In situ hydrothermal transformation of titanium surface into lithium-doped continuous nanowire network towards augmented bioactivity

Journal Pre-proofs Full Length Article In Situ Hydrothermal Transformation of Titanium Surface into Lithium-Doped Continuous Nanowire Network towards Augmented Bioactivity Abdalla Abdal-hay, Karan Gulati, Tulio Fernandez-Medina, Ma Qian, Saso Ivanovski PII: DOI: Reference:

S0169-4332(19)33420-8 https://doi.org/10.1016/j.apsusc.2019.144604 APSUSC 144604

To appear in:

Applied Surface Science

Received Date: Revised Date: Accepted Date:

5 July 2019 1 November 2019 4 November 2019

Please cite this article as: A. Abdal-hay, K. Gulati, T. Fernandez-Medina, M. Qian, S. Ivanovski, In Situ Hydrothermal Transformation of Titanium Surface into Lithium-Doped Continuous Nanowire Network towards Augmented Bioactivity, Applied Surface Science (2019), doi: https://doi.org/10.1016/j.apsusc.2019.144604

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© 2019 Published by Elsevier B.V.

In Situ Hydrothermal Transformation of Titanium Surface into Lithium-Doped Continuous Nanowire Network towards Augmented Bioactivity

Abdalla Abdal-hay1, Karan Gulati1, Tulio Fernandez-Medina1, Ma Qian2, Saso Ivanovski1*

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The University of Queensland, School of Dentistry, Herston QLD 4006, Australia; RMIT University, School of Engineering, Centre for Additive Manufacturing, Melbourne, VIC

3000, Australia.

Keywords: Ionic exchange; Titanium implants; Surface modifications; Bone Tissue Engineering; Lithium.

*Corresponding Author: Prof. Sašo Ivanovski ([email protected]) School of Dentistry, University of Queensland, 288 Herston Road, Herston QLD 4006, Australia

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Abstract: Herein, we present a new strategy to enable the fabrication of a highly stable lithium nanowire network on titanium (Ti) surfaces. The Ti surfaces were chemically modified by an alkali treatment, followed by in situ transformation of alkali-titanate into a Li-nanowire network (TiLi) via ionic exchange of Li+ ions during the hydrothermal reaction. The physicochemical characterization of the as-prepared Ti-Li substrates were analyzed using FE-SEM, XRD, LAICP-MS, and XPS techniques, in order to confirm the successful deposition of Li+ ions onto the Ti substrates. In-depth topographical and chemical characterization revealed that the stable continuous nanowire network is composed of fine Li-based nanoparticles (~7 nm) and exhibits high surface wettability, high mechanical stability and a sustained release of Li+ ions over 21 days at 37 °C under vigorous shaking in Milli-Q water, simulated body fluid (SBF) and proteincontaining fluids. Despite the coverage of the Ti-Li-treated surface with nanocrystals layer from the surrounding SBF media, Li release was not impaired. Human osteoblasts-derived cells cultured on the resultant Ti-Li surfaces indicated good viability, strong adhesion and attachment into nanowires. In conclusion, this novel Li-incorporated nano-scaled surface modification approach holds great promise towards the fabrication of bone/titanium dental implants with superior bone-forming ability.

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1. Introduction Skeletal disorders are a major health and economic burden which is costing around 254 billion dollars to society [1, 2], a figure expected to rise considerably with an ageing population worldwide. Severe skeletal disorders like osteoporotic fractures and non-unions require surgical correction using implants to assist in restoring the function of the compromised structures. Currently, more than 80% of the bone implants are made from titanium and titanium alloys [1, 3, 4]. As the most commonly used bone implant material, Ti is, however, characterized as a biopassive material [5]. The low bioactivity of Ti implants can result in poor bone-implant integration (loosening) and ingress of bacteria [6-9]. Further, the treatment regime becomes less effective in patients with chronic diseases, such as diabetes mellitus, immune deficiencies and rheumatoid arthritis [1, 10, 11]. Surface chemistry and topography can play crucial roles in determining the rate and extent of osseointegration, which in turn dominates early stability and long-term success [10, 12]. Accelerated osseointegration (OI) can improve outcomes as a result of enhanced load distribution and a more stable bone-implant interface. Natural bone is composed of various hierarchal organizations in the range of nano-, micro- and macro-scales, and attempts to augment OI have been directed at tailoring surfaces within these size scales [13, 14]. Recently, nano-scaled surface modifications of Ti-based implants have been recognized as the most promising strategy to achieve desirable bioactivity and therapeutic outcomes while maintaining mechanical stability [13, 15]. Various studies have established that surface topography and chemistry are key influencing factors in the wound healing process leading to OI, influencing aspects ranging from the initial protein adsorption to downstream cell functions, including immuneinflammatory and osteogenic responses [1, 6, 12, 16]. This in turn has been shown to determine

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the rate and extent of integration at the implant-bone interface in both human and animal models [12, 17-19]. Incorporation of various metallic ions (Ag, Au, Fe, Se, Zn, etc.) into nano-scaled titanium surfaces has been researched to obtain enhanced bioactivity and osseointegration, as well as antibacterial properties [13]. More recently, lithium (Li) has been identified as a biologically active ion that can stimulate osteoblast cell activity, and hence Li doped biomaterials have attracted considerable attention [20-24]. The pro-osteogenic properties of Li+ ions can be attributed to activation of the canonical Wnt signaling pathway by inhibiting glycogen synthase kinase-3β (GSK-3β) [22], which has been shown to promote osteoblasts activity, such as proliferation and differentiation in vitro [25, 26] and bone mineralization and formation in vivo [27-29]. More and more pieces of evidence indicated that Li has an efficient effect on bone formation[30]. Thus, incorporation of Li ions on Ti implant surfaces represents a viable means of enhancing cellular functionality. To this end, Liu et al [28] and others[30] attempted to incorporate Li ions onto Ti surface via chemical deposition techniques. In their studies, in vitro cell tests showed improved osteoblast morphology, adhesion, and viability in a lithium-containing nanoporous coating titanium scaffold. However, there was limited evidence about the Li formation, binding mechanism, stability, sustainable release and adhesive strength to the Ti surface. Therefore, it is highly desirable to develop novel strategies for incorporating Li ions onto Ti implants with augmented bone cell functions and appropriate mechanical stability. In this study we have discovered a facile and reproducible method for the synthesis of Li-incorporated Ti, achieved via ion exchange during a low temperature (90 °C) hydrothermal treatment, which led to the formation of a highly stable Li-doped nanowire network on the Ti surface. Briefly, sodium titanate was formed post NaOH treatment on the Ti surface, which was dissolved during a hydrothermal reaction with Li solution, thereby forming a Li-titanate

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layer with high chemical stability and Li density. Our established modification technique has been proved as a versatile way to prepare nano structures of a controlled size. The chemical composition and morphology of the titanium surface could be easily regulated by changing the concentration of the Li+ ions. Furthermore, upon culture of human osteoblasts, enhanced proliferation rates and strong attachment were observed, suggesting favorable surface features towards osteogenesis. Additionally, Li-titanate formed an apatite-like layer on its surface in a physiological environment [31, 32]. A thorough literature survey indicates that no study has ever verified the formation and binding mechanism of Li on Ti surface. The surface properties of Ti treated by Li+ ions were characterized by a variety of techniques such as X-ray diffraction (XRD), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and, Xray photoelectron spectrometer (XPS) in order to confirm the successful deposition of Li ions onto the Ti substrates. Furth more, the topographical structure, surface wettability, adhesive strength, sustainability of Li ions release, human osteoblasts viability and attachment and calcium phosphate nucleation capacity in vitro after incorporation of Li+ ions onto Ti implants were assessed. The results suggest that Li-incorporated nano-scale surface modification on Ti implants holds great promise towards the fabrication of the next generation of bone/dental implants, aimed at superior bone-forming ability. 2. Materials and methods

2.1.Surface modification of titanium substrates using lithium Ti flat foil (99.5% purity, Nilaco, Japan) with a thickness of 0.20 mm was mechanically prepared using abrasive SiC papers [33]. The prepared Ti foil was cut into 10 mm  10 mm squares, cleaned in distilled water and an ethanol in ultrasonic bath and subsequently dried in air. The alkali treatment using NaOH solution was applied on Ti substrates as described previously [34-36]. Briefly, Ti discs were etched in an acid mixture (equal volumes of concentrated H2SO4: HCl: H2O) at 80 °C for 1 h to remove the natural oxide layer and increase surface roughness. Then, the samples were immersed in 200 mL of 5.0 M NaOH aqueous 5

solution at 60°C for 24 h and rinsed in distilled water. To introduce Li ions, the alkali treated Ti samples were first immersed in lithium chloride (LiCl: 0.025, 0.1 and 0.2 M), and then hydrothermally treated in a Teflon container at 90 °C for 24 h. After Li-containing compound precipitation, the Ti substrates were rinsed in distilled water and dried at 45°C for 24 h. The alternative name of untreated and treated Ti samples are summarized in Table. 1. 2.2.Surface topography characterization of Li-Ti network Surface (Ti1-Ti5) topographies were examined using a scanning electron microscope (SEM, Jeol, 7001F). The samples were mounted on a SEM holder with double-sided conductive tape and coated with a 5 nm thick layer of platinum. Images with a range of scan sizes at normal incidence and at a 30° angle were acquired from the top/bottom surfaces and cross-sections. The SEM images were analyzed using the ImageJ software package (https://imagej.nih.gov/ij/). The phase composition of the Ti discs was characterized using an X-ray diffractometer (XRD; Rigaku, Japan). The surface roughness [root-mean-square (Rq) and arithmetic mean (Ra)] measurements of all untreated and treated Ti samples (Ti1-Ti5) were checked at 8 different locations using a Surface Roughness Tester (SURFTEST SV-3000, Mitutoyo, Japan) with the stylus moving in one direction along a 2.5 mm length at a speed of 0.2 mm/s (Surface roughness profiles are shown in Fig. S1 of the supporting information). In order to confirm the surface wettability, Hank’s balanced salt solution (HBSS, mimicking the simulated body fluid solution) was dropped on sample surfaces, and the contact angle was measured by contact angle analyzer (SEO Phoenix, Surface & Electro Optics Co., Ltd, Korea) [37]. 2.3 Investigating Li distribution on Ti using LA-ICP-MS and XPS The Li distribution deposited on Ti surfaces was analyzed using a Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) system at the Radiogenic Isotopic Facility at the University of Queensland. This system includes a Resolution ATL

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193 nm excimer Ar-F laser system coupled with a Thermo Fisher Scientific Icap RQ ICPMS. Full details of the method can be found in the literature [38-40]. The samples were mounted and secured in the sample holder and then scanned and placed into the sample cell. The sample holder was coordinated with the XYZ stage of the sample cell. The ablation location of the samples was visualized and controlled by Geostar software. A sequence of the ablation tracks including line and spot profile was created. Three line and four spot analyses were conducted for each sample and the operating parameters of the LA-IC-PMS are shown in Table S1 of the supporting information. Next, the samples were ablated in brackets of a set of standard materials (Glass standards NIST610, NIST612, BHVO-2, and BCR-2) and the sample aerosols were analyzed using a mass spectrometer for 7Li, 23Na, 31P, 43Ca, 44Ca and 49Ti. The data acquisition for each line/spot was performed with Qtegra v2.8 software in time-resolved analysis mode as a single csv file. The raw data file was exported from Qtegra and imported into Iolite software. X-ray photoelectron Spectrometer (XPS) incorporating a 165 mm hemispherical electron energy analyzer. The incident radiation was Monochromatic Al Kα X-rays (1486.6eV) at 150W (15kV, 10ma). Survey (wide) scans were taken at an analyser pass energy of 160 eV and multiplex (narrow) high resolution scans at 20 eV. Survey scans were carried out over a 1200-0 eV binding energy range with 1.0 eV steps and a dwell time of 100 ms. Narrow highresolution scans were run with 0.05 eV steps and 250 ms dwell time. Atomic concentrations were calculated using the CasaXPS version 2.3.14 software and a Shirley baseline with Kratos library Relative Sensitivity Factors (RSFs). Peak fitting of the high-resolution data was also carried out using the CasaXPS software. All data has been charge corrected using the Ti 2p3/2 from Ti (IV) at 458.7 eV. 2.4 Evaluation of mechanical stability of Li-Ti

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The adhesion strength between the precipitated Li layer and the Ti substrate was tested using a tensile testing machine (Instron model 3353) with a tensile rate of 0.5 mm/min at room temperature. In this test, thicker (~3 mm) Ti substrates were used to avoid buckling during the tensile test. At least three samples were tested for each group and an untreated Ti sample (Ti1) was used as a control. The tensile test specimen was prepared by gluing the Ti1, Ti2 and Ti3 samples to two aluminum alloy supports. The Al alloy supports were vertically fixed between the machine grips and strain was applied perpendicular to the surface of the samples. The load at which the support-Ti samples became separated from the samples was recorded as the failure load. From the stress-strain curve analysis, tensile strength (the maximum load that the samples can withstand during the test) was considered as the adhesion strength between the Ti samples and the supports. 2.5 Release quantification of Li ions in vitro Li+ ions were forced release via shaking speed of 100 RPM of incubated treated substrates in three different solutions: Milli-Q water, Hank’s Balanced Salt Solution (HBSS) and Bovine calf serum (BCS) for 3, 7, 14 and 21 days at 37±0.5 °C. Milli-Q water is suitable for comparative studies on the hydrolytic release. HBSS (pH=7.4) has ion concentrations essentially equal to those of human blood plasma [41, 42] and is a buffered solution. Bovine calf serum (BCS, Thermo Fisher Scientific, Waltham, MA, USA, pH=7.45) was employed to be an alternative to synovial fluid which has been widely used as a simulator fluid of joint studies [43, 44]. Penicillin-Streptomycin (10 000 U ml−1, Sigma-Aldrich) was added to BCS to inhibit bacterial growth. During the shaking process, Ti-Li specimen were individually placed into a 15 ml centrifugal tube which contains 6 ml of solution. The tubes were closed to avoid evaporation and then were kept horizontal during the whole soaking time. For Li+ release, the samples were analyzed at 670.783 nm wavelength using a Varian (brand, manufactured

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Melbourne Australia) Vista Pro (model) radial ICP-OES instrument. Standards from 0-5 mg/L Li were prepared from Fluka, TraceCERT 1000 mg/L stock standard. The changes of pH in the solution containing Ti-Li treated specimens were monitored in Milli-Q water, SBF and BCS under the same conditions. The pH electrode was calibrated before every measurement. Results reported are the average of three repeated measurments. The results correspond to average values of three specimens. The microstructure analysis of the Ti-Li samples were performed twice: at 3 and 21 days. Data are presented as mean value ± standard deviation. 2.6. Apatite formation Li-treated Ti substrates were soaked in 40 mL of HBSS which was used to simulate human body conditions and as a simulated body fluid (SBF) [45, 46]. Additional details were reported in our previous publication [45]. HBSS was refreshed every day and the ratio of the HBSS solution volume to the Ti substrates mass was 200  mL g-1. The Ti substrates were collected from HBSS after incubation for 5 days, rinsed with distilled water 5 times and dried at room temperature for a week. The apatite formation on the surfaces of scaffolds was observed by SEM, EDS and XRD, as described elsewhere [45]. 2.7 Culture of human osteoblasts on various substrates and Osteoblast Viability Alveolar-bone derived human osteoblast (OB) cells at a density of 50 ×103/well were seeded in a 24-well plate. The primary cell cultures were established as described previously [47-49] (section 1 of supporting information). A LIVE/DEAD assay® (Life Technologies, Australia) was performed after 24 and 72 hours of culture. At predetermined time intervals, fluorescence images of the cells on the substrates were collected using Confocal Microscopy (Nikon Eclipse Ti-E. Nikon Instruments INC. U.S.A) at maximum excitation/emission wave lengths 493/510 nm for FDA and 540/625 nm for PI. Representative fluorescence images were taken and processed with ImageJ® free software (ImageJ, National Institutes of Health,

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Bethesda, USA) to determine the percentage of live and dead cells. Alamar blue test (Thermo Fisher Scientific Inc. Australia) was processed to measure the metabolic activity and growth of untreated cells and Li-treated Ti samples on day 1, 3 and 7. Absorbance was measured at 570/600 nm wavelength and the values were normalized to obtain the percentage of AlamarBlue reduction. 2.8. Characterization of cellular spread morphology Cells were fixed after culturing for various periods on various substrates surfaces for the purpose of imaging using SEM. Briefly, the samples were immersed in fixation buffer (3% Glutaraldehyde in 0.1M Cacodylate buffer), washed with Cacodylate buffer (0.1M), treated with Osmium Tetroxide (1% in Cacodylate buffer) and rinsed with ultra-high quality (UHQ) water. Thereafter, the samples were dehydrated in ethanol (50-100%), followed by treatment with hexamethyl disilazane (HMDS) for 1 h. The samples were then air-dried and mounted on SEM holders as described previously [50]. Cell morphology was assessed as previously described, with some modifications [47]. After 24 h of incubation on the various surfaces, the cells were fixed (4% w/v paraformaldehyde/PBS, 20 min) and the samples were then blocked with goat serum (2.5% in PBS) for 1 h. This was followed by permeabilization with Triton X-100 (0.5% in PBS) on ice for 5 min and staining with Phalloidin-TRITC (10 μg/ml in PBS; Sigma Chemical, St Louis, MO, USA) at room temperature in the dark for 1 h. After washing (3 × PBS), the cells were incubated with DAPI (4′,6-diamidine-2′- phenylindoledihydrochloride; 1 μg/ml in methanol;Roche Diagnostics, Castle Hill, NSW, Australia) for 10 min. After washing with PBS (3 ×), the samples were mounted in glycerol (50% in PBS) and examined by confocal microscopy (Leica). Images were taken using water immersion at 10× objective. The emission was viewed through a long-pass barrier filter (E570P). The images were analyzed using Confocal Assistant software (Todd Clarke Brelje, USA).

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2.8 Statistical Analysis All data are shown as means with standard deviation. The standard deviation (S.D.) is the same as the “combined standard uncertainty of the mean” for the purposes of this work. To test for statistically significant differences, a t-test was used for pairwise comparisons (p < 0.05) and one-way analysis of variance (ANOVA) with Tukey's post hoc testing was used for comparison of 3 or more treatments (p < 0.05). 3. Results and Discussion 3.1.Surface Characterization of lithium incorporated titanium substrates SEM images of the Ti substrates before (Ti1) and after alkali (Ti2) treatment are shown in Fig. 2a-f. The micrograph of the as-polished Ti substrate (Fig. 2a-c) shows a smooth surface with few defects and without topographical features. After the alkali treatment, oriented (vertical) nanowire arrays are formed on the Ti surface (Fig. 2d-f). These nanostructures are similar to those reported previously [6], resulting from the reaction of metallic Ti with NaOH. Further, hydrothermal reaction with Liresulted leads to the formation of a unique continuous nanowire network layer on Ti as shown in Fig. 3a-m. These unique interconnected nanowires were uniformly distributed across the substrate surface. Next, the effect of incorporating different Li+ ion concentrations during the hydrothermal reaction on the surface topography of Ti substrates was investigated. A gradual change in the porous structure of Ti samples treated with different concentrations of Li+ ions can be observed from these images (Fig. 3a-m). It has been reported that like other osteogenic ions, Li has the capacity to increase bone tissue cell responses and may improve the rate of bone [24]. The porosity and pore size look dissimilar between the different treated Ti samples. At low Li+ ions concentration (Ti3), pronounced and interconnected nanowires were formed (Fig. 3a-c). The interconnected porous structure with nanowire topography interacts closely with the pseudopodia that extend from the cellular membranes of the osteoblasts that surround the implant. It is worth noting that an increased surface area with unique chemical characteristics 11

can allow for enhanced interaction between the surface of the implant and adjacent cells [51]. Furthermore, nano-scale surface topography has the potential to orchestrate osteogenesis and achieve early stability and long term success of implants [13]. The ECM-like surface features with increased porosity make our nanotextured titanium surface promising towards increased bioactivity. Importantly, this nano-engineered titanium surface can easily be extended onto current titanium-based bone and dental implants. A reduction in porosity resulted from the further loading of Li+ ions, and further nucleation (Fig. 3f) leading to isolated and agglomerated particles on the early formed nanowires network layer (Ti4, Fig. 3d-m) could be observed. Indeed, the Ti5 samples treated with 0.2 M LiCl started to congregate and short/dense and non-connected nanowires were observed (Fig. S2), and some agglomerated particles were formed on top of the nanowire layers, as shown in Fig. 3i-m. From the SEM images, the average calculated nanowire diameter is around 62±8.5 nm which is assembled from fine nanoparticles with diameter 7±3.5 nm (Fig. S2). These results show that the porosity of the synthesized continuous nanowire network layers is influenced by the concentration of the LiCl solution, while the formed nanowire maintains its physical structure with minor growth. The XRD patterns of the resulting chemically treated surfaces are shown in Fig. 4. The X-ray diffraction matches well with previous reports [52, 53] of the Ti1 (hexagonal α-Ti phase) and Ti2 (sodium titanate) surfaces. From the XRD patterns, weak broad shoulders at around 2theta= 11°, 20-30°, 47-50° and around 58°of Ti2 corresponds to sodium titanate hydrogel layer formed on Ti surface after alkali treatment (which is likely to have a composition of Na2Ti6O13 or Na2Ti5O11 [6, 54]) . Most of the sodium titanate peaks did not disappear after hydrothermal treatment with pure water (supporting information, Fig. S3) and Li ions (Fig. 4) solution at 100 °C for 24 h. This can be attributed to Na+ ions remaining in the structure and not being completely released into the Li+ solution [53]. Several studies have 12

shown that the sodium titanate gel formed on Ti substrates is unstable after hydrothermal treatment (from 40 °C [53]) and the gel layer releases from the treated surface during the reaction process [32]. The common chemistry at play here is the ion exchange between Na+ and H+ during hydrothermal reaction, resulting in the formation of Ti-OH [32]. Moreover, no obvious diffraction peaks corresponding to Li compounds at Ti3, Ti4 and Ti5 were detected by XRD (Fig. 4), which may be due to the low XRD technique sensitivity to Li compounds or/and the low content of the Li element in the formed composite layer on Ti substrate in relation to the Ti substrate itself. Since LA-ICP-MS is a direct (in situ) sampling analytical technique that enables highly sensitive elemental and isotopic analyses of solid materials [38, 55], the Li content on the Ti surface was assessed by this technique. The analysis could be performed without any sample preparation. In addition, a spatial analysis was undertaken, with micron range resolution in general both in terms of depth and lateral directions. Fig. 5 and Fig. S4 present a typical ablation profile distribution of line and spot scans of 7Li+ measured on continuous nanowire network composite layers formed onto Ti substrates using the proposed LA-ICP-MS procedure. In conjunction with the ablation line shots, a strong 7Li+ ion intensity was observed (Fig. 5a) with an average concentration of about 3101±74.5 ppm (Table. 2) of Ti3. The results did not show any significant (p >0.05) difference in Li concentrations onto the Li-treated Ti substrates (Table. 2), which ca be attributed to the formation of a stable lithium composite layer on Ti substrates. In general, from the line shots analysis, the Li content shows a homogeneous distribution over the scanned Ti surface (Fig. 5a and Fig. S4). These results correlate with the spot profile data which is presented in Fig. 5b. Furthermore, these findings indicate that Li is successfully incorporated onto the surface of Ti substrates during the hydrothermal process. Together, this is the first report to investigate the formation of Li ions on Ti surfaces by LAICP-MS as a quantitative analysis technique.

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To gain more insight into the sample surface composition at the atomic level, XPS measurements were performed and the corresponding spectra of Ti3 are presented in Fig. 6 (XPS patterns of all samples are shown in Fig. S5 of supporting information). Fig. 6a represents the full-scale XPS spectra composition of the formed nanowire network layer on Ti3. All the binding energies (BEs) in the XPS analysis were corrected for specimen charging by referencing them to the Ti 2p3/2 from Ti (IV) at 458.7eV. A typical O1s spectrum of the Li-Ti treated samples (Fig. 6b) contains three oxygen species, evidenced by the corresponding O 1s peaks at 528.36, 530.16 and 532.09 eV, respectively (Fig. 6b). These results suggest that oxygen in the surface layer exists in the form of oxide and hydroxide [56]. The O1s peak at 530.16 eV shown in the O1s spectrum corresponds with the Ti-O/Li-O metallic band in the nanowire layer, whereas the other peak centered at 532.09 eV can be ascribed to the absorbed oxygen by carbonate species and/or hydroxyl oxide [56, 57]. The Ti 2p3/2, Ti 2p1/2 spin orbit doublet (Fig. 6c) and Li 1s (Fig. 6d) characteristics peaks of the Li-Ti treated sample are located at 458.7 eV, 464.4 eV and 55.58 eV, for the predominant oxidation states of Ti4+, and Li+1, respectively [31, 56, 58]. Hence, Li was found to be in its ionic form Li+1 and Ti is in its fourvalent state, as confirmed by the Li 1s signal at and Ti2p3/2 signal (which is likely to be a lithium titanate [31, 59]). This experiment demonstrated that the deposited Li onto Ti substrates is combined with Ti and O elements to form a continuous nanowire network composite layers. However, due to Li exhibiting a low atomic ratio (inset of XPS curves) and a low sensitivity detection in XPS (0.025 relative sensitivity factor (R.S.F)) compared to Ti (2.001 R.S.F) and O (0.78 R.S.F), it is difficult to identify the formed chemical structure on Ti substrates after the hydrothermal process. Furthermore, a new Ti 2p peak of Li-Ti samples is observed at 457.37 (2p3/2) and 462.57 (2p1/2) eV, 456.4 (2p3/2) and 462 (2p1/2) eV in Fig. 6c, and it may be assigned to the formation of Ti3+ and Ti2+ [60] due to ionic exchanges during the hydrothermal reaction, which is absent in Ti2 samples (Fig. S1a). The C 1s peak at 284.6 eV may be attributed to the

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C-H and C-C bonds of hydrocarbons caused by carbon contamination. Hence, for Ti treated samples with a continuous nanowire network layer, the main component (Fig. 6 and Fig. S5) of the coated layer are O, Ti and a small amount of Li (Table. 2 and inset of XPS curves). The state of O, Ti and Li on the surface of Ti3 is identical to that on Ti4 and Ti5, but a slightly higher Li content (at %) on Ti5 was detected compared to the other substrates, in good agreement with the LA-ICP-MS data (Table. 2). The fabrication of a Li nanowire layer on Ti involved the growth of a sodium titanate hydrogel layer on the surface of titanium substrates after alkali treatment [6, 61, 62], and subsequent composite layer formation through ion-exchange during a hydrothermal reaction[63]. Although Li+ cations are not suitable to replace Na+ in the reaction in question, the ion exchange of H+ by Li+ was possible, as reported by Izawa et.al [64]. However, the ionic radius of Li+ is smaller than that of Na+. Thus, a Ti surface exhibiting a more stable chemical structure [31, 65] is expected when Li+ is substituted for Na+. Furthermore, owing to its small ionic radius, the field strength of Li+ is the greatest among the alkali metals. The sodium titanate layer formed as an amorphous phase [6, 61], which is unstable in hot water (< 100 °C) [32] and even unstable during low heat treatment (40 °C [52]) or in practical use [66]. When exposed to Li+ solution during hydrothermal treatment, the alkali titanate layer is again hydrated to transform into TiO2 hydrogel via release of Na+ ions from the alkali titanate layer into the Li solution (Fig. 1). Hence, immediately after immersion of the alkali-Ti samples in the Li solution, they release Na+ ions from the surface of sodium titanate, thus forming Ti-OH groups on the Ti surface [52]. The Ti-OH groups, immediately after their formation, incorporate the Li+ ions in the fluid to form a Li-Ti-O-H composite, which is later nucleated into fine NPs and assembled into a regular nanowire network structure [67]. There are Ti-O-H bonds in the Litreated specimen, and ion exchange of H+ by Li+ [63] takes place during the hydrothermal reaction after Na+ is freed from the sodium titanate into the Li solution, which may form a

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chemically stable amorphous lithium titanate (LTO) [31, 32], according to the expected overall chemical reaction as shown below in Eq. 1 [68]. Thus, Li+ has strong affinity for bonding to oxygen [24] and tends to contract the free spaces in the Ti network. This kinetically reduces the rate of TiO2 dissolution and improves chemical durability. Once the LTO nuclei are formed, they spontaneously grow, consuming the Li+ ions from the surrounding Li solution resulting in assembled nanowire-like structures from nucleated fine particles from the surrounding solution (Fig. S2). Clearly, the nanowire network composite layers were formed via ions exchange during the hydrothermal reaction. Explanation of such behavior is difficult and requires additional complete structural characterization and understanding of the ionic exchange mechanism.

Li+ + Cl− + TiO2 (gel layer) + OH − + H + + Na+ (released into Li solution) → Li2 TiO3 + 2NaCL + 𝐻2 𝑂 𝐸𝑞. 1 [68]

3.2. Mechanical stability of lithium modified titanium In a load bearing bone/dental implant setting, inappropriate mechanical stability leading to release of disintegrated implant surface material can cause severe immunotoxicity and lead to complete implant failure [69]. Clearly, the durability and the stability of coatings depends intimately on the degree of adhesion between the substrate and coating [69, 70]. For example, for regeneration, the coating must be physically stable in the defect site during healing [69]. The adhesion strength average values between the coating and Ti substrates is shown in Fig. 7. The highest adhesion strengths were found for Ti3 samples. The adhesion strength of the Ti3 coated layer is 11.23±1.12 MPa, which is higher than that of Ti1 (3.63±0.45 MPa) and Ti2 (6.45 ±1.12 MPa). It is well known that surface properties, such as surface roughness and wettability, play an important role in enhancing the adhesion strength [6, 71]. Hence, rough 16

and wettable surfaces increase the interfacial strength between the glue and Ti substrates. It was observed that since Ti1 has a smooth surface and low wettability (Table. 1), the adherent layer was completely detached from Ti1 and attached onto the two Al alloy supports (data not presented) resulting in a low pull-off resistance during the adhesion test. In comparison, both Ti2 and Ti3 coatings have a higher surface roughness and wettability compared to Ti1 substrates, resulting in higher resistance strength, as demonstrated in Table. 1. On the other hand, physical bonding may result from mechanical interlocking between the penetrated adherent (epoxy resin) molecules, achieved by diffusion during sample preparation and interlocking with the formed grooves on Ti2 and Ti3 samples [6]. However, the results raised the question as to why Ti3 presented higher adhesion strength, even though both Ti2 and Ti3 samples showed almost similar surface roughness and wettability (Table. 1), which is likely due to the low stability of the sodium titanate hydrogel layer formed on the Ti surface [32, 52, 54]. 3.3. In vitro release of Li+ ions release in various media To investigate the coating stability and the sustained release of Li+ ions from the Ti3 treated surfaces, Ti3 samples were incubated in Milli-Q water, HBSS and BCS solutions for 3, 7, 14 and 21 days at 37 °C under vigorous shaking at 100 rpm. Overall, Ti3 samples incubated in BCS media showed the lowest Li ions release (Fig. 8a-c). During the 3 days of incubation, the released amount of Li ions from the samples into the three solutions was in the following order: HBSS (0.437±0.03 mg/l) > Milli-Q water (0.376±0.023 mg/l) > BCS (0.198±0.02 mg/l), as presented in Fig. 8a-c. At day 7 of incubation, the Li ions release in did not show a significant decrease in both Milli-Q water and BCS media, whereas SBF showed a significant decrease (abrupt change) of the ions release. For a longer incubation time, the release demonstrated a sustained pattern, particularly after 14 days of soaking under vigorous shaking. The higher rate of Li ions release after 3 days, can be attributed to agglomerated particles on the Ti3 treated

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surfaces, which leached out during the in situ precipitation. Subsequently, a sustained release rate of Li ions into the three different media was observed until 21 days. These results indicate that a very low amount of Li ions is released into the different solution compared to the total amount detected by LA-ICP-MS (Table 2), as mentioned above. Notably, the concentration of Li on the Ti3 surface as detected by LA-ICP-MS was about 3101±74.5 ppm (mg/l), which was over 8k-fold the amount of Li ions released from the same surface after 3 days in both Milli-Q water and HBSS. Fig. 8 shows the measured pH values in the three presented solutions. Clearly, as compared to the control group (solution free samples), Li ions release did not change the pH of the Milli-Q water, HBSS and BCS solutions. This indicates that the pH of the buffered and unbuffered solutions was not affected by the Li ions release. From SEM images (Fig. 8a), there were apparent difference between the surface morphologies of Ti3 at day 3 and 21 of incubation in Milli-Q water. Surprisingly, at 21 days of incubation in HBSS, although the surface of Ti3 treated sample was fully covered with bonelike apatite layer (discussed in following section), the surface continued releasing Li ions into the solution (Fig. 8b). This is likely the reason for abrupt decrease of Li ions release at day 7 of incubation in HBSS. Further, the surface of Ti3 after incubation in BCS was partially covered by a new layer (Fig. 8c) which was formed from the surrounding BCS solution. Likewise, the layer did not restrict the release of Li ions. However, the release was decreased after 14 days of incubation in BCS. It is noteworthy that BCS contains comparable amounts of Na, K, Ca, Mg and organic spices (e.g. proteins, amino acids, etc.) [43, 44], which can affect the diffusion gradient and hence the release of ions from Ti surfaces. These findings suggest that the lithium titanate nanowire network layers formed on Ti substrates have high coating stability and enables low sustainable release, thus allaying any biosafety concerns upon implantation [69]. 18

3.4.Assessment of in vitro apatite-forming ability Previous studies demonstrated that apatite mineralization on the surface of biomaterials plays an important role in improving osteoblast growth and enhanced osteogenic ability [37, 42, 52, 72-74]. In the current study, we found that the Ti3 coating induced apatite formation on their surfaces (Fig. 9). A possible mechanism is that existent Na+ cation [6, 52] exchanges or/and the OH- group (as documented from XPS results) favors deposition of (Ca, P)containing apatite on the surface of the Ti3 nanowire network layer in SBF. This observation correlates with the cauliflower-like structures (Fig. 9a, b) that are characteristic of calcium phosphate formation (Fig. 9c,d) [37, 42]. Further investigations are needed to reveal the exact composition/crystallinity of this layer. Another possible explanation is that the nano-scale features of the Ti3 coating, including the interconnected nanowire layer, can facilitate biomineralization because of their resemblance to native collagen and hydroxyapatite (HAP), the main constituents of bone [42, 75, 76]. Hartgerink’s results confirmed that these interconnected nanowire materials promote deposition of HAP [77], while our study shows that the Li+ release from Ti3 coating induced the formation of apatite minerals on the biomaterial, including Li crystals. Our previous publication also showed that Ti implants coated with an interconnected nanowire/nanofiber layer could enhance apatite mineralization on the coating surface [78], although the fibers lacked bioactivity. This indicates that the Litreated Ti surfaces can bond to living bone [42, 75, 76]. The apatite coating achieved by biomimetic deposition can make Ti-based implanted materials osteoconductive, leading to improved bone tissue repair by metal implants. It is noteworthy that the implant modification demonstrated in this study on pure Ti implants may be easily translated onto Ti-alloy based implants, thereby applicable to a wider implant market. 3.5.Human osteoblast functions in vitro: viability and spread morphology

19

In vitro testing of cell viability and cellular activity on these novel biomaterials is fundamental for the quantification of any changes to cell function provoked by the formation of Li-O engineered nanotopography and/or Li+ release, which may be ultimately translated into improved bone formation in vivo [21, 75, 79]. To evaluate the potential cytotoxic effects, alveolar-bone derived primary human osteoblasts (OB) were cultured on the untreated and treated Ti substrates, and live/dead staining was performed after 1 and 3 days. The live/dead stain images revealed no difference in the percentage of live cells with and without Li treatment (Fig. 10a). The fluorescent images confirmed that nearly all cells were viable and that they maintained an osteoblastic morphology during cultivation. However, the percentage of live cells was higher on the Ti3 and Ti4 samples, particularly Ti3, than the Ti1 and Ti2 substrates, although this differences were not statistically significant (Fig. 10b, p > 0.05). The metabolic activity of OB cells cultured on the Ti1, Ti2 and Ti3 samples is shown in Fig. 10(c). The percent reduction of AlamarBlue values for all samples increased continuously with incubation time, which indicates a normal growth trend and confirms good cytocompatibility. It should be noted that the Ti3 samples induced higher growth than the Ti1 and Ti2 samples, and Ti3 caused the highest cellular metabolic activity rate compared to the other groups (P < 0.05) at the longer incubation time period (day 7). This means that surface modification by Li on treated Ti surfaces is supportive of osteoblast functions [28]. To further evaluate the effect of the Ti surface modification by Li on the cell growth and adhesion behavior, the attached cells were stained 7 days after seeding. Confocal laser microscopy scanning images (Fig. S6) after 7 days of culture showed superior attachment and spreading on Ti3 compared to Ti1 and Ti2, confirming that cells were firmly anchored on the surface of the Li-Ti treated surface. In addition, Fig. 10d shows that the Ti3 samples were mostly covered with confluent OB that were attached and spread, and exhibiting clear lamellae. A higher magnification observation (Fig 10d) revealed cell-matrix interactions with an intimate 20

contact of the cells with the Ti3 surface. Further, numerous cells presented a wide cellular membrane spread with flattened morphology, as demonstrated in Fig. 10d. On the high magnification SEM images (Fig. 10d and Fig. S7), it could be observed that the filopodia of the cells on the Ti3 samples were more developed than those on the Ti1 substrates. Our findings correlate with Dalby et al. [80] who reported that cells use filopodia to interact with nanostructures with interconnected pores. Additionally, filopodia-nanostructure interactions were also clearly visible (Fig. S7 of supporting information). Long, thin filopodia (∼120 nm diameter) interacted with the Ti3 nanowire network layer at a distance from the cells (Fig. S7), whereas, shorter filopodia were observed close to the cell bodies. Filopodia are responsible for detecting favorable locations for attachment and for inducing subsequent reorganization of the cytoskeleton to allow further cell migration. Hence, once filopodia have adhered to the desired locations, focal adhesions from at the leading edges of the cells, which create linking of the actin cytoskeleton to the ECM as illustrated in Fig. 10d and Fig. S7 [15]. Furthermore, the significant number of interconnected cells on the Ti3 coatings confirmed a favorable growth behavior. Hence, Ti3 coatings provide a bioactive environment that supports cell adhesion and growth. Numerous studies have confirmed that a hierarchical nanostructure similar to human bone could influence the conformation of typical adhesive proteins (fibronectin and vitronectin) present in serum that have been shown to influence osteoblasts behavior [1, 28, 52, 80, 81]. The complex extracellular matrix (ECM) that surrounds bone cells exhibits highly interconnected fibers at the nanoscale. Evidence also suggests that surfaces with patterns that mimic ECM can lead to enhanced metabolic and osteogenic activity [82]. In addition, the interconnected nanowire-like structure that mimics the ECM has proved to be very effective in enhancing cellular interaction with biomaterials [1, 80, 82]. Our preliminary cell culture data revealed that in comparison with the untreated Ti samples (Ti1), the nanowire Li-Ti treated 21

surface with nanoporous structure accelerates cell attachment and growth [15, 52, 76]. The impact of nanotopographical cues on osteoblast adhesion was investigated initially by Dalby et al [83] and later by Lamers et al. [84]; they demonstrated that porous nanotopographical structures result in significant increases in cells adhesion. It is also likely that the Li ions release from Ti3 surfaces (Table. S2, supporting information) might enhance cellular activity [28]. The release of Li ions from scaffolds was no more than 0.1 mg after soaking for 7 days, which would have no cytotoxicity effect at these low amount [21, 28]. Consistent with our findings, Chen et.al [79] investigated a range of Li ion concentrations, and shows that Li ions significantly stimulated cellular functionality and simultaneously promoted cell attachment and growth, as compared to Li-free controls. Zhang’s group [28] demonstrated

improved

osteoblast morphology, adhesion, and viability in Ti implants containing Li. Further investigations are needed to study Li-Ti treated surfaces, including the effects of both nanotopography and Li ions release on cellular function in both in vitro and in vivo models. To summarize, the Li-incorporated Ti-based implant surfaces fabricated in this study belong to a growing family of metal-ion doped Ti implants, exhibiting improved biocompatibility (with in vitro apatite-forming ability) and augmented osteoblast functions, and therefore comprehensive biological studies on Li-Ti-based implants will be the focus of our future work. Cell attachment and proliferation on Li-Ti treated substrates was enhanced compared to the untreated Ti substrate. These observations suggest that the osteoblasts were responding positively to the Li contained in the nanowire network matrix. This strong response suggests that the presentation of structural and chemical components together may stimulate increased osteoblast proliferation and attachment. Future studies will aim to clarify the specific role of Li ions release from the Li-Ti treated surfaces on the osteoblasts activity over a longer culture period (30 days). 4. Conclusion 22

In a pioneering attempt, this proof-of-concept study reports the fabrication of a stable lithium-doped nanowire network on alkali-treated Ti surfaces via ion-exchange during a low temperature hydrothermal process, towards enhancing the bioactivity of conventional Ti-based orthopedic/dental implants. This cost-effective and scalable technology can easily be extended to the current implant market, and results in the synthesis of a Li-Ti nanowire network with outstanding high-performance mechanical stability. Additional investigations revealed favorable osteoblast functions, including proliferation and adhesion, as well as apatite formation in artificial body fluid and sustained release of potent Li ions, translating into an attractive surface modification option for next generation of Ti implants. The formation mechanism of such novel nano-architecture on Ti surfaces is elucidated with the support of indepth chemical and surface characterizations, indicating ion-exchange during the hydrothermal process, with final topography and chemistry influenced by Li+ ions concentration. Allowing for tailored bioactivity and augmented osteoblast functions based on varying the hydrothermal concentration, our proposed Li-doped continuous nanowire network on current Ti implants can potentially enable enhanced local bioactivity and therapeutic effect from the surface of the implant.

Conflict of interests The authors declare no conflicts of interest of this work.

Acknowledgments The authors thank the assistance of Dr Barry J Wood from the Centre of Microscopy and Microanalysis at the University of Queensland in the data processing (curve fitting) of the XPS spectra using the CasaXPS software. Karan Gulati is supported by the National Health and

23

Medical Research Council (NHMRC) Early Career Fellowship (ECF), Peter DohertyAustralian Biomedical Fellowship (APP1140699).

Appendix A. Supplementary data Supplementary data related to this article can be found at

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Figure captions

Table 1. Surface roughness and water contact angle measurements of various titanium substrates. Samples Samples description

Surface roughness (Ra)

Water Contact

(µm)

Angle (WCA) (Degree)

Ti1

Polished Ti

0.144±0.01

85±3.2

Ti2

Ti1 treated with NaOH

0.703±0.03

14±0.5

Ti3

Ti2 treated with 0.025M LiCl 0.612±0.07

12±1.4

Ti4

Ti2 treated with 0.1M LiCl

0.795±0.06

15±1.5

Ti5

Ti2 treated with 0.2M LiCl

0.644±0.01

16±1.2

28

Table 1 shows that the TI2, Ti3, Ti4 and Ti5 coated Ti samples have an average surface roughness of about 0.688±0.1 nm, which is much higher than that of Ti1 sample (about 0.144±0.01 nm). The average contact angle is about 85° on the surface of Ti1 substrate, whereas it decreases significantly (P< 0.05) to 14° for the Ti2 substrate. The same phenomenon is observed on the Ti3, Ti4 and Ti5, indicating that the incorporation of Li on Ti surface did not significantly affect the surface wettability of NaOH treated samples.

29

Table 2. Quantification of lithium content on Li-modified Ti substrates. Data represents the average values of LA-ICPMS and XPS analysis. Samples

LA-ICP-MS

XPS

Li (ppm=mg/l)a

Li (At%)b

Ti3

3101±74.5

3.22±0.1

Ti4

2772±256

3.18±0.12

Ti5

3712±188

4.12±0.09

a

average Li concentration in ppm measured by LA-ICPMS a line (raster) analysis.

b

average Li concentration in atom% which was detected by XPS

30

Li+ aqueous solution

NaOH aqueous solution Na

Filopodia

HTIO3-

+

nucleus

Na+

LTO layer STH

TiO2 hydrogel

TiO2

TiO2

Migratory cell

Cell attachment on Li-Ti treated surface CaP minerals

Ti

Formation of sodium titanate hydrogel (STH) layer by alkali treatment

Ti

Formation of lithium titanate (LTO) layer by hydrothermal treatment process

Ti substrate

CaP formation on Li-Ti treated surface

Fig. 1 Schematic representation of the lithium nanowire network formation on titanium implants towards augmented bioactivity.

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Fig. 2 Top-view SEM images showing the topographical features of various titanium substrates: (a-c) polished Ti (Ti1) and (d-f) NaOH treated Ti (Ti2).

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Fig. 3 Lithium nanowire network formation post LiCl treatment of NaOH-treated Ti substrates. Top-view SEM images showing LiCl treatments with various concentrations: (a-c) 0.025 M (Ti3), (d-f) 0.1M (Ti4) and (i-m) 0.2 M (Ti5).

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Fig. 4 X-ray diffraction (XRD) patterns of various Ti substrates: Ti1 (polished Ti), Ti2 (NaOH treated Ti1), Ti3 (T2 treated with 0.025M LiCl), Ti4 (T2 treated with 0.1M M LiCl), and T5 (T2 treated with 0.2M M LiCl).

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Fig. 5 Evaluation of Li distribution on Li treated Ti using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The plots show: (a) line and (b) spot laser ablation.

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Fig. 6 X-ray photoelectron spectroscopy (XPS) of Ti3 (NaOH-treated polished Ti, modified with 0.025M LiCl) showing binding energies of (a) total survey, (b) oxygen, (c) titanium, and (d) lithium.

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Fig. 7 Mechanical stability and Li ion releasing performance of Li nanowire modified Ti substrates. (a) Adhesion strength of Li modification on Ti determined by tensile testing (Ti1: bare polished Ti, Ti2: NaOH treated Ti1, and Ti3: T2 modified with 0.025M LiCl).

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a

3 day

21 day

b

3 day

21 day

c

3 day

21 day

20 μm

Fig. 8Li+ ions release, pH measurements and SEM observation of TI3 samples achieved by constant shaking at 37C in (a) Milli-Q water; (b) HBSS and (c) BCS solutions for different time points.

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Fig. 9 Induced apatite mineralization on Li-treated Ti substrate (Ti3: NaOH-treated polished Ti, modified with 0.025M LiCl) in simulated body fluid (SBF) for 5-days. (a-b) SEM images at low and high magnifications showing top-view surface features. The newly formed apatite layer was composed of aggregates of nanocrystals with cauliflower-like morphology (b). (c) Electron dispersive spectroscopy (EDS) analysis profile showing the presence of CaP on the newly formed apatite crystals. (d) X-ray diffraction (XRD) analysis of Ti3 samples before and after immersion in SBF. XRD analysis further confirmed that the newly formed crystals on the surface of the Ti3 sample were apatite (2θ = 26°) microcrystals (JCPD card No. 09-0432).

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c

d Ti1

Ti3

* 50 μm

10 μm

50 μm

10 μm

Fig. 10. In vitro alveolar-bone derived human osteoblast (OB) functions on various treatments of Ti substrates. (a) Live/dead fluorescence imaging, (b) Live/dead quantifications, (c) AlamarBlue assay, and (d) top-view SEM observations of attached cells after 7 days culture. The viability/cytotoxicity assay was performed after 1 and 3 days of culture. Live cells were stained green by calcein acetoxymethyl (calcein AM) and dead cells were stained red by ethidium homodimer-1 (EthD-1). Ti1: polished Ti), Ti2: NaOH treated Ti1, Ti3: T2 treated with 0.025M LiCl, and Ti4: T2 treated with 0.1M M LiCl.

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Highlights

Li-doped nanowire layer was formed on the Ti surface via ionic exchange of Li+ ions. The physiochemical analysis confirms the deposition of Li+ ions onto the Ti substrates. Li-doped nanowire layer on Ti showed a high wettability and strong adhesive strength. A sustained release of Li+ ions over 21 days indicates a high stability of Ti modified surface. The Li treated Ti substrates had better cell response and apatite-like formation capability.

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