 nm.

[0134] Calcium ion release profiles: ICP-OES -derived 30-day Ca2+ion cumulative release profile from the CFOJ oxide group is provided in Figure 13. The profile revealed an initial burst release followed by a sustained release over a period of 30 days, with a cumulative amount reaching an average of 76 ppm by day 30.El Discussion

[0135] Titanium anodization processes incorporating Ca and P dopants into CaP oxide layers have been extensively explored previously with the goal of improving osseointegration. [82,84,90,103,166] Most of these CaP oxide studies have continued to use anodization electrolytes similar to the original P-glycerophosphate and calcium acetate-based electrolyte formula that was shown to obtain oxide surface Ca / P ratios within the commonly reported range of human bone. [23,84, 101]Here, we developed novel anodization electrolytes based on combinations of juiced citrus fruit and commercially available calcium additives, which readily formed oxides that exhibited hierarchical micro and nano-scale surface topographies and surface Ca / P ratios within the range of human bone. Anodization in citric acid-based electrolytes has been previously explored for other purposes, such as the hard anodization of aluminium alloys and improving the corrosion resistance and oxide crystallinity of titanium alloys. [167-170] However, the use of the fruit juice-based anodization process of embodiment 1 has never been explored for the purposes of forming Ca and P doped titanium oxides on implant surfaces. Calcium acetate and monobasic calcium phosphate additives are commonly used as food stabilizers and leavening agents in baking products. [88,171]

[0136] While a number of previous CaP oxide studies have successfully achieved surface Ca / P ratios above 1.0 and even within the range of human bone, the Ca / P ratio alone does not provide sufficient information as to the actual amounts of Ca and P incorporated into the oxide layers. Earlier anodization studies incorporating either Ca or P dopants revealed oxide groups showing higher dopant uptake to exhibit increased alkaline phosphatase activity in cell culture studies and increased removal torques from anodized screws implanted into rabbits. [47,51,52] Nonetheless, relatively few CaP oxide studies have reported the actual Ca and P dopant uptake percentages along with the surface Ca / P ratios. One study combining EDTA with a pure calcium phosphate powder and calcium acetate electrolyte showed that the Ca dopant uptake and the surface Ca / P ratio increased with electrolyte pH.

[0092] Specifically, increasing the electrolyte pH from 6 to 14 by adding EDTA increased the Ca dopant uptake from 10 to 16%.

[0092] Another study anodizing in a P-glycerophosphate disodium and calcium acetate electrolyte showed approximately5% Ca dopant uptake.

[0048] Our EDS and XPS analyses for oxides formed in the citrus fruit-based electrolytes showed a similar range of Ca dopant uptake of 6% to 8%. Additionally, there was a sustained release of substantial amounts of Ca2+ions from the CFO J oxide group over a 30-day period.

[0137] It has been commonly known that the adhesion strength of anodized titanium oxides generally decreases with increasing oxide cross-sectional thickness values. [52,80,83] Many previous Ca and CaP-doped oxide studies have used galvanostatic waveforms with low current densities of up to 50 mA / cm2,resulting in oxide thickness values ranging from 5 pm to 200 pm. [84,88,89,103,172] One recent CaP-oxide study used galvanostatic waveforms with higher current densities ranging from 400 to 1200 mA / cm2,which also resulted in oxide thickness values between 3 pm and 8 pm.

[0091] In contrast, the fruit juice-based electrolytes in El were formed using galvanostatic waveforms with current densities of 700 or 1000 mA / cm2and revealed much thinner oxides of approximately 0.5 pm. These comparatively thin citrus-based CaP oxides were still shown to exhibit multiscale micro and nano surface topographies, high levels of Ca and P dopant uptake, Ca / P ratios within the range of human bone, and the formation of titanate compounds that have been shown to be an important precursor for apatite formation. Thus, fruit juice-based CaP oxides of embodiment 1 in the present invention, exhibiting calcium titanate formation show much promise for hydroxyapatite formation during future bioactivity assessments.

[0138] Since most CaP oxide studies have used electrolytes similar to the [3- glycerophosphate and calcium acetate combinations, the resulting oxides have, almost exclusively, shown micro and nanoscale porous surface topographies and the formation of mixed anatase and rutile phase oxide crystallinity. [82,84] However, oxides formed using this anodization electrolyte composition have shown difficulty forming apatite in SBF bioactivity testing methodologies similar to the method of the ISO 23317 standard.

[0166] In fact, the originally formed CaP oxides required subsequent hydrothermal autoclaving treatments at extreme temperatures of 300 °C for a two-hour duration prior to SBF soaking to form apatite on the oxide surfaces. [82,84]

[0139] The surface topography and the chemical composition of an implant surface have both been shown to play a critical role in the resulting biological response.

[0023] Several research groups have explored alterations in the electrolyte chemistries with the goal of improving the bioactivity of CaP oxides. [101,102,125] Previous Ca and CaP-doped oxide studies have shown titanate compound formation to be an important precursor forfuture apatite formation. [94,102,103] In keeping with that recent CaP oxide studies have used microwave treatments or UV after anodization treatments in order to form beneficial titanate compounds. [48,173] One research group formed oxides using anodization at 350 V and 70 mA / cm2in a 50% P-glycerophosphate and calcium acetate and 50% NaOH electrolyte to form a nanoflower surface topographies with an anatase, rutile, and sodium titanate oxide crystallinity.

[0102] The nanoflower oxide layers containing the titanate crystalline compounds were shown to precipitate apatite on the surfaces after only a seven-day soak using SBF bioactivity testing.

[0102] Interestingly, the fruit juice-based anodization process in El exhibited pH values between 5 and 5.15 and formed unique cauliflower-like nano-scale surface topographies with anatase and calcium titanate oxide crystallinity as evidenced by SEM, AFM, XPS, and XRD analyses.

[0140] Conclusions of Embodiment 1: Anodization processes allow modifications of the oxide surface topography and the incorporation of beneficial dopant elements, such as Ca and P, into the oxide layers with the goal of improving osseointegration. Previous studies have used anodization electrolytes with combinations of P-glycerophosphate and calcium acetate to form CaP-doped oxides with similar surface Ca / P ratios to human bone. In Contrast, El includes a novel anodization process based on juiced citrus fruits and commercially available calcium additions to form CaP oxides. The citrus-based CaP oxides of the present invention exhibit a hierarchical micro and nano-scale surface topography with a unique cauliflower-like surface morphology on the nanoscale. In addition, the citrus-based CaP oxides of the present invention revealed significant Ca and P dopant uptake into the oxide layers, leading to a Ca / P ratio in the range of human bone. Finally, these oxides revealed anatase and calcium titanate formation, which both have been shown to be favorable precursors for apatite formation on the oxide surfaces. Moreover, the citrus-based CaP oxides revealed sub-micron oxide thickness values which have been suggested to correlate with high oxide adhesion strengths to titanium substrate materials. Thus, the citrus-based CaP oxides of the present invention can be utilized to provide implants with improved osseointegration potential.Embodiment 2: Anodization Process Using Citrus Fruit Juice-Based Electrolyte to Form an Oxide Coating that Includes Tri-calcium Phosphate and Hydroxyapatite Crystalline Compounds

[0141] Embodiment 2 (E2) of the present invention is a fruit juice-based anodization process for modifying titanium implant surfaces to form an oxide layer containing tri-calcium phosphate and hydroxyapatite crystalline compounds and hierarchical micro and nanoscale surface roughness profiles.E2 Materials and methods

[0142] Specimen preparation: Commercially pure titanium grade 4 (CPTi4) discs were cut to 3 mm thickness from a 12.7 mm diameter bar stock. The CPTi4 discs were then briefly wet ground with 320 grit SiC paper to remove any rough edges generated during cutting, ultrasonically cleaned with laboratory detergent (Alconox®, White Plains, NY, USA), and rinsed with distilled water. The cleaned discs were then dipped in a nitric acid-hydrofluoric acid (10: 1 ratio) solution (TURCO NITRADD, Henkel Corporation, Madison Heights, MI, USA) for 30 s to activate the surface for anodization.

[0143] Anodization: The citrus fruit-based anodization electrolytes used are provided in Table 2 and Figure 14. Each electrolyte consisted of a mixture of commercially available calcium-fortified orange juice (Calcium-Fortified Orange Juice, Kroger, Cincinnati, OH, US), monobasic calcium phosphate (90%, Thermo Fisher Scientific, Waltham, MA, US), and calcium acetate (99% Spectrum chemical, New Brunswick, NJ, US). The calcium phosphate and calcium acetate powders were dissolved into the calcium-fortified orange juice to produce the final molarities listed in Table 2. The anodization reaction cell consisted of a 500 mL beaker of each electrolyte, the CPTi4 disc anode specimen, and two CPTi4 strip cathodes. Activated disc specimens were anodized using a DC rectifier (350 V, 10 A, Dynatronix, Amery, WI) using a pulsed-galvanostatic waveform. Pulse current densities (700 mA / cm2, 29% duty cycle, frequency of 7.2 Hz) with a 40 ms on time / 99 ms off time were applied for a period of 120 seconds in each electrolyte. We recently used the same anodization waveform in another hydroxyapatite-containing oxide study.

[0061] Table 2. Composition of anodization electrolytes for forming calcium-rich oxides.Group Calcium Fortified Orange Calcium Phosphate Calcium AcetateJuice (M) (M)Oxide A 500 mL - 0.15Oxide B 500 mL 0.15 0.2Oxide C 500 mL 0.15 0.25Oxide D 500 mL 0.15 0.275

[0144] Oxide surface characterization: Thin-film X-ray diffraction (XRD, XDS 2000, Scintag, Franklin, MA) was used to determine the crystalline phases present within the CPTi4 titanium substrate material and each of the anodized oxides. Representative specimens (n=4) were rotated 1° away from the copper X-ray source (0.154 nm Cu-Ka)to improve the interaction volume of X-rays. XRD scans were performed over a 20° to 80° two-theta range. Optical imaging (Keyence, Osaka, Japan, VHX-1000) and Scanning electron microscopy (SEM, Zeiss, Jena, Germany, Supra 40) were used for characterizing the topography of representative specimen surfaces. SEM imaging was done using a 12 kV accelerating voltage at 5,000X to capture the surface topography at micron-level, while a lower 9 kV accelerating voltage was used at 75,000X to avoid surface charging effects. A 3D optical profilometer (Keyence, Osaka, Japan, VK-X3000) was used to measure the surface roughness values from three randomized areas of each oxide specimen. The average roughness (Sa) values and peak-to-valley roughness (Sz) values for each oxide were calculated using Gwyddion software from the 3D profiles. Energy dispersive X-ray spectroscopy (EDS, ED AX, Mahwah, NJ, APEX EDS Software Suite) was used to collect the oxide surface chemistries using an accelerating voltage of 12 kV at 500X. Triplicate areas on each specimen were used to collect the representative EDS spectra. The average surface concentrations of each element and the respective surface Ca / P ratios were calculated for each oxide. Finally, attenuated total reflectance fourier transform infrared spectroscopy (ATR FTIR) was used on representative specimens from each group to determine molecular differences. A Spectrum 100 FTIR (Perkin-Elmer, Waltham, MA) was used over the range of 650-4000 cm'1at a 100 cm'1spectral resolution.

[0145] Oxide cross-sectional characterization: Representative oxide specimens were cross-sectioned and mounted in a conductive epoxy (Polyfast, Struers, Cleveland, OH, USA), wet ground with 220 grit SiC paper, polished with a 9 pm diamond suspension, and then to a final surface finish in a 0.02 pm colloidal silica suspension (Struers, Cleveland, OH, USA). Ten cross-sectional images were collected from each oxide, and five thickness measurements were carried out on each image. This produced a combination of 50 cross-sectional thickness measurements per oxide. Triplicate EDS line scans were collected on each oxide using a 12kV accelerating voltage and a magnification range of 5000-10000X in order to determine the compositional variations throughout the oxide cross-sections. A scan resolution level of 10 data points per micron for thinner oxide layers and 3 data points per micron for thicker oxide layers was used to estimate the chemistry of the outermost oxide surfaces. We recently used these same oxide cross- sectional characterization methods in another hydroxyapatite-containing oxide study.

[0061]

[0146] Oxide adhesion quality: The VDI 3198 standard Rockwell C indentation test was used for the evaluation of the adhesion quality of all the anodized oxides.

[0174] This is an indentation-based adhesion strength assessment and has been used frequently in other implant coating studies. [23,59,175,176] Three indentations were performed on representative specimens from each of the oxides using a Rockwell C indenter with a load value of 150 kg, followed by imaging using an optical microscope. The obtained images were compared to the VDI 3198 standard adhesion quality maps. [23,59,174-176]

[0147] Oxide microhardness assessment: The anodized oxide microhardness was evaluated using a Vicker’s microhardness tester (Clark CM-400AT, Sun-Tec, Novi, Michigan, USA) with a 300 g load and a dwell time of 12 s (HV 0.3). Ten indentations were placed into representative oxide specimens and evaluated. This microhardness method has also recently been used in another anodization study.

[0059]

[0148] XPS surface analyses: X-ray photoelectron spectroscopy (XPS, Thermo Scientific, Waltman, MA, K-Alpha XPS System Avantage v5.9911 Software Suite) characterization was also performed to further characterize the outermost surface layer of the hydroxyapatite containing oxide. The XPS system having a monochromatic X-ray source at 1486.6 eV, conforming to the Al Kaline, and an X-ray power of 75 W at 12 kV with a spot size of 400 pm2was used for all experiments. The base pressure of the K- Alpha instrument was at 9.0 x 10'10mbar. The instrument was calibrated to give a binding energy of 84.0 eV for Au 4f? / 2 and 284.8 eV for the Cis line of aliphatic carbon present on the non-sputtered samples. The photoelectrons were collected at a take-off angle of 90° relative to the sample surface, and a series of XPS spectra were performed in the Constant Analyzer Energy mode. Triplicate survey spectra were collected from different areas of a representative specimen (n=l) using a pass energy of 200 eV and a 1.0 eV step size. Triplicate high-resolution (HR) core level spectra of C Is, O Is, P 2p, Mg Is, Ca 2p, and Ti 2p were also taken using a 40 eV pass energy, 0.1 eV step size, and an average of 50 scans.

[0149] Ion release rates: Inductively coupled plasma optical emission spectrometry (ICP-OES, SPECTRO AMETEK, SPECTROGREEN, Kleve, Germany, SPECTRO ICP Analyzer Pro Software) was used to determine the release profiles and cumulative release profiles of Ca and magnesium (Mg) ions from the hydroxyapatite forming oxide group. Representative anodized disc specimens (n=3) were immersed in 10 mL of calcium and magnesium-free phosphate-buffered saline (PBS) solution (MP Biomedicals, Santa Ana, CA, USA). The specimens were transferred to fresh PBS at time points 2, 4, 6, 8, 10, 12,14, 18, 22, and 30 days. 300 pL of nitric acid and 50 pL of hydrochloric acid were added to each collected solution to dissolve any larger particulate matter. The solutions were then filtered using 0.4 pm syringe filters and analysed for Ca and Mg release concentrations in parts per million (ppm).

[0150] Statistical analyses'. Welch's one-way ANOVA (a = 0.05) with post hoc Dunnett' s T3 analyses were used to determine significant differences in surface roughness values and the cross-sectional thickness values amongst all the oxide groups due to the observance of unequal variance within the datasets.E2 Results

[0151] Oxide crystallinity analyses: The representative crystallinity results from the CPTi4 substrate material and each anodized oxide are provided in Figure 15. 20° to 80° two-theta scans are shown to the left side of Figure 15 to show all crystalline phases that are present. A zoomed-in 24° to 36° two-theta region is also provided to the right side of Figure 15 to emphasize the formation of crystalline calcium compound phases. The CPTi4 substrate material and each oxide group exhibited alpha phase titanium peaks. The oxide groups also exhibited a transition in crystallinity from oxides A through D evolving through various calcium compounds. Oxide A showed anatase phase titanium dioxide and calcium titanate (CaTiOs) formation, whereas oxide B revealed predominantly a- tricalcium phosphate with calcium diphosphate and calcium titanate phases. Oxides C and D showed a combination of calcium diphosphate, calcium titanate, a-tricalcium phosphate, and hydroxyapatite phase formation. The relative intensities of calcium diphosphate and a-tricalcium phosphate were shown to decrease in transitioning from oxides B, C, and D. Oxide C was the first to show hydroxyapatite formation in combination with calcium diphosphate and a-tricalcium phosphate, while oxide D showed predominantly hydroxyapatite.

[0152] Oxide surface topographies: Oxide optical microscopy and SEM images are compiled in Figure 16. Low-magnification optical microscopy images are shown in the left column of Figure 16, and SEM images of the micro and nano-scaled surfaces are provided in the rightmost two columns of Figure 16. The CPTi4 un-anodized specimens showed evident markings remaining from the cutting and grinding specimen preparation. For oxide A, optical images revealed a uniform greyish-white appearance and micron- scaled SEM images showed dispersed small white deposits across the surfaces. Nanoscale SEM images for oxide A exhibited surface nanopores. The optical images for oxide B revealed somewhat larger white deposits across the surfaces and some small localizeddarker areas. Micron-scaled oxide B images revealed pores in the oxide layer and a significant increase in the white deposits compared to those for oxide A. Nanoscale SEM images for oxide B showed fine white deposits uniformly distributed across the oxide surfaces. Optical images for oxide C revealed an increase in larger localized white deposits and some dark deposits remaining on the surfaces. Micron-scaled SEM images showed a significant morphology change to petal-like features while some white deposits remained visible. It should be noted that oxide surface pores were no longer visible at this scale. Nanoscale SEM images again showed petal-like surfaces with some nanoscale porosity still present. Optical images for oxide D revealed a similar distribution of white and dark deposits as compared to oxide C. Micron-scaled SEM images showed petal-like surfaces with very few remaining white deposits. Similar to oxide C, the surface pores were no longer visible in the micron-scale oxide surfaces. Nanoscale SEM images also showed larger petal-like surface features but without the remaining nanoscale porosity.

[0153] Oxide surface roughness: Representative oxide surface roughness results are compiled in Figure 17. Average oxide surface roughness, Sa, values are shown in Figure 17A, while peak-to-valley roughness, Szvalues, are compiled in Figure 17B. Oxide C was shown to exhibit significantly higher average Sa(p < 0.05) and Sz(p < 0.001) values compared to the other oxides. Additionally, Oxide D had significantly higher Szvalues compared to oxide A (p < 0.01) and oxide B (p < 0.05).

[0154] Oxide surface compositions: The EDS surface compositions for each oxide group are compiled in Table 3. The corresponding oxide average Ca / P ratios are provided in Figure 18. Each oxide group showed the presence of titanium (Ti) and oxygen (O), with the amount of Ti decreasing in transitioning from oxide A through D. 4% Ca and 3% P and some Mg (<1%) dopants were incorporated into oxide A. Oxide B showed substantially higher dopant uptake, with approximately 13% Ca, 9% P, and <1% Mg. Oxides C and D exhibited similar dopant uptake levels of at least 10% Ca, 5% P, and <1% Mg. The surface EDS-derived Ca / P ratios were also shown to generally increase with the transitions from oxide A to oxide D.Table 3. EDS surface chemistry for each oxide group.Elements GroupOxide A Oxide B Oxide C Oxide D (At.%)Titanium 12 ± 4 4 ± 1 <1 <1Oxygen 58 ± 4 60 ± 5 51 ± 3 47 ± 2Calcium 4 ± 2 13 ± 3 10 ± 1 10 ± 2Phosphorus 3 ± 1 9 ± 2 5 ± 1 5 ± 2Magnesium <1 <1 <1 <1

[0155] Oxide molecular structure analyses: Representative FTIR scans for the CPTi4 substrate material and each oxide group are compiled in Figure 19. The CPTi4 substrate material showed no visible FTIR peaks. Oxide A had some poorly defined absorption peaks around 1050 cm I 1450, and 1570 cm’1. Oxides B, C, and D showed intensive absorption peaks at 1050 cm corresponding to phosphate (PO43) groups, indicating the presence of calcium phosphate compounds formed on the surfaces of the respective oxides, as shown in Figure 1. [59,177] Additionally, oxide B also had some poorly defined carbonate (CO32) (1450 and 1570 cm’1) peaks, whereas oxides C and D showed well-defined characteristic absorption peaks for CCE2' (875, 1450, and 1570 cm’1). [59,123,177] These peaks are more pronounced in oxide D compared to oxides B and C. Furthermore, a broad O-H band between 3000-3600 cm1is evident in oxide D and is attributed to the bending mode of adsorbed water molecules. [59,123,177] However, the characteristic hydroxyapatite OH peak at 3570found in synthetic hydroxyapatite coatings, is absent in the oxides C and D. [123,177] The presence of CO32substitution peaks (875, 1450, and 1570 cm ') instead of the characteristic hydroxyapatite peak (3570 cm ') confirmed the formation of bone-like carbonated apatite. [59,123,177] Oxide D exhibits more pronounced high-intensity peaks at these positions, indicating higher degrees of carbonate substitution compared to the oxide C counterparts.

[0156] Oxide thickness evaluation: Representative cross-sectional images of each oxide are provided in Figure 20 and the measured oxide cross-sectional thickness values are compiled in Figure 21. Oxide A showed a single layer oxide and Oxides B, C, and D were found to exhibit bi-layered oxides as shown in Figures 17 and 18. The analyses of the inner and outer layer portions of the oxide thicknesses were evaluated separately, as compiled in Figures 21 A and 2 IB. The total oxide thickness for each oxide group is compiled in Figure 21C. Interestingly, Oxides C and D revealed significantly thicker inner oxides of approximately 1.5 pm compared to 1.2 pm for oxide B (p<0.01) and 1m for oxide A (p<0.0001). Of the bi-layered oxides, oxide C revealed a significantly thicker outer layer of approximately 26 pm, compared to 14 pm for oxide D (p<0.001) and approximately 4 pm for oxide B (p<0.0001). Similarly, the oxides C and D revealed significantly thicker total oxide thickness values of 27.6 pm and 15 pm compared to 5.5 pm for oxide B (p<0.0001) and 1 um for oxide A (p<0.0001).

[0157] Oxide cross-sectional compositional analyses: Figure 22 shows representative line scans for each oxide cross-section in order to evaluate the distribution of the incorporated elements. For clarity in the complex figure, the distribution of elements across the oxide cross-sections was divided into two sub-groups. Ti and O distributions are shown in the left column, while the Ca, P, and Mg dopant elements are shown in the right column. Each window of Figure 22 shows the substrate material / oxide interface to the left side, followed by the inner and outer layer oxides moving left to right. The cross- sectional EDS line scans confirmed the bi-layered structures for the oxides B, C, and D. The inner oxide layers for each oxide were titanium dioxide-rich. Since the oxide A was only a single layer, Ca, P, and Mg dopants were also present within this same oxide layer. In contrast, the Ca, P, and Mg dopants were predominately incorporated into the outer oxide layers in oxides B, C, and D. The concentrations of Ti in the outer layers of the B, C, and D oxides were substantially reduced compared to that in the same inner layer oxides. Thus, the outer layers in oxides B, C, and D correspond to the calcium phosphate compounds, including tricalcium phosphate and hydroxyapatite, which were confirmed in the XRD analyses in Figure 19.

[0158] EDS Ca, P, and Mg dopant uptake levels within the outermost 0.5 microns of oxide A, and the outermost 2 microns of oxides B, C, and D are compiled in Figure 23 A. The corresponding oxide Ca / P ratios are compiled in Figure 23B. The corresponding Ca / P ratio from these outermost layers of each bi-layered oxide ranged from 1.3 to 1.7, which is much closer to the known ranges for tricalcium phosphate and hydroxyapatite compounds.

[0159] Oxide layer adhesion results: Representative VDI 3198 standard oxide layer adhesion results are compiled in Figure 24. The oxide adhesion results for oxide B revealed some microcracking and delamination. However, the oxide layer formed in oxides A, C, and D showed good adhesion to the titanium substrate with no evidence of delamination in either oxide, but mild microcracking was observed in oxides C and D.

[0160] Additional characterization of oxide D: Since oxide D predominately contained the hydroxyapatite phase within the outermost oxide layer, additional microhardness andXPS characterizations of this oxide group was performed. Ca and Mg release profiles from these promising oxide D surfaces were also produced.

[0161] Oxide microhardness: Vicker’s microhardness dataset generated for oxide D is provided in Figure 25. The individual indentation values are included within the bar chart containing the average and standard deviation HV 0.3 values. The ten micro-hardness indentations exhibited HV0.3 values of 329.2 ± 40.9.

[0162] XPS surface chemistry: Representative XPS spectra for oxide D are shown in Figure 26. XPS survey spectra revealed Ti, O, Ca, P, Mg, and C peaks, as shown in Figure 26A. The high-resolution spectra for the Ti2Ppeaks are shown in Figure 26B. The Ti2P3 / 2 peaks with a binding energy of 459 eV represent the formation of TiO2. [48,92] The Ois peaks with a binding energy of 531.05, as shown in Figure 26C, are representative of calcium phosphate-containing oxides. [75,92] The Cis peaks exhibited approximately 284.96 eV and 288.8 eV binding energies, as shown in Figure 26D, and indicate the presence of C-C bonds and carbonates. [48,75] This finding is in good agreement with the carbonate substitution peaks found in oxide D FTIR analysis (Figure 16). The 284.96 eV binding energy peaks for Cis are suggestive of carbon from surface contamination and may also be due to the presence of organics from the calcium fortified orange juice component of the electrolyte. [51,165] The Ca2P3 / 2 and Ca2Pi / 2peaks revealed binding energies of approximately 347.3 eV and 350.85 eV, as shown in Figure 26E, and are indicative of the formation of calcium titanate (CaTiCE) and calcium phosphates. [48,52,75,92,128,164,178] The P2Ppeaks exhibited binding energies of 133.6 eV, as shown in Figure 26F, indicative of phosphorus-containing titanium oxides and phosphates. [48,75,128] Finally, the Mgispeaks exhibited binding energies of 1303.9 eV, as shown in Figure 26G, which represents the presence of Mg containing metal oxides.

[0179]

[0163] Calcium and magnesium ion release profiles: ICP-OES derived 30-day Ca and Mg ion release profiles from the predominately hydroxyapatite containing oxide D are provided in Figure 27. Both Ca and Mg ions showed a sustained release over the entire 30-day duration with an initial burst release. The Ca ion release reached a cumulative amount of approximately 120 ppm, whereas the Mg release was much lower at approximately 4 ppm over a period of 30 days.E2 Discussion

[0164] Hydroxyapatite and tricalcium phosphate are important calcium phosphates found in bone and have been widely used to form surface coatings on titanium implants toimprove osseointegration. [39,109] Previously, a-tricalcium phosphate coatings on titanium implants created by magnesium-sputtering have increased BIC and peri-implant bone volumes in rabbit femurs.

[0112] Hydroxyapatite-coated titanium implants have a long history of use in dental and orthopedic implants. These coatings have shown improved osseointegration abilities both in vitro and in vivo. Hydroxyapatite implant coatings have previously been shown to have superior osteoblast cell viability as well as enhanced differentiation and mineralization. [23,59,125,180] These coatings have demonstrated early new bone induction surrounding the implant and improved BIC in rat and rabbit femur models in comparison to non-anodized implants. [180,181] However, the conventional techniques used for forming these coatings, like plasma spraying, have certain drawbacks, such as requiring specialized and expensive processing equipment, long preparation times, and lower than desirable adhesion strengths. [44-46] The present embodiment makes use of citrus-based electrolytes for the purpose of anodization to produce hydroxyapatite coatings, which is unique and has never been explored before. The present embodiment used an electrolyte combining calcium fortified orange juice with monobasic calcium phosphate and calcium acetate. Interestingly, calcium acetate and monobasic calcium phosphate are also both commonly used as food stabilizers and leavening agents in baking products. [88,171]

[0165] The anodization oxide groups formed in the present study revealed distinct transitions in terms of crystallinity, topography, molecular structure, chemistry, and cross-sectional composition with changes in the citrus fruit-based electrolyte chemistry (Table 3). Oxide A showed crystalline compounds anatase and calcium titanate compounds within the single-layered oxide. Early CaP oxide studies showed oxide surface Ca / P ratios within the range of human bone and hydroxyapatite. [23,82] However, the same early CaP oxide studies showed difficulty forming crystalline hydroxyapatite during subsequent in vitro bioactivity testing. [23,82,102] It was later discovered that the formation of calcium titanate compounds during anodization facilitated the formation of hydroxyapatite during subsequent bioactivity testing. [23,94,101-105] Adding additional calcium acetate into the electrolyte resulted in the formation of a-tricalcium phosphate compounds in oxide B. Further increases of the calcium acetate concentrations in the electrolyte resulted in oxides C and D forming combinations of a-tricalcium phosphate and hydroxyapatite. While some research groups have formed hydroxyapatite-containing anodization coatings on titanium, few have formed the combination of hydroxyapatite, tricalcium phosphate, and calcium titanate as observed in the present study.[23,59,124,125] Additionally, it has been suggested that tricalcium phosphate is more bioresorbable and degrades first, leaving surface porosity, which is subsequently used by calcium titanate to promote further bone growth.

[0096] In addition to anodization studies, a sol-gel study revealed calcium titanate in the interlayer between the implant substrate and the hydroxyapatite-coated layer improved the adhesion strength between the two layers.

[0097]

[0166] The surface topography of the implants plays an important role in its interaction with bone cells. The surface topographies of each citrus fruit-based oxide group exhibited multiscale micro and nanoscale features. Since each oxide was anodized using the same galvanostatic pulsed anodization process, the electrolyte chemistry changes were responsible for the changes in surface topography between the different oxide groups. Increases in the electrolyte calcium acetate concentration in oxides C and D correlated with an oxide surface topography transition to micro and nano-scaled petal-like surface features. While the petal-like hydroxyapatite topography formation has been reported in some other anodization studies that formed hydroxyapatite, the anodization process in Embodiment 2 of the present invention formed oxide surfaces containing morphological combinations of white particle deposits and the petal-like hydroxyapatite structures as shown in oxide groups C and D which have not been reported elsewhere. [123-125]

[0167] FTIR analyses on oxide A in the present study revealed some poorly defined absorption peaks. In contrast, oxide B revealed small absorption peaks for PCh3' (1050 cm’1) in agreement with a-tricalcium phosphate formation in addition to some poorly defined CCh2' (1450 and 1570 cm’1) peaks. Oxides C and D showed characteristic absorption peaks for PO43' (1050 cm’1) and COs2' (875, 1450, and 1570 cm’1), which agreed with the combinations of a-tricalcium phosphate and hydroxyapatite shown in the XRD analyses. Therefore, the oxides C and D confirmed the presence of carbonated or bone-like apatite, which was more prominent in oxide D. The embodiment 2 anodization process of the present invention revealed oxides with the presence of bone-like carbonated apatite (Oxides C and D).

[0168] Previous papers have mentioned that an electrolyte pH range of 5-6, while some others reported a pH range of 9-11 is needed for the formation of hydroxyapatite on the surface. [34, 101 , 182] In contrast, the pH of the fruit-based electrolytes that formed oxides C and D in the present study was substantially more acidic, averaging 4.5 for oxide C and 4.3 for oxide D. These more acidic electrolyte pH ranges may have also contributed to the unique oxide topographies and structures shown in these novel oxides.

[0169] Some research groups have successfully been able to form bi-layered hydroxyapatite coatings on titanium surfaces directly using a single-step anodization process. [23,59,124,125] Some of these oxides demonstrated improved adhesion strengths between the titanium substrate and the hydroxyapatite coatings than those of conventional hydroxyapatite spray coatings due to the interlocking at the interface between the inner titanium oxide layer and the outer hydroxyapatite layers. [59,124] Similarly, the oxides B, C, and D in the present embodiment were shown to form a bilayered oxide consisting of a titanium dioxide-rich inner layer and a tricalcium phosphate / hydroxyapatite-rich outer layer. This was further reflected in oxide D XPS analysis, wherein the TiCh peak exhibited lower intensities, indicating the presence of an outer layer. At least oxides A, C, and D showed good adhesion to the titanium substrate as evidenced in the VDI indentation testing. Oxide D also showed HV 0.3 microhardness values that were comparable to a previous study on hydroxyapatite-containing anodized oxides.

[0059]

[0170] Like previous CaP anodization oxide studies that formed hydroxyapatite, our study also showed significant uptake of Ca and P into each oxide layer, as evidenced by the EDS surface analyses. [59,119,121,122,124,125,129] The average EDS-derived surface Ca / P ratios for oxides A and C were found to be in the range of bone and hydroxyapatite (1.5 - 1.7). [82,84] Thus, the embodiment 2 anodization process also formed oxides having a significant uptake of Ca and P, as evidenced by the EDS analyses with Ca / P ratios close to the range found in natural bone. Oxide B revealed a surface Ca / P ratio slightly below 1.5, while oxide D revealed an average surface Ca / P ratio slightly over 2.0. However, surface EDS analysis results are known to average values from a substantial interaction volume of the SEM electron beam with the subject material. The below surface depth and lateral dimension of this interaction volume depend greatly on the accelerating voltage used as well as the elements present and crystalline structures present in the subject material. In contrast, the use of cross-sectional EDS line scan analyses of these oxides facilitates a clearer examination of the actual Ca / P ratios shown near the surface of these coatings because the below-surface depth interaction volume is more consistent. An examination of the data from the outermost two microns of the B, C, and D oxides revealed oxides B and C to exhibit of Ca / P ratio below 1.5, while oxide D exhibited a ratio of 1.7. Thus, the oxide D ratio showed very good agreement with the known 1.67 stoichiometric ratio of hydroxyapatite. [82,84] Another recent study revealed that a-tricalcium phosphate having Ca / P ratios below 1.5 appeared as 1-2 pm denseagglomerates under SEM, similar to the deposits observed in the oxides B and C in the present study.

[0184] Therefore, the EDS analysis of these near oxide surface areas from oxides C and D in the present study helps to elucidate the greater hydroxyapatite formation shown within the oxide D. Since the outermost two microns of the oxide would likely be in constant contact with bodily fluids and tissues, this was an effective and surprising result.

[0171] Each citrus fruit-based oxide in the present study also showed some amount of Mg uptake. This was initially a somewhat surprising result since Mg was not intentionally added other than the amounts contained within the calcium fortified orange juice component of the anodization electrolyte. Mg is one of the topmost abundant elements in the body and the most prevalent in cells.

[0138] In its ionic form, Mg plays a vital role in bone growth and cellular functions such as proliferation, signaling, and metabolism.

[0138] Mg has also been shown to inhibit osteoclast differentiation and bone resorption, promoting bone growth and regeneration. [133,139] Mg-doped titanium surfaces revealed enhanced promotion of osteogenesis, cell adhesion, and angiogenesis through pathways like PI3K, ERK, and BMP-4. [140,141] A few previous studies have successfully incorporated Mg dopants into anodized oxides. [140,143,144] Mg -incorporated anodized titanium surfaces enhance osteogenic differentiation and rapid bone integration, as evidenced by increased removal torque values in animal models. [140,143] Moreover, coatings combining Mg and hydroxyapatite have shown improved adhesion, trabecular bone formation, and osseointegration, with superior interfacial strength and push-out forces compared to hydroxyapatite-only coatings.

[0142] The oxide D in the present study exhibited a Mg-doped predominantly bone-like carbonated hydroxyapatite outer oxide structure. Furthermore, a sustained Mg release was shown over a 30-day period in our ICP-OES study for this oxide D. This oxide coating shows much promise to improve the osseointegration characteristics of future implants.

[0172] The presence of Ca and Mg ions surrounding the implant microenvironment play a critical role in enhancing osseointegration. [140,185] Ca ions promote fibrin clot formation, which serves as a chemotactic scaffold for recruiting osteogenic cells to the implant site, further supporting cell attachment, proliferation, and differentiation, thereby accelerating the bone healing and remodeling process. [70,71] Additionally, Ca ions contribute to the later stages of the coagulation cascade, reducing complement activation and fostering a more favorable environment for bone regeneration during the healing phase. [70-72] Mg ions, on the other hand, activate the transient receptor potentialmelastatin 7 (TRPM7) / phosphoinositide 3 -kinase (PI3K) signaling pathway, which induces osteogenic differentiation in osteoblasts and mesenchymal stem cells.

[0185] Additionally, Mg ions promote the stabilization of P-catenin, which is essential for the early stages of mesenchymal stem cell osteogenic differentiation.

[0185] Recently, another research group studied the effects of Ca and Mg ion releases from titanium implant surfaces that were individually doped by these elements via wet chemical treatment.

[0185] Interestingly, it was found that the Mg-doped surfaces showed earlier cell spreading, focal adhesion, and osteogenic differentiation of the primary bone marrow mesenchymal stem cells compared to the Ca-doped surfaces. In terms of ion release, this study revealed Ca- ion concentrations to be about 3.6 ppm on day 1, with cumulative concentrations reaching 7.3 ppm by day 8, whereas that for Mg was 0.7 ppm by day 1 and 1.9 ppm by day 8. In the present study, the Ca-ion release was significantly higher, with concentrations of about 14 ppm on day 1 and a cumulative release of about 56 ppm by day 8. Whereas for Mg-ions, it was 1.3 ppm on day 1 and 2.7 ppm by day 8. Additionally, the present study shows potential for a synergistic effect of prolonged Ca and Mg ion release over a period of 30 days. The anodization process in embodiment 2 of the present invention used citrusbased electrolytes to form calcium and magnesium-releasing, carbonated hydroxyapatite and tricalcium phosphate-containing surfaces in a single-step anodization process, which is unique to the present invention. Thus, the oxides formed with the embodiment 2 anodization process are capable of improving implant stability and osseointegration.

[0173] Conclusions for Embodiment 2 The citrus-fruit anodization process of embodiment 2 produced a predominately bone-like carbonated apatite oxide exhibiting good adhesion strengths in VDI indentation testing and HV 0.3 hardness values above 300. This oxide also exhibited sustained release of Ca and Mg over a 30-day period in the dissolution assessment. The anodization process of embodiment 2 is the first to form a combination of calcium and magnesium releasing carbonated hydroxyapatite and tri calcium phosphate oxides on titanium implant materials using a single-step anodization process in a citrus-fruit based electrolyte. Given the usefulness of hydroxyapatite coatings toward improving osseointegration of dental and orthopedic implants and considering the poor adhesion strengths of some hydroxyapatite coatings, which result in delamination and potential loosening of implant devices, the novel oxides prepared herein are capable of improving future patient outcomes with titanium implants.Embodiment 3: Anodization Process Using Commercial Chemical-Based Electrolyte to Form an Oxide Coating that Includes Tri-calcium Phosphate and Hydroxyapatite Crystalline Compounds

[0174] Embodiment 3 (E3) utilizes a commercially available laboratory chemi cal -based synthetic recipe anodization process for modifying titanium implant surfaces to form an oxide layer containing tri -calcium phosphate and hydroxyapatite crystalline compounds and a hierarchical micro and nanoscale surface roughness profile. E3 replaces the citrus fruit juice component in the E2 group D anodization process electrolyte with synthetic commercially available laboratory chemicals and produces similar hydroxyapatite containing oxides.E3 Test 1 - Materials and methods

[0175] Initial testing aimed to identify the active ingredients present within the E2 anodization process. First, the citrus fruit juice component was removed from the group D electrolyte used in the E2 anodization process and replaced with distilled water. The group D electrolyte from the E2 anodization process served as a control group. Table 4 lists the anodization process electrolytes used in this testing. It should be noted that the anodization waveform used for this E3 testing was also the same as that used in the E2 anodization process. XRD analyses were utilized to identify the crystalline compounds produced for each of the test anodization process trials.Table 4 - Anodization electrolyte trials to test the importance of citrus-based electrolyte components in the E2 anodization process.*Electrolyte D from the E2 anodization processE3 Test 1 - Results

[0176] The anodization process in Table 4 that did not include the citrus fruit juice component was unable to form crystalline hydroxyapatite compounds. This finding demonstrated that a citrus component would be included in the E3 synthetic anodization process electrolyte.E3 Test 2 - Materials and methods

[0177] It was then hypothesized that a citric acid solution could be utilized in place of the citrus fruit juice electrolyte component that was used in the E2 anodization process toform hydroxyapatite coatings on titanium. Accordingly, citric acid concentration trials were conducted based on a calculation using the manufacturers label information off the CFOJ juice, adjusting to the equivalent of a 500 ml serving. The citric acid solution concentration within the E3 electrolyte was then adjusted over a range of molarities (0.05- 0. IM) until the XRD spectra for the generated oxide revealed similar hydroxyapatite and a-tricalcium phosphate peaks to those shown for the group D oxide obtained using the E2 anodization process. The citric acid-based electrolyte chemistries used in these trials are listed in Table 5. The most effective oxide obtained from the E3 anodization process trials was characterized further using optical microscopy, SEM, EDS and FTIR. The methodology used for these characterization techniques was identical to those used in the E2 and E3 anodization processes.Table 5 - Citric acid concentration trials with calcium additions equivalent to those used in the E2 Group D anodization process.E3 Test 2 - Results

[0178] The oxides resulting from the 0.1 M citric acid E3 anodization process successfully formed tri-calcium phosphate and hydroxyapatite on the titanium surface as shown in the XRD graph in Figures 29 and 30. This finding confirmed that citric acid may be utilized in the E3 synthetic anodization process, in replacement of the citrus fruit juice electrolyte component used in the E2 citrus fruit anodization process for the formation of hydroxyapatite and a-tricalcium phosphate combination coatings via anodization. The optical microscopy results for the CA-Citric group showed formation of small white connecting patches on the specimen surfaces as shown in Figure 31. SEM results on both micro and nanoscale revealed uniformly distributed petal-like structures with numerous white patch deposits surrounding the petal-like structures (Figure 31). It was hypothesized that the petal-like structures are hydroxyapatite as shown in previous studies [158,186,187], and the white crystalline deposits were evidence of a-tricalcium phosphate. Interestingly, the CA-Citric oxide surface morphology from the 0.1M citric acid E3 anodization process was very similar to what was observed in the E2 group D anodization process.

[0179] A representative EDS spectrum from the oxide surface is provided in Figure 32. EDS dopant uptake levels for Ca and P are shown in Figure 33A. The resulting average EDS Ca / P ratios is shown in Figure 33B, was approximately 1.8 ± 0.5. A representative FTIR spectrum is shown in Figure 34. The E3 anodization process oxide revealed intensive absorption peaks for PO43' (1050 cm’1) [59,177], which agreed well with the hydroxyapatite and a-tricalcium phosphate formation which was shown in the XRD analyses. OH- band representing bending mode of adsorbed water was present between 3000-3600 cm’1[59,123,177], However, the characteristic OH- peak at 3570 cm’1was not very clear [123,177], Instead, there was presence of sharp high intensity COs2' absorption peaks (880, 1450 and 1570 cm’1) which substituted and caused disturbance in the OH- absorption peak at 3570 cm’1[59,123,177], Such a substitution confirms the presence of bone-like carbonated apatite in these groups

[0188] , Previously, it has been shown that such sharp peaks indicate higher crystallinity for hydroxyapatite and such highly crystalline hydroxyapatites have been shown to have better mechanical stability [59, 128], Thus, the E3 anodization process formed oxides containing highly crystalline bone-like carbonated apatite. Representative cross-sectional thickness values for the oxides are shown in Figure 35. As the oxide was determined to be a bi-layered oxide, thickness values for the inner and outer layers were measured separately as compiled in Figure 35 A. The average values for the inner and outer layer were then combined to approximate the representative total cumulative thickness of the oxide as shown in Figure 35B.

[0180] The E3 anodization process formed oxides containing hydroxyapatite with carbonate substitutions that are similar to the apatite found in natural human bone. Thus, the oxides formed using the E3 anodization process can improve implant stability and osseointegration.Embodiment 4: Anodization Process Using Commercial Chemical-Based Electrolyte & Calcium Carbonate as Phosphate Binder to Form an Oxide Coating that Includes Tri-calcium Phosphate and Hydroxyapatite Crystalline Compounds

[0181] Embodiment 4 (E4) of the present invention is another commercially available laboratory chemi cal -based synthetic recipe anodization process for modifying titanium implant surfaces to form an oxide layer containing tri-calcium phosphate and hydroxyapatite crystalline compounds and a hierarchical micro and nanoscale surface roughness profile. E4 replaced the calcium acetate phosphate binder component in the E3 anodization process with calcium carbonate and produced similar hydroxyapatite containing oxides.E4 Test 1 - Materials and methods

[0182] The anodization set-up and waveforms used in the current embodiment of the present invention were identical to those used in the E2 and E3 anodization processes. First, the function of the calcium acetate phosphate binder in oxide hydroxyapatite formation was tested. For this test the calcium acetate component used in E3 was removed and anodization processes were carried out solely using the other electrolyte components, namely, the commercially available monobasic calcium phosphate and citric acid as listed in Table 6. XRD analyses were utilized to identify the crystalline compounds produced for each of the test anodization process trials.Table 6 - Anodization electrolyte trials to test the importance of the calcium acetate phosphate binder component in the E3 anodization process.E4 Test 1 - Results

[0183] The oxide group formed without calcium acetate phosphate binder electrolyte component showed small amounts of calcium titanate phase but did not form any tricalcium phosphate or hydroxyapatite compounds as evidenced on the XRD analysis. A phosphate binder component was therefore also included in the E4 synthetic anodization process electrolyte.E4 Test 2 - Materials and methods

[0184] It was then hypothesized that calcium carbonate could be used in place of the calcium acetate phosphate binder used in the E3 anodization electrolyte to generate hydroxyapatite oxide coatings with multi-scaled roughness profiles. The calcium acetate containing electrolyte from the E3 anodization process served as the control group as listed in Table 7. The anodization setups and waveforms used for this test were identical to those used in the E2 and E3 anodization processes. The most effective oxide obtained from the E4 anodization testing was characterized further using optical microscopy, SEM, EDS and FTIR. The methodology used for these characterization techniques was identical to those used in the E2 and E3 anodization processes.Table 7 - Anodization electrolyte trials using calcium carbonate as the phosphate binder in place of the calcium acetate that was used in the E3 anodization process.E4 Test 2 - Results

[0185] Both the E3 anodization process utilizing the calcium acetate electrolyte phosphate binder and the E4 anodization process utilizing the calcium carbonate phosphate binder formed oxides containing tri-calcium phosphate and hydroxyapatite crystalline compounds. This finding demonstrated that calcium carbonate may be utilized as a phosphate binder to form hydroxyapatite and a-tricalcium phosphate combination coatings via anodization. The optical microscopy results for the CaCCE-Citric group oxides group showed formation of uniform small white connecting patches on the specimen surfaces as shown in Figure 39. SEM results on both micro and nano scale revealed uniformly distributed petal-like structures with numerous white patch deposits surrounding the petal-like structures (Figure 39). It was hypothesized that the petal-like structures are hydroxyapatite as shown in previous studies [158,186,187], and the white crystalline deposits were evidence of a-tricalcium phosphate. Interestingly, the CaCCE- Citric oxide surface morphology from the calcium carbonate E4 anodization process in the present invention was very similar to what was observed in the E2 group D and E3 anodization process in the present invention.

[0186] A representative EDS spectrum from the oxide surface is provided in Figure 40. EDS dopant uptake levels for Ca and P are shown in Figure 41A. The resulting average EDS Ca / P ratios is shown in Figure 41B, was approximately 2.0 ± 0.8. A representative FTIR spectrum is shown in Figure 42. The E3 anodization process oxide revealed intensive absorption peaks for PCh3' (1050 cm’1) [59,177], which agreed well with the hydroxyapatite and a-tricalcium phosphate formation which was shown in the XRD analyses. OH- band representing bending mode of adsorbed water was present between 3000-3600 cm’1[59,123,177], However, the characteristic OH- peak at 3570 cm’1was not very clear [123,177], Instead, there was presence of sharp high intensity CO32' absorption peaks (880, 1450 and 1570 cm’1) which substituted and caused disturbance in the OH- absorption peak at 3570 cm’1[59,123,177], Such a substitution confirms the presence of bone-like carbonated apatite in these groups

[0188] , Previously, it has been shown that such sharp peaks indicate higher crystallinity for hydroxyapatite and such highlycrystalline hydroxyapatites have been shown to have better mechanical stability [59, 128], Thus, the E4 anodization process also formed oxides containing highly crystalline bonelike carbonated apatite. Representative cross-sectional thickness values for the oxides are shown in Figure 43. As the oxide was determined to be a bi-layered oxide, thickness values for the inner and outer layers were measured separately as compiled in Figure 43 A. The average values for the inner and outer layer were then combined to approximate the representative total cumulative thickness of the oxide as shown in Figure 43B.

[0187] The E4 anodization process formed oxides containing hydroxyapatite with carbonate substitutions that are similar to the apatite found in natural human bone. Thus, the oxides formed using the E4 anodization process can improve implant stability and osseointegration.Embodiment 5: Anodization Process Using Commercial Chemical-Based Electrolyte to Form a Magnesium-Enhanced Oxide Coating that Includes Tri-calcium Phosphate and Hydroxyapatite Crystalline Compounds

[0188] Embodiment 5 (E5) includes a commercially available laboratory chemi cal -based anodization process for modifying titanium implant surfaces to form a magnesium enhanced oxide layer containing tri-calcium phosphate and hydroxyapatite crystalline compounds and a hierarchical micro and nanoscale surface roughness profile. The embodiment 5 anodization process exhibited magnesium uptake as shown in the hydroxyapatite containing oxides formed using the E2 group D citrus fruit-based anodization process, but with a synthetic laboratory chemi cal -based electrolyte. The electrolytes used in the embodiment 5 anodization process included magnesium phosphate as the phosphate component in place of the calcium phosphate component utilized in the E3 and E4 anodization processes. Both the calcium acetate (as in E3) and calcium carbonate (as in E4) phosphate binder electrolyte components were tested with the new magnesium phosphate component in the E5 anodization process.E5 Materials and methods

[0189] The calcium phosphate component from both the calcium acetate (as in E3) and calcium carbonate (as in E4) phosphate binder electrolytes was replaced by magnesium phosphate as listed in Table 8. The anodization setups and waveforms used in E5 were identical to those used in E2, E3 and E4. The oxides obtained from the E5 anodization processes were characterized further using optical microscopy, SEM, EDS, XPS, and FTIR. The methodology used for these characterization techniques was identical to those used in E2, E3, and E4.Table 8 - Anodization electrolyte trials with magnesium component to the calcium acetate and calcium carbonate phosphate binder electrolytes.

[0190] Validation of use of chelating acids including multiple carboxylic acid groups'. The citrus fruit juice component of the anodization electrolyte used in E2 and the citric acid component of the anodization electrolytes used in E3, E4, and E5 represent a chelating acid that includes multiple carboxylic acid groups.

[0148] Ethylenediaminetetraacetic acid (EDTA), malic acid, tartaric acid, ascorbic acid, oxalic acid, and succinic acid are chelating acids that include multiple carboxylic acid groups that may be used in addition to or alternatively to citric acid. [148-153] Thus, anodization oxides formed in E3, E4, and E5 can also be formed using one or more chelating acids that include multiple carboxylic acid groups. In the present embodiment, we show a representative anodization process using example chelating acids EDTA and malic acid. Figure 52 shows the anodization electrolyte composition and anodization process used in E3, E4, and E5, and also shows carboxylic acid containing chelating acids that can be used in addition to or as an alternative to citric acid (e.g., EDTA and / or malic acid). This embodiment shows anodization processes using magnesium phosphate as the phosphate component and calcium acetate as the phosphate binder component in electrolytes containing either citric acid, EDTA, or malic acid as the chelating acid including multiple carboxylic acid groups. The compositions of the tested E5 electrolytes are listed in Table 9. The resulting oxides obtained from the three anodization processes were characterized further using XRD and FTIR. The methodology used for these characterization techniques was identical to those used in E2, E3, and E4.Table 9 - Anodization electrolyte trials with multiple carboxylic acid containing chelating acids.E5 Results

[0191] Figure 44 shows the representative oxide crystallinity results for the oxides obtained from both the phosphate binder electrolytes containing magnesium phosphate anodization electrolyte trials as listed in Table 8. Each oxide group revealed a-tricalcium phosphate phase formation as shown by the diffraction peaks located at 30.7° and 22.9° 29 angles, and hydroxyapatite phase formation as exhibited by the diffraction peaks at 31.8°, 33.0°, and 25.7° 29 angles. It should be noted that both of these oxide groups had equivalent intensity hydroxyapatite XRD peaks compared to those shown for E4. The presence of each of these compounds has been previously shown to improve osseointegration. Figure 45 shows representative oxide surface morphology results for each oxide groups. Macroscale optical microscopy images are shown in the left column and micro and nano-scaled SEM images are shown in the middle and right columns. Each of the groups revealed macro-scaled surface morphologies with a greyish-white general surface appearance with some darker areas also present. Micro-scaled SEM images revealed comparatively rougher and porous surfaces for each of the oxide groups. Nanoscale SEM images revealed white deposits surrounded by nanoscale surface porosity with some petal-like structured appearance present on the MP-CaCCE oxide group. This petal-like surface appearance has been previously associated with hydroxyapatite formation. However, the lack of petal-like surface appearance on the micro-scale of both groups and nanoscale of the MP-CA oxide group suggests that the petal-like structures are buried under the white deposits observed on the surface. Similar multi-scale surface morphologies with micro and nanoscale features have previously been shown to improve mechanical bone interlocking, protein absorption, osteoblast cell attachment and differentiation, and osseointegration.

[0192] Figure 46 shows the representative EDS spectra of each oxide group. The bulk surface Ca and P uptake levels, and the corresponding Ca / P ratios for each oxide are compiled in Figure 47 A and 47B. The average surface Ca / P ratio for both the oxide groupswas approximately 2.2. Both the oxide groups also showed some uptake of magnesium (Mg) as shown in Figure 48. Interestingly, the embodiment forming oxides from MP- CaCCh group revealed a substantially higher Mg uptake, at an average of approximately 1.1%, compared to the MP-CA group. Representative XPS spectra for both the oxide groups are shown in Figure 49. XPS survey spectra revealed Ti, O, Ca, P, Mg, and C peaks, as shown in Figure 49A. The high-resolution spectra for the Ti2Ppeaks are shown in Figure 49B. The Ti2P3 / 2 peaks with a binding energy of 459 eV represent the formation of TiC>2. [48,92] The Ois peaks with a binding energy of 531.24, as shown in Figure 49C, are representative of calcium phosphate-containing oxides. [75,92] The Cis peaks exhibited approximately 284.84 eV and 228.6 eV binding energies, as shown in Figure 49D, and indicate the presence of C-C bonds and carbonates. [48,75] The 284.84 eV binding energy peaks for Cis are suggestive of carbon from surface contamination. [51,165] The Ca2P3 / 2 and Ca2Pi / 2peaks revealed binding energies of approximately 347.2 eV and 350.87 eV, as shown in Figure 49E, and are indicative of the formation of calcium titanate (CaTiCh) and calcium phosphates. [48,52,75,92,128,164,178] The P2Ppeaks exhibited binding energies of 133 eV, as shown in Figure 49F, indicative of phosphorus- containing titanium oxides and phosphates. [48,75,128] Finally, the Mgispeaks exhibited binding energies of 1302.4 eV, as shown in Figure 12G, which represents the presence of Mg containing metal oxides.

[0179] Representative FTIR spectra for both the oxide groups are presented in Figure 50. Both oxides revealed intensive absorption peaks for PCU3' (1050 cm’1) which agreed well with the hydroxyapatite and a-tricalcium phosphate formation which was shown in the XRD analyses. [59,177] OH- band representing bending mode of adsorbed water was present between 3000-3600 cm’1. [59,123,177] However, the characteristic OH- peak at 3570 cm’1was not very clear. [123,177] Instead, there was presence of high intensity COs2' absorption peaks (880, 1450 and 1570 cm’1) which substituted and caused disturbance in the OH- absorption peak at 3570 cm’1. [59,123,177] Such a substitution confirms the presence of bone-like carbonated apatite in these groups.

[0188] Previously, it has been shown that such sharp peaks indicate higher crystallinity for hydroxyapatite and such highly crystalline hydroxyapatites have been shown to have better mechanical stability. [59,128] The E5 anodization oxides exhibited oxides containing magnesium enhanced highly crystalline bone-like carbonated apatite.

[0193] Representative cross-sectional thickness values for each of the oxide groups are shown in Figure 51. As the oxides were determined to be bi-layered oxides, thickness values for the inner and outer layers were measured separately as compiled in Figure 51 A.The average values for the inner and outer layer were then combined to approximate the representative cumulative thickness values for each oxide as provided in Figure 5 IB. Each oxide was shown to be bi-layered, which has been commonly shown in other CaP oxides that have formed hydroxyapatite. The inner layer of these previous CaP oxides has been shown to be predominately TiCh, while the outer layer was predominately calcium- containing compounds. These bi-layered oxides containing hydroxyapatite have shown increased adhesions strengths compared to their plasma sprayed hydroxyapatite counterparts. The reason the increased adhesion strength is believed to be due to the stronger adhesion of hydroxyapatite to the TiCh inner oxide layer. The anodization oxides were found to be having substantially lower total oxide thickness values, below 20 pm compared to the previous E2 and E3 anodization process oxides. Specifically, the E5 MP- CA oxides revealed an average thickness of approximately 12 pm. Interestingly, the inner oxide layer oxide thickness for MP-CA group was the thinnest averaging to about 1 pm. The oxide thickness of both the E5 anodization process oxide groups was found to be significantly lower than those observed in E3 and E4. The E5 anodization process adds magnesium enhancements to the carbonated apatite anodization oxides created using the E3 and E4 anodization processes.

[0194] Figure 53 shows the representative XRD results from each of the anodized oxide surfaces formed using the MP-CA formula (citric acid-MPCA) by replacing citric acid with other chelating acids that include multiple carboxylic acid groups (here, EDTA and malic acid as listed in Table 9). Each oxide group revealed a-tri calcium phosphate phase formation as shown by the diffraction peaks located at 30.7° and 22.9° 29 angles, and hydroxyapatite phase formation as exhibited by the diffraction peaks at 31.8°, 33.0°, and 25.7° 29 angle. Representative FTIR spectra for all the oxide groups are presented in Figure 54. All three oxides revealed carbonate substitutions, confirming the formation of bone-like carbonated apatite in all three oxides.

[0195] The E5 anodization process demonstrated that other chelating acids that include multiple carboxylic acid groups, such as EDTA and malic acid, may be used as chelating acids in the anodization process to form hydroxyapatite and a-tricalcium phosphate combination coatings via anodization.Embodiment 6: Anodization Process Using Commercial Chemical-Based Electrolyte to Form an Oxide Coating on Titanium Alloys Used in Implants

[0196] Embodiment 6 (E6) includes a commercially available laboratory chemi cal -based synthetic recipe anodization process for modifying different wrought titanium alloyimplant surfaces to form a magnesium enhanced oxide layer containing tri-calcium phosphate and hydroxyapatite crystalline compounds and a hierarchical micro and nanoscale surface roughness profile. The E6 anodization processes implemented the E5 anodization process (with calcium acetate phosphate binder) on commonly used titanium implant alloy substrate materials.E6 Materials and methods

[0197] Specimen preparation: Commercially pure titanium grade 4 alpha-phase (CPTi) alloy, Ti-15Mo beta phase (TiMo P) alloy, Ti-15Mo duplex alpha + beta-phase (TiMo a+P) alloy, and Ti-6A1-4V ELI duplex alpha + beta-phase (TAV) alloy disc specimens were cut from 12.7 mm diameter bar stock to a 3 mm thickness. The discs were then ultrasonically cleaned using laboratory detergent (Alconox®, White Plains, NY, USA) and rinsed with distilled water. Cleaned discs were dipped in a nitric acid-hydrofluoric acid solution in a 10: 1 ratio (TURCO NITRADD, Henkel Corporation, Madison Heights, MI, USA) for a period of 30 seconds to activate the surface for anodization. Activated disc specimens were then rinsed with distilled water and dried with laboratory forced air.

[0198] Anodization: Each anodization reaction cell consisted of a titanium alloy disc anode and two CPTi strip cathodes submerged in the electrolyte. The composition of the anodization electrolyte consisted of citric acid (99+%, Fisher Scientific, Waltham, MA, USA), magnesium phosphate dibasic trihydrate (96.0%, Spectrum chemical, New Brunswick, NJ, US), and calcium acetate (99% Spectrum chemical, New Brunswick, NJ, US) mixed in distilled water in concentrations shown in Table 10. Disc specimens were anodized using a DC rectifier (350 V, 10 A, Dynatronix, Amery, WI) using a pulsed galvanostatic waveform. Multiple disc specimens (n = 4) of each titanium alloy were anodized at current densities of 700 mA / cm2using a 28% duty cycle (40 ms on time / 99 ms off time) at a frequency of 7.2 Hz for 120 seconds.

[0199] Oxide surface characterization: Thin-film X-ray diffraction (XRD, XDS 2000, Scintag, Franklin, MA) was utilized to determine the crystalline phases present within the anodized oxides. Representative disc specimens (n=4) from each oxide group were rotated 1° away from the copper X-ray source (0.154 nm Cu-Ka) to enhance the X-ray interaction volume. XRD scans were conducted over a two-theta angle range from 20° to 90°. Optical imaging (Keyence, Osaka, Japan, VHX-1000) and Scanning electron microscopy (SEM, Zeiss, Jena, Germany, Supra 40) were utilized to characterize the surface topography of each oxide surface. A 12 kV accelerating voltage was used for SEM imaging at 5,000X to capture the micron-level surface topography, while a lower 9kV accelerating voltage was utilized at 75,000X to mitigate the intensified surface charging effects exhibited by the anodized oxides at higher magnifications. A Laser confocal microscope (LCM, Keyence, Osaka, Japan, VK-X3000) was used to measure the oxide surface roughness values from triplicate areas of four specimens of each anodized oxide (n=4). Gwyddion software was used to calculate each oxide group’s average roughness (Ra) values and peak-to-valley roughness (Rz) values from the 3D profiles. Energy dispersive X-ray spectroscopy (EDS, ED AX, Mahwah, NJ, APEX EDS Software Suite) was used to collect surface chemistry spectra from each oxide group using an SEM accelerating voltage of 12 kV and a magnification of 500X. Representative EDS spectra were collected from triplicate areas of each oxide. The average surface concentrations of each element and the surface Ca / P ratios were calculated for each oxide group. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR FTIR) was also performed on each oxide group to assess any molecular differences. For these analyses, a Spectrum 100 FTIR (Perkin-Elmer, Waltham, MA) was used over the range of 650-4000 cm'1at a spectral resolution of 100 cm'1.

[0200] Oxide cross-sectional characterization: Representative specimens from each oxide were sectioned, mounted in conductive epoxy (Polyfast, Struers, Cleveland, OH, USA), and rotary-polished to a 0.02 pm surface finish in colloidal silica suspension (Struers, Cleveland, OH, USA) in order to visualize the anodized oxide layer crosssections for each group. Ten cross-sectional images were acquired from each specimen, and five oxide thickness measurements were performed on each of these images. This combination provided 50 cross-sectional oxide thickness measurements for each group. Representative EDS line scans were also collected using an accelerating voltage of 12 kV and a magnification of 3500X to determine the composition variations across the oxide cross-sections. A resolution level of 3 data points per micron was utilized for the line scan analyses.

[0201] Oxide adhesion quality: The quality of adhesion of the anodized oxides to the titanium alloy substrates was evaluated using the VDI 3198 standard Rockwell C indentation test.

[0174] This indentation-based adhesion strength test has been utilized in a number of recent implant coating studies. [23,59,175,176] Indentations were performed on each alloy group using a Rockwell C indenter with a load of 150 Kg. Indentations were then examined using an optical microscope and compared to the VDI 3198 standard adhesion strength quality reference maps. [23,59,174-176]

[0202] Statistical analysis: Welch's one-way ANOVA (a = 0.05) with post hoc Dunnett's T3 analyses were utilized to determine significant differences in the oxide surface roughness values and the oxide cross-sectional thickness values for the oxide groups since unequal variances were shown.Table 10. Anodization electrolyte composition.Solution component Citric Acid Magnesium Phosphate Calcium Acetate(M) (M) (M)500 mL Distilled water 0.05 0.15 0.275E6 Results and Discussion

[0203] Oxide crystallinity analyses: Representative crystallinity results for each oxide group are provided in Figure 55. A full range of 20° to 90° two-theta scans are shown on the left side of Figure 55 to show all of the crystalline phases formed in each titanium alloy surface coating. The CPTi oxides showed alpha-phase titanium peaks, and the duplex TAV alloy oxides revealed both alpha and beta-phase peaks, as expected. The thin-film XRD scans of the TiMo P and TiMo a + P oxides, however, also revealed evidence of omega (co) phase formation. TiMo alloys have been previously reported to be subject to omega phase formation when Mo levels are not sufficient to fully stabilize the beta-phase. [189,190] The co phase, an embrittling transformation that reduces the ductility of the alloy, may occur within the beta-phase of Ti-15Mo alloys under specific cooling rates. [189-191] While most of the peak positions of beta-phase titanium and omega-phase diffraction database files show substantial overlap, making the phases difficult to distinguish, the omega phase has a relatively high-intensity diffraction peak at a 79° two-theta angle. The presence of a small peak at this two-theta diffraction angle for both the TiMo P and TiMo a+p oxides confirmed the omega phase presence. In the present study, the formation of the omega phase at the TiMo substrate surfaces was attributed to the localized depletion of molybdenum from the substrate surfaces, as some molybdenum migration was shown into the inner oxides, as shown below. The TiMo P oxides also exhibited an a-phase peak at a two-theta diffraction angle of approximately 39° 2-theta, which is indicative of alpha-case formation within the surface oxide layer. Alpha-case is another surface embrittling phase that can form on titanium alloys in oxygenating environments. [192,193] A zoomed-in 24° to 36° two-theta diffraction angle region is also provided on the right side of Figure 55 to emphasize the formation of various crystalline calcium compound phases within the oxides. Each oxide exhibitedevidence of hydroxyapatite, a-tricalcium phosphate, and calcium titanate formation. The highest intensity diffraction peaks for each of these calcium compounds are contained within the 24° to 36° two-theta diffraction angle range. Hydroxyapatite has been extensively explored for the formation of coatings on the surface of dental and orthopedic titanium implants to improve osseointegration.

[0039] However, the conventional techniques of forming hydroxyapatite coatings have limitations, necessitating the use of specialized or expensive processing equipment, long preparation periods, and inadequate adhesion strengths. [44-46] Anodization has previously been used to create hydroxyapatite coatings on titanium substrates using a single-step process. [23,59,124,125] Some of these anodized oxides showed improved adhesion strengths between the titanium substrate and the hydroxyapatite coatings. [59,124] Hydroxyapatite and tricalcium phosphate are the most studied calcium phosphates and have been commonly used in bone cement and for bone substitution.

[0109] Hydroxyapatite-coated titanium implants have demonstrated superior osteoblast viability in MTT assays, enhanced differentiation in ALP assays, and increased mineralization in Alizarin Red assays in vitro. [23,59,125,180] Furthermore, these coatings have been shown to promote earlier induction of new bone around the implant and improved BIC in rat and rabbit femur models in vivo, compared to their non-anodized counterparts. [180,181] Previously, magnesium-sputtered a-tricalcium phosphate coatings on titanium have shown increased bone-to-implant contact and peri -implant bone volume in rabbit femurs.

[0112] While numerous studies have formed anodized hydroxyapatite coatings on titanium surfaces, only a few have reported the combination formation of hydroxyapatite, tricalcium phosphate, and calcium titanate, as observed in the present study.

[0023] A solgel coating study revealed that the formation of calcium titanate in an interlayer improved the adhesion strength between titanium alloy substrates and an outer hydroxyapatite layer.

[0097] Furthermore, evidence of calcium diphosphate formation was also shown in the oxides formed on the TiMo a + P and TAV.

[0204] Oxide surface topographies'. Figure 56 shows representative oxide surface topography results for each oxide group. Optical microscopy images are shown in the left column, and micro and nano-scaled SEM images are shown in the middle and right columns. Optical images revealed a relatively uniform distribution of darker and lighter areas for the CPTi, TiMo P, and TAV oxide groups. Contrastingly, the TiMo a + P oxide showed localized areas with a white patchy appearance surrounded by darker areas. Micro-scaled SEM images revealed a similar rough surface appearance on each oxidegroup, while nano-scaled SEM images revealed white deposits surrounded by nano-scale surface porosity. However, no compositional differences were shown between the chemistries of the surface white deposits and the surrounding darker areas in subsequent EDS spot analyses at high magnification. Previously, anodized oxide surfaces with microscale roughness topographies have shown improved mechanical bone interlocking [51,52], while surfaces with nano-scale roughness features have shown increased protein absorption, osteoblast cell attachment, differentiation, and ultimately faster osseointegration. [48,49] More recent anodization studies have shown surfaces exhibiting complex hierarchical surface topographies integrating micro and nano-scale surface features to promote mechanical interlocking, cell proliferation, differentiation, and extracellular matrix formation. [48,49]

[0205] Oxide surface roughness'. Figure 57 shows the representative surface roughness results for each oxide group. LCM oxide surface profile images are compiled in Figure 57A, and the respective surface Raand Rzvalues are compiled in Figures 57B and 57C. The CPTi, TiMo a+P, and the TAV oxide groups were shown to exhibit statistically similar average Ravalues (p > 0.99) and Rzvalues (p > 0.98). The TiMo P oxide group exhibited significantly lower average Ravalues (p = 0.03) and Rzvalues (p = 0.0002) compared to the CPTi oxide group. However, the TiMo P oxide surface roughness values were not statistically different compared to the TAV and TiMo a + P duplex alloy oxides.

[0206] Oxide surface compositions'. Table 11 compiles the EDS surface compositions for each oxide group. The corresponding average Ca / P ratios for each oxide group are provided in Figure 58. The alpha and beta-phase stabilizing alloying elements Al, V, and Mo within each titanium alloy substrate were found to be largely absent from the oxides in these EDS surface analyses. The resulting Ca / P surface ratios for each oxide group were approximately 2.0, which is close to the range observed in bone tissue (1.5 - 1.7). [82,84] Recent studies in our laboratory added P oxide dopants through anodization and subsequently added Ca onto the anodized surfaces using hydrothermal soaking treatments.

[0083] The two-step oxides exhibited surface Ca / P ratios similar to those found in hard tissues like bone and were shown to significantly improve osteoblast mineralization compared to non-anodized CPTi substrates.

[0083] Titanium oxide surfaces containing Ca and P dopants have been shown to improve bone-implant interface bone formation by increasing the local mineral content, and these coatings are thus considered osteoinductive. [194,195]

[0207] For magnesium, the TiMo P and TiMo a+P oxides exhibited higher uptake than the CPTi and TAV oxides, as shown in Table 11. Previously, Mg-hydroxyapatite combination sol-gel dip coatings on titanium surfaces showed improved interfacial coating adhesion strengths and push-out forces compared to the hydroxyapatite-only counterparts.

[0142] Furthermore, micro-CT analyses of these Mg-hydroxyapatite combination coatings showed improved trabecular bone formation and osseointegration abilities. In a rabbit tibia model study, Mg-doped titanium anodization coatings showed rapid integration into the bone and improved removal torque values compared to nonanodized implants.

[0143] Table 11 - EDS surface chemistry of each oxide group.Elements Titanium alloysTitanium 0.52 ± 0.42 0.05 ± 0.02 0.05 ± 0.05 0.56 ± 0.83Aluminium - - - 0.03 ± 0.04Oxygen 53.40 ± 0.92 51.58 ± 1.35 52.19 ± 2.51 57.00 ± 0.99Calcium 14.30 ± 1.10 11.41 ± 1.45 11.65 ± 1.73 14.78 ± 0.76Phosphorus 6.42 ± 0.25 5.25 ± 0.50 5.15 ± 0.52 6.96 ± 0.54Magnesium 0.63 ± 0.17 1.13 ± 0.22 0.92 ± 0.25 0.75 ± 0.27

[0208] Oxide molecular structure analyses'. Representative FTIR scans for each oxide group are compiled in Figure 59. Each oxide group revealed intensive absorption peaks for PO43' (1050 cm'1), which agreed well with the hydroxyapatite and a-tricalcium phosphate formation on the surfaces, as shown in the XRD analyses (Figure 55). [59,177] A weak broad band O-H representing the bending mode of adsorbed water was also present between 3000-3600 cm'1. [59,123,177] Synthetic hydroxyapatite coatings typically show a characteristic FTIR hydroxyapatite OH' peak at 3570 cm'1. [123,177] In contrast, the anodized hydroxyapatite containings in the present study revealed a weak OH' peak in combination with high-intensity COs2' absorption substitution peaks at 875, 1450, and 1570 cm'1. [59,123,177] These COs2' substitution peaks confirm the presence of bone-like apatite within each anodized oxide group.

[0177] Biological apatite or bonelike apatite is often called carbonated hydroxyapatite because it contains carbonate substitutions that may cause changes in the lattice parameters, crystallinity, crystal symmetry, thermal stability, morphology, solubility, physical, chemical, and biological characteristics. [115-117,196,197] The characteristic groups most commonly shown on the FTIR spectrum for carbonated apatite include PCU3', OH', COs2', and HPO42.Recently, some research groups have successfully formed hydroxyapatite on titanium surfaces directly using anodization. [23,59, 124, 125] Most of these studies found that their surfaces had a disturbance in the characteristic OH' hydroxyapatite absorption peak in FTIR analysis due to CCE2' substitution resulting from the formation of carbonated apatite similar to oxides in the present study. [59,125]

[0209] Oxide groups thickness evaluation: Representative cross-sectional thickness values for each oxide group are shown in Figure 60. This examination revealed each oxide group to exhibit bi-layered oxides. Therefore, we separated the analyses of the oxide thickness values for the inner and outer layer portions, as compiled in Figures 60A and 60B. Interestingly, the TiMo P and TiMo a+P oxides had significantly thicker inner oxide values compared to CPTi and TAV oxides (p<0.0001). Furthermore, the CPTi oxides showed significantly thinner outer oxide values compared to each of the other titanium alloy oxides (p<0.0001). Overall, the total thickness of the CPTi oxide group was found to be the thinnest among the oxide groups in the study. Previous anodization studies that have formed hydroxyapatite studies have shown similar bi-layered oxide formation, consisting of a titanium dioxide-rich inner layer and a hydroxyapatite-rich outermost layer. [121,123,124,132,198]

[0210] Oxide cross-sectional compositional analyses'. Representative cross-sectional EDS line scans for each oxide group are compiled in Figure 61. Given the high number of dopant elements incorporated into these oxides, the element distributions across the cross-sectional thicknesses were divided into two sub-groups for clarity. The Ti, O, and substrate alloying element distributions are shown in the left-most column, while the distributions of elements incorporated from the anodization electrolyte (Ca, P, and Mg) are shown in the right-most column. Within each of the Figure 61 images, the substrate material / oxide interface is shown to the left side, followed by the inner layer oxide, and then the outer layer oxide to correspond with the oxide thickness descriptions provided in Figure 60. The cross-sectional EDS line scans clarified the presence of bi-layered structures for each oxide. The inner oxide layers were shown to be titanium dioxide-rich, and the outer oxide layers to contain calcium compounds and magnesium dopant uptake levels that explain the hydroxyapatite and tricalcium phosphate formation shown in the XRD analyses in Figure 55. Furthermore, the inner layer oxides in each group revealed the incorporation of some of the substrate alloying elements. For the two TiMo oxides, the substrate oxide interfaces clearly show a drop in the Mo levels as it is incorporated as a dopant into at least the inner oxide layer. This finding explains the formation of theomega phase on the substrate surfaces as the beta-stabilizing Mo levels are no longer sufficient to stabilize the beta-phase titanium. The concentration of Ti within the oxide cross-sections continues to decrease, moving away from the substrate, and is almost completely absent in the outer oxide layer for most of the oxides. Another interesting note is that the wider inner oxide layer regions in the TiMo alloys, based on the inner layer thickness measurements, revealed substantial Ca and P uptake profiles on the outer or right-most sides of these inner layer oxides. However, given the formation of the embrittling omega phase at the alloy substrate-oxide interfaces, the use of these anodization processes on TiMo would not be recommended.

[0211] The Ca, P, and Mg dopant uptake was shown to be predominately in the outer layers of each oxide formed through our anodization processes. Representative EDS Ca, P, and Mg dopant uptake levels within the outermost two microns of the outer oxide layers and the corresponding Ca / P ratios are compiled in Figures 62A and 62B. The chemistries in these outermost two microns are particularly relevant as they would approximate the chemistries in contact with most of the bodily fluids and tissues. The TiMo P, TiMo a+P, and TAV oxide groups exhibited slightly higher Ca, P, and Mg dopant uptake compared to the CPTi oxides. The corresponding Ca / P ratios from these outermost layers of each oxide group were within the range of 1.3 - 1.7, which more closely aligns with ratios in bone tissue. However, the Ca / P ratios measured from the oxide surfaces were higher, around 2. This discrepancy suggests that the elevated Ca / P ratios from the oxide surfaces may result from increased Ca incorporation in the inner regions of the outer oxide layer, as shown in the cross-sectional line scan profiles.

[0212] Oxide layer adhesion results: Representative VDI 3198 standard layer adhesion results for each oxide group are compiled in Figure 63. The cross-sectional oxide analyses revealed each oxide group to be bi-layered. Previously, bi-layered hydroxyapatitecontaining anodized oxides have been shown to have higher adhesion strengths compared to plasma-sprayed hydroxyapatite coatings. [121,124] The higher strengths of the bi- layered coatings were attributed to stronger interlocking at the interface between the inner titanium dioxide-rich layer and the outer hydroxyapatite-rich layer. [121,124] The CPTi oxide group revealed the thinnest oxide and exhibited only a small number of microcracks in the adhesion quality testing, as shown in Figure 8A. It has been widely reported that the adhesion strengths of anodized titanium oxides generally decrease with increasing oxide cross-sectional thickness values. [52,80,83] However, the thicker oxides shown on the duplex TiMo a+P and the TAV oxides still exhibited acceptable oxide adhesionqualities, as shown in Figures 8C and 8D. This finding was especially surprising for the TiMo a+P oxide, which was shown to contain substantial omega phase formation in the XRD analyses (Figure 1). While some microcracking and interface chipping were shown after the indentation test for the TiMo a+P oxide, this image is still an acceptable adhesion level according to the VDI adhesion quality reference images. [23,59,174-176,199,200] In contrast, the thicker TiMo P oxide, which was shown in the XRD analyses to contain evidence of both omega phase and alpha-case embrittling phases, was shown to almost completely delaminate from the substrate surface during the VDI adhesion testing, as shown in Figure 8B. The presence of the omega phase indicates embrittlement caused by thermal or stress-induced transformation occurring during the anodization process as the molybdenum concentration is reduced near the substrate / oxide interface. Furthermore, the alpha-case layer formed on the substrate surface at the oxide interface is also a brittle layer, and it occurs in titanium alloys when exposed to higher temperatures in oxidizing environments.

[0192] The combination of the co phase and the alpha-case layer formation at the substrate-oxide interface, the TiMo P oxide group in the present study may have weakened the substrate-oxide adhesion strength of the anodized coating. As stated earlier, the formation of the omega phase in the TiMo alloy anodization processes would likely weaken implant ductility of the substrate alloys and thus would not be desirable for implant applications, even though the TiMo a+P oxides showed good adhesion strengths. However, the CPTi and TAV oxides also showed good adhesion strengths, and these two substrate alloys make up the vast majority of titanium alloys used for commercial implants.

[0213] Conclusions'. Hydroxyapatite with carbonate substitutions is similar to the apatite in natural bone and has shown comparatively improved bioactivity. The disclosed anodization processes formed Mg-doped carbonated hydroxyapatite and tricalcium phosphate coatings on a series of implant grade titanium alloy substrates. The oxides formed on each alloy were shown to exhibit a combination of micro and nano-scale surface topography and substantial dopant uptake of essential bone minerals, including calcium, phosphorus, and magnesium. The CPTi and TAV alloy oxides showed acceptable adhesion strengths to the substrate materials. Given that the CPTi and TAV alloys make up the vast majority of titanium alloys commercially used for implants, the novel anodization coatings can be effectively utilized to promote osseointegration and, ultimately, improve patient outcomes in future titanium implant devices.

[0214] The results demonstrate that the magnesium enhanced anodization processes disclosed herein are applicable to and effective for a wide variety of commonly used titanium implant alloy substrates.

[0215] Preliminary Animal study on E6 (TAV): We implanted TAV screws (4 mm long, 1.4 mm diameter, MCT Titanium Bone and Membrane Screws, Dental Implant Technologies) that were coated using the magnesium enhanced anodization processes (E5) into one femur of two 14-week-old Sprague-Dawley albino rats (Charles River Laboratories) and observed them for 4 weeks. The animals were given a long-lasting analgesic and carprofen (5 mg / kg SC once daily) just prior to surgery. Animals were observed for 72 hours post-surgery, and pain was evaluated by noting their activity and physical state. Animals were sacrificed after 4 weeks and the screw containing femur bones were extracted to evaluate bone growth. Micro-CT analyses was conducted to measure the Bone-to-implant contact (BIC).

[0216] Results of preliminary animal study. Post-bone extraction micro-computed tomography (Micro-CT), as shown in Figure 64, revealed new trabecular bone formation surrounding the implants, seamless bone-implant interfaces, and a high BIC value of 47.7%. These results demonstrate in vivo efficacy of the coatings formed using the novel anodization processes disclosed herein.Embodiment 7: Anodization Process Using Commercial Chemical-Based Electrolyte to Form an Oxide Coating on 3D Printed Porous Titanium Alloys used in Implants

[0217] Embodiment 7 (E7) includes a commercially available laboratory chemi cal -based synthetic anodization process for modifying 3D printed titanium alloy implant surfaces to form a magnesium enhanced oxide layer containing tri-calcium phosphate and hydroxyapatite crystalline compounds and a hierarchical micro and nanoscale surface roughness profile. The E7 anodization processes utilized the E5 anodization process with the calcium acetate phosphate binder on commonly used 3D printed titanium implant alloy substrate materials.E7 Materials and methods

[0218] Two 3D printed gyroid porous titanium alloy lattice substrates were included in the E7 anodization processes, namely, CPTi gyroid and TAV gyroid. The electrolyte used was the same as the calcium acetate phosphate binder electrolyte used in the E5 anodization process. The anodization setups and waveforms used in the E7 anodization process of the present invention were identical to those used in E2 - E6. The anodization oxides obtained on these 3D printed porous titanium alloys were characterized furtherusing optical microscopy, SEM, EDS and FTIR. The methodology used for these characterization techniques was identical to those used in E2 - E6.E7 Results

[0219] Figure 65 shows examples of contrasting solid and 3D printed porous gyroid lattice titanium substrates. Figure 66 shows the representative oxide crystallinity results for each oxide group of the 3D printed porous gyroid titanium alloy anodization trials. Each oxide group revealed a-tricalcium phosphate phase formation as shown by the diffraction peaks located at 30.7° and 22.9° 29 angles, and hydroxyapatite phase formation as exhibited by the diffraction peaks at 31.8°, 33.0°, and 25.7° 29 angles. The presence of each of these compounds has been previously shown to improve osseointegration. Figure 67 shows representative oxide surface morphology results for each oxide group of the 3D printed porous gyroid titanium alloy anodization trials. Macroscale optical microscopy images are shown in the left column and micro and nanoscaled SEM images are shown in the middle and right columns. Both the oxide groups revealed macro-scaled surface morphologies with thick white patchy appearance surrounding darker areas. Micro-scaled SEM images revealed comparatively rougher and porous surfaces for each of the oxide groups while nano-scaled SEM images revealed white deposits surrounded by nanoscale surface porosity.

[0220] The bulk surface EDS Ca, P and Mg uptake levels, and the corresponding Ca / P ratios for each oxide are compiled in Figure 68A and 68B, respectively. The average surface Ca / P ratio for all TAV gyroid group (3.5) was found to be higher than that of the CPTi gyroid oxide group (2.5). Interestingly, the Mg uptake for both the oxide groups was pretty much similar averaging to about 1 atomic %. Representative FTIR spectra for both the oxide groups are presented in Figure 69. Both oxides revealed intensive absorption peaks for PO43' (1959 cm’1) which agreed well with the hydroxyapatite and ethical cium phosphate formation which was shown in the XRD analyses [59,177], OH- band representing bending mode of adsorbed water was present between 3999-3699 cm’ 1 [59,123,177], However, the characteristic OH- peak at 3579 cm’1was not very clear [123,177], Instead, there was presence of sharp high intensity COs2' absorption peaks (889, 1459 and 1579 cm’1) which substituted and caused disturbance in the OH- absorption peak at 3579 cm’1[59,123,177], Such a substitution confirms the presence of bone-like carbonated apatite in these groups

[0188] , Previously, it has been shown that such sharp peaks indicate higher crystallinity for hydroxyapatite and such highly crystalline hydroxyapatites have been shown to have better mechanical stability [59, 128],Such a substitution in the oxides confirms the presence of highly crystalline bone-like carbonated apatite in these groups.

[0221] These results demonstrate that at least the magnesium enhancing anodization processes can be utilized with 3D printed porous titanium alloy lattice implant substrate materials.Example Aspects

[0222] The following clauses represent a non-exhaustive list of example aspects of the present disclosure.

[0223] Clause 1. A titanium composition, comprising: an oxide coating formed via an anodization process, wherein an anodization electrolyte used in the anodization process comprises (1) a chelating acid component, (2) a phosphate component, and (3) a phosphate binder component, wherein the resulting oxide coating comprises a-tricalcium phosphate and / or hydroxyapatite crystalline compounds with a calcium to phosphate (Ca / P) ratio within a range of human bone.

[0224] Clause 2. The composition of clause 1, wherein the oxide coating is generated via a single step anodization process.

[0225] Clause 3. The composition of any preceding clause, wherein the Ca / P ratio of the oxide coating is 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, or is within a range using any combination of the foregoing as endpoints.

[0226] Clause 4. The composition of any preceding clause, wherein the oxide coating is bi-layered, with an inner layer comprising titanium oxide and calcium titanate, and an outer layer comprising hydroxyapatite and a-tricalcium phosphate, optionally wherein the inner layer has a thickness of at least about 500 nm and the outer layer has a thickness greater than the inner layer.

[0227] Clause 5. The composition of any preceding clause, wherein hydroxyapatite in the oxide coating includes carbonate substitutions.

[0228] Clause 6. The composition of any preceding clause, wherein the chelating acid component comprises multiple carboxyl groups.

[0229] Clause 7. The composition of clause 6, wherein the chelating acid component comprises citric fruit juice, citric acid, malic acid, tartaric acid, succinic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), or a combination thereof.

[0230] Clause 8. The composition of clause 7, wherein the citric fruit juice comprises orange juice, mandarin juice, grapefruit juice, or a combination thereof.

[0231] Clause 9. The composition of any preceding clause, wherein the chelating acid component comprises a solution with a concentration of at least 0.05M, or at least 0. IM, or at least 0.15M, or at least 0.2M, or has a concentration within a range that uses any combination of the foregoing as endpoints.

[0232] Clause 10. The composition of any preceding clause, wherein the phosphate component comprises calcium phosphate, magnesium phosphate, strontium phosphate, or combination thereof.

[0233] Clause 11. The composition of clause 10, wherein the oxide coating comprises: up to 5.0, or up to 10.0, or up to 15.0, or up to 20.0, or up to 25.0 atomic % calcium, or within a range that includes any combination of the foregoing as endpoints; up to 1.0, or up to 2.0, or up to 3.0, or up to 4.0, or up to 5.0 atomic % magnesium, or within a range that includes any combination of the foregoing as endpoints; and / or up to 1.0, or up to 2.0, or up to 3.0, or up to 4.0, or up to 5.0 atomic % strontium, or within a range that includes any combination of the foregoing as endpoints.

[0234] Clause 12. The composition of any preceding clause, wherein the phosphate binder component comprises calcium acetate, calcium carbonate, calcium citrate, or a combination thereof.

[0235] Clause 13. The composition of any preceding clause, wherein the titanium composition comprises an alloy that includes titanium.

[0236] Clause 14. The composition of clause 13, wherein the alloy comprises a pure titanium alloy, a titanium-molybdenum alloy, a titanium-aluminum-niobium alloy, a titanium-aluminum-vanadium alloy, or combination thereof.

[0237] Clause 15. The composition of clause 13 or clause 14, wherein the titanium composition comprises a wrought alloy and / or an additively manufactured alloy, optionally comprising a 3D printed porous lattice structure, optionally comprising a strutbased or triply periodic minimal surfaces (TPMS), optionally comprising a gyroid lattice structure.

[0238] Clause 16. The composition of any preceding clause, wherein the oxide coating has a sub-micron thickness.

[0239] Clause 17. An implantable device comprising the titanium composition as in any preceding clause, optionally wherein the implantable device is configured as a dental implantable device, an orthopedic implantable device, or a spinal implantable device.

[0240] Clause 18. A method of anodizing a titanium composition to form an oxide coating, the method comprising: subjecting the titanium composition to an anodizationprocess, wherein an anodization electrolyte used in the anodization process comprises (1) a chelating acid component, (2) a phosphate component, and (3) a phosphate binder component, wherein the oxide coating comprises a-tricalcium phosphate and / or hydroxyapatite crystalline compounds with a calcium to phosphate (Ca / P) ratio within a range of human bone.

[0241] Clause 19. The titanium composition of any one of clauses 1-16, wherein the anodization process comprises the method of clause 18.References

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Claims

CLAIMS1. A titanium composition, comprising: an oxide coating formed via an anodization process, wherein an anodization electrolyte used in the anodization process comprises(1) a chelating acid component,(2) a phosphate component, and(3) a phosphate binder component, wherein the resulting oxide coating comprises a-tricalcium phosphate and / or hydroxyapatite crystalline compounds with a calcium to phosphate (Ca / P) ratio within a range of human bone.

2. The composition of claim 1, wherein the oxide coating is generated via a single step anodization process.

3. The composition of claim 1, wherein the Ca / P ratio of the oxide coating is 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, or is within a range using any combination of the foregoing as endpoints.

4. The composition of claim 1, wherein the oxide coating is bi-layered, with an inner layer comprising titanium oxide and calcium titanate, and an outer layer comprising hydroxyapatite and a-tricalcium phosphate.

5. The composition of claim 1, wherein the hydroxyapatite in the oxide coating includes carbonate substitutions.

6. The composition of claim 1, wherein the chelating acid component comprises multiple carboxyl groups.

7. The composition of claim 6, wherein the chelating acid component comprises citric fruit juice, citric acid, malic acid, tartaric acid, succinic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), or combination thereof.

8. The composition of claim 7, wherein the citric fruit juice comprises orange juice, mandarin juice, grapefruit juice, or combination thereof.

9. The composition of claim 1, wherein the chelating acid component comprises a solution with a concentration of at least 0.05M, or at least 0.1M, or at least 0.15M, or at least 0.2M.

10. The composition of claim 1, wherein the phosphate component comprises calcium phosphate, magnesium phosphate, strontium phosphate, or combination thereof.

11. The composition of claim 10, wherein the oxide coating comprises:up to 5.0, or up to 10.0, or up to 15.0, or up to 20.0, or up to 25.0 atomic % calcium, or within a range that includes any combination of the foregoing as endpoints; up to 1.0, or up to 2.0, or up to 3.0, or up to 4.0, or up to 5.0 atomic % magnesium, or within a range that includes any combination of the foregoing as endpoints; and / or up to 1.0, or up to 2.0, or up to 3.0, or up to 4.0, or up to 5.0 atomic % strontium, or within a range that includes any combination of the foregoing as endpoints.

12. The composition of claim 1, wherein the phosphate binder component comprises calcium acetate, calcium carbonate, calcium citrate, or combination thereof.

13. The composition of claim 1, wherein the titanium composition comprises an alloy that includes titanium.

14. The composition of claim 13, wherein the alloy comprises a pure titanium alloy, a titanium-molybdenum alloy, a titanium-aluminum-niobium alloy, a titanium-aluminum- vanadium alloy, or combination thereof.

15. The composition of claim 13, wherein the titanium composition comprises a wrought alloy and / or an additively manufactured alloy, optionally comprising a 3D printed porous lattice structure, optionally comprising a strut-based or triply periodic minimal surfaces (TPMS), optionally comprising a gyroid lattice structure.

16. An implantable device comprising the titanium composition as in any preceding claim.

17. The implantable device of claim 16, wherein the implantable device is configured as a dental implantable device.

18. The implantable device of claim 16, wherein the implantable device is configured as an orthopedic implantable device.

19. The implantable device of claim 16, wherein the implantable device is configured as a spinal implantable device.

20. A method of anodizing a titanium composition to form an oxide coating, the method comprising: subjecting the titanium composition to an anodization process, wherein an anodization electrolyte used in the anodization process comprises(1) a chelating acid component,(2) a phosphate component, and(3) a phosphate binder component,wherein the oxide coating comprises a-tricalcium phosphate and / or hydroxyapatite crystalline compounds with a calcium to phosphate (Ca / P) ratio within a range of human bone.

21. The titanium composition of any one of claims 1-15, wherein the anodization process comprises the method of claim 20.

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