Materials Science and Engineering C 60 (2016) 308–316

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Materials Science and Engineering C

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Preparation and characterization of TiO2/silicate hierarchical coating ontitanium surface for biomedical applications

Qianli Huang a, Xujie Liu a,b, Tarek A. Elkhooly a,d, Ranran Zhang a, Xing Yang a, Zhijian Shen a, Qingling Feng a,c,⁎a State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, Chinab Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Chinac Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, Chinad Biomaterials Department, Inorganic Chemical Industries Division, National Research Centre, Dokki, 12622 Cairo, Egypt

⁎ Corresponding author at: School of Materials ScienUniversity, Beijing 100084, China.

E-mail address: biomater@mail.tsinghua.edu.cn (Q. Fe

http://dx.doi.org/10.1016/j.msec.2015.11.0560928-4931/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 July 2015Received in revised form 20 October 2015Accepted 20 November 2015Available online 25 November 2015

In the currentwork, TiO2/silicate hierarchical coatingswith various nanostructuremorphologieswere successful-ly prepared on titanium substrates throughmicro-arc oxidation (MAO) and subsequent hydrothermal treatment(HT). Moreover, the nucleation mechanism and growth behavior of the nanostructures, hydrophilicity, proteinadsorption and apatite-inducing ability of various coatings were also explored. The novel TiO2/silicate hierarchi-cal coatings comprised calcium silicate hydrate (CSH) as anouter-layer and TiO2matrix as an inner-layer. Accord-ing to the morphological features, the nanostructures were classified as nanorod, nanoplate and nanoleaf. Themorphology, degree of crystallinity and crystalline phases of CSH nanostructures could be controlled by optimiz-ing theHT conditions. The nucleation of CSHnanostructures is caused by release and re-precipitationmechanism.The TiO2/CSH hierarchical coatings exhibited some enhanced physical and biological performances compared toMAO-fabricated coating. The improvement of the hydrophilicity, fibronectin adsorption and apatite-inducingability was found to bemorphological dependent according to the following trend: nanoleaf coating N nanoplatecoating N nanorod coating N MAO coating. The results indicate that the tuning of physical and morphologicalproperties of nanostructures coated on biomaterial surface could significantly influence the hydrophilicity, pro-tein adsorption and in vitro bioactivity of biomaterial.

© 2015 Elsevier B.V. All rights reserved.

Keywords:Micro-arc oxidationHydrothermal treatmentTitanium implantSurface topographySurface chemistryCalcium silicate hydrate

1. Introduction

Titanium and its alloys are widely used in the manufacturing of or-thopedic and dental implants due to their desirable characteristics,such as good mechanical properties, corrosion resistance and biocom-patibility [1]. The successful fixation of titanium-based implants ismainly determined by the establishment of integration between the im-plant and the surrounding bone [2]. However, the bio-inert nature of ti-taniumand its alloysmay lead to the formation of a fibrous layer aroundthe implant and finally result in the failure of implantation [3].

In order to make titanium directly bond to bones, surface modifica-tion methodologies including chemical, physical and biologicaltreatments have been explored to improve osteoconductivity orbioactivity of titanium in the past decades [4–6]. Most of the surfacemodification strategies aim at modifying the surface topography andchemistry of titanium-based implants, which are considered astwo critical factors that influence osteointegration [7]. Surface topo-graphical modification is considered as an effective approach to

ce and Engineering, Tsinghua

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improve biological performance of titanium-based implants. It hasbeen reported that micro-scale and nano-scale topographies could sig-nificantly affect protein absorption andmodulate cell adhesion, prolifer-ation, differentiation and mineral deposition [8,9]. Micro- and nano-structured TiO2 coatings are commonly applied to enhance the topo-graphical complexity of implant surface [10–12]. Biggs et al. reportedthat nanotopography could be a regulator of cell functions throughfocal adhesions [13]. Rani et al. reported that leaf-like nanopatternsshowed enhanced osteogenic performance both in vitro and in vivocompared to other nanostructures such as nanotube, nanoscaffold andnanoneedle, indicating that osteointegration is sensitive to specificnanostructure morphology [12]. Surface chemical modification is an-other important approach to achieve improved osteointegration. Hy-droxyapatite (HA), a native component of bone, is widely applied tocoat the surfaces of joint and dental implants due to its structural resem-blance to the inorganic mineral of the natural bone holding great prom-ise for osteointegration [14,15]. Many processing techniques, such asplasma spraying, electrophoretic deposition and magnetron sputtering,have been developed to fabricate HA coatings on titanium surface [14,16,17]. However, few of these techniques can develop tunable topo-graphical features on titanium surface and simultaneously altering itssurface chemistry significantly. The development of strategies for an

Fig. 1. Schematic diagram showing the experimental setup used for the MAO treatment.

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appropriate surfacemodification in order to develop tunable surface to-pography on titanium surface and simultaneously control its surfacechemistry still remains a challenge.

Micro-arc oxidation (MAO) is a recently developed techniquewhichcould produce a porous and firmly adherent ceramic coating on titani-um surface, at the same timemodify the surface topography and chem-istry [18]. Through this technique, various kinds of bioactive elements,such as Ca, P, Si, Sr and Ag, can be incorporated into interfacial coatedlayer formed by MAO to enhance the biological performance of the re-constructed layer [19–22]. Recently, it has been reported that Ca- andP-incorporated MAO coating could in situ nucleate nanostructured HAon coating surface after hydrothermal treatment, finally lead to amicro/nano hierarchical structure on titanium surface [23,24]. The hier-archical micro/nano-topography, has been proven useful to mimic thehierarchical characteristics of the bone, acting as signal for osteoblast at-tachment, proliferation and differentiation [25–27]. These findings sug-gest that combing both MAO and HT (MAO-HT) could be an effectivestrategy to develop hierarchical surface topography on titanium surfaceand simultaneously control the surface chemistry.

Silicates, including silicate bioglass and silicate bioceramics, havebeen reported to stimulate osteogenic differentiation and bone deposi-tion in many studies [28,29]. CaSiO3 ceramic has been reported to showgreater ability to support cell attachment, proliferation, and differentia-tion compared to β-tricalcium phosphate (β-TCP) ceramic [30]. Recent-ly, calcium silicate hydrates (CSH) have attracted a great deal ofattention due to their good cytocompatibility and bioactivity [31,32].Wang et al. reported that tobermorite (Ca5(Si6O16)(OH)2·4H2O) couldpromote alkaline phosphatase (ALP) activity and expression of osteo-genic genes and angiogenic growth factors of rat bone marrow stromalcells (rBMSCs) [32]. Another study reported that the addition ofxonotlite (Ca6(Si6O17)(OH)2) into poly(L-lactide) (PLLA) could greatlyenhance the apatite-forming ability of PLLA films, as well as the attach-ment and proliferation of rBMSCs [33]. Moreover, calcium silicate hy-drates are commonly synthesized by HT technique and exhibitstypical nano-dimensional features [34,35]. In our previous study, we re-ported the possibility of fabricating TiO2/CSH hierarchical coating on ti-tanium surface through MAO-HT strategy [36]. In order to preciselycontrol the surface topography and chemistry of TiO2/CSH hierarchicalcoatings, more in-depth understanding of the nucleation mechanismand growthbehavior of CSH nanostructures onMAO coating is required.

Therefore, in the present study, a detailed investigation of the nucle-ation mechanism and growth behavior of CSH nanostructures on MAOcoating was conducted under various HT conditions. Additionally, thehydrophilicity, protein adsorption and apatite-inducing ability of vari-ous TiO2/CSH hierarchical coatings were also investigated.

2. Experimental procedure

2.1. Micro-arc oxidation of pure titanium

Grade 2 commercially pure titanium (Ti) disks (diameter: 15 mm;thickness: 1 mm) were used as a substrate material. The surfaces ofsamples were abraded with emery paper up to grid 1500 and then suc-cessively cleaned in acetone, ethanol and distilled water in ultrasonicbath. For MAO treatment, A MAO system (WHD-20, Harbin, China;shown in Fig. 1)was employed. The electrolytewas prepared as follows:firstly, Na2(EDTA) (0.10 M), Ca(CH3COO)2·H2O (0.10 M) and NaOH(0.25 M) were orderly dissolved in ultra pure water with continuousstirring. After that, the solution was stirred for 24 h in order tocompletely transform Ca2+ into Ca-chelate (Ca(EDTA)2−). Finally,Na2SiO3·9H2O (0.02 M) was added into the solution. During the MAOprocess, an applied voltage was fixed at 270 V and a pulse frequency,a duty cycle and the duration time set 50 Hz, 50% and 5min, respective-ly. After MAO treatment, such MAO treated samples (referred as MAOcoating) were washed with distilled water and then air dried.

2.2. Hydrothermal treatment of MAO coating

For HT process of MAOed coating, Teflon-lined autoclaves with vol-ume of 20 ml were used and 8 ml ammonia aqueous solution (pH 11)for each sample was added to them. The autoclaves were heated at200 °C for 4, 24 and 120 h. Each time of the hydrothermal treatmentof MAO coatings will be then referred to as MAO-HT4, MAO-HT24,and MAO-HT120 coatings, respectively. They were washed with dis-tilled water and then dried in air. The concentrations of Ca and Si ionswhich released fromMAO coating to ammonia aqueous solution duringHT process were measured by an inductively coupled plasma-opticalemission spectroscopy (ICP-OES; Varian Vista MPX, Varian, Palo Alto,CA, USA). The pH values of the ammonia aqueous solutions after HTprocess were measured using a pH meter (FE20K, Mettler Toledo,Switzerland).

2.3. Physicochemical properties analysis

The specimen surface morphology was observed by field emissionscanning electron microscopy (FESEM; JSM-7001F, JEOL, Japan)equipped with an energy dispersive X-ray spectroscopy (EDS)system. 3D roughness profiling of MAO, MAO-HT4, MAO-HT24 andMAO-HT120 coatings were evaluated using 3D profiling system(MicroXAM-3D Phase Shift, ADE Co., USA). The phase composition ofthe coatings was characterized with an X-ray diffractometer (Rigaku,Tokyo, Japan) using Cu Kα radiation. Themicrostructure of the CSH crys-tallites, which were carefully scratched from the surface of MAO-HT24and MAO-HT120 coatings, then was investigated using a transmissionelectron microscopy (TEM; JEM-2100, JEOL, Japan) operated at200 kV. Static water contact angle of the coatings wasmeasured by ses-sile drop using an optical tensiometer (JC2000C, Powereach, China). Atleast three samples were used for each group.

2.4. Protein adsorption assay

Rhodamine labeled fibronectin (Rhodamin-FN) from bovine plasma(Cytoskeleton, USA) was used as protein model to investigate the pro-tein adsorption. For protein adsorption assay, the substrates were incu-bated with 20 μg/ml Rhodamin-FN (in PBS) at 37 °C for 4 h, andfollowed bywashingwith PBS for three times. Fluorescencemicroscope(Leica, Germany) was used to monitor the fluorescence signal ofadsorbed FN on the coated surfaces. All photos were taken using thesame parameters such as time of exposure, saturation and contrast. Atleast 6 images were analyzed and semi-quantified by Image J softwareto obtain the optical density (OD) values.

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2.5. Surface mineralization

In vitromineralization was carried out following a protocol reportedby Liu et al., which has been identified as amore efficient heterogeneousmineralization process than the simulated body fluid (SBF) method [37,38]. Mineralization stock solution was prepared by suspending 7.37 ghydroxyapatite (Institute of Nuclear and New Energy Technology,Tsinghua University) in 500 ml urea aqueous solution (2 M), followedby the addition of concentrated hydrochloric acid under continuousstirring until a clear solution was obtained (final pH 2.5–3.0). Eachsample was vertically placed in a 50 ml plastic tube containing 10 mlof themineralization solution and covered with a perforated aluminumfoil. Then, the tubes were heated from 37.0 to 95.0 °C at a heating rateof 0.2 °C/min in a muffle furnace (Tianjin Zhonghuan, China). Mineral-ized samples were bath-sonicated for 30 s in distilled water to ensurethe removal of loosely boundmineral precipitates and dried under vac-uumbefore characterizations. The number and size of HA crystalswhichnucleated on various coated surfaces were quantified in randomly se-lected 340 μmsquares using ImageJ software. At least 6 imageswere an-alyzed by ImageJ software and all experiments were carried out intriplicate.

3. Results and discussion

3.1. Physicochemical properties analysis

A series of unique nanostructures have been fabricated on MAO-fabricated TiO2 coating surfaces through HT technique. Fig. 2 shows

Fig. 2. SEM images of MAO (a–c), MAO-HT4 (d–f), MAO-HT24 (g–

the surface morphology of MAO, MAO-HT4, MAO-HT24 and MAO-HT120 coatings using SEM micrographs at different magnifications.MAO coating shows macro-porous surface morphology with numerousmicro-sized and crater-shaped protuberances on its surface (Fig. 2a–b).The asperities and pores of MAO coating are considered to be beneficialfor the mechanical interlocking between the implant and bone [1].Compared to MAO coating with single-scale surface topography,MAO-HT-fabricated coatings possess dual-scale hierarchical surfaces.The micro-scale protuberances are still visible at low magnification, es-pecially on MAO-HT4 and MAO-HT24 coating surfaces (Fig. 2d, e, g andh). However, the porous feature is not that obvious on MAO-HT120coating surface (Fig. 2 j and k). At higher magnifications, it is observedthat various nanostructures have been obtained homogeneously onMAO coating surface (Fig. 2e–f, h–i and k–l). According to the morpho-logical features of the nanostructures which can be seen at high magni-fication micrographs (Fig. 2f, i and l), they can be classified as nanorod(MAO-HT4 coating), nanoplate (MAO-HT24 coating) and nanoleaf(MAO-HT120 coating). The dimensional features of the nanostructuresare listed as following: nanorod (diameter: ~40 nm, length:~100 nm), nanoplate (thickness: ~40 nm, length: ~400 nm, width:~130 nm) and nanoleaf (thickness: ~40 nm, length: ~800 nm, width(widest axis): ~220 nm). It indicates that the nanostructures tend togrow in length and width with prolonged HT duration but not in diam-eter or thickness. It is obvious from SEM micrographs that the surfacearea of the coatings increases with HT duration due to the continuousgrowth of the nanostructures (MAO-HT120 coating N MAO-HT24coating N MAO-HT4 coating N MAO coating). Semi-quantitative EDSspot analysis was used tomeasure the chemical compositions of various

i) and MAO-HT120 (j–l) coatings at different magnifications.

Table 1Chemical composition of various coatings measured by EDS.

Elemental atomic percentage (%) Ti Ca Si Na O

MAO 23.24 7.71 9.72 1.54 57.79MAO-HT4 22.25 6.18 6.11 0.67 64.79MAO-HT24 17.39 5.17 5.21 0.86 71.37MAO-HT120 24.26 7.07 3.26 0.33 65.08

Fig. 4. XRD patterns of various coatings.

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coatings (Table 1). It is revealed that bothMAO and TiO2/CSH hierarchi-cal coatings are composed of Ti, Ca, Si, Na and O.

The arithmetic mean surface roughness (Ra) of various coatings ex-hibited close values in the range of 600 to 700 nm (as shown in Fig. 3). Itindicates that the superposition of nanostructures with different mor-phologies to micro-scale topography does not significantly changethe surface roughness. All coatings have submicron-scale surfaceroughness.

In order to detect the change in the crystallinity of surface coatingsupon increasing of hydrothermal treatment time, X-ray diffraction(XRD) was used. The XRD patterns of MAO and various hierarchicalcoatings are shown in Fig. 4. Additional to the peaks of Ti belongs tothe pristine substrate (JCPDS # 44-1294), feature peaks of anatase(JCPDS # 21-1272) and rutile (JCPDS # 21-1276) are detected for allcoatings. Anatase peaks detected at 25.3, 37.8 and 48.0° aremuch stron-ger than those of rutile at 2θ = 27.4 and 36.1°, indicating that anatasephase is more predominant than rutile. The patterns of MAO-HT24and MAO-HT120 coatings show typical peaks of CSH [31]. However,the locations of these peaks are found to be different. It indicates thatthe CSH phase of the nanoplate layer is different from that of thenanoleaf layer (C1: nanoplate, C2: nanoleaf; Fig. 4). No extra peaks arenoticed for MAO-HT4 coating besides those of Ti, anatase and rutile, in-dicating the amorphous nature of the CSH layer in the nanorodstructure.

In order to determine the exact crystalline phases of the nanoplateand nanoleaf layers, TEMwas employed. The selected area electron dif-fraction (SAED) pattern of the nanoplate of MAO-HT24 coating

Fig. 3. 3D toughness profile of MAO (a), MAO-HT4 (b

demonstrates that the nanoplate is polycrystalline Ca6Si3O12·H2O ac-cording to the diffraction rings (JCPDS # 14-0035, Fig. 5b). The phaseof the nanoleaf of MAO-HT120 coating is characterized to bemonocrys-talline gyrolite (Ca4Si6O15(OH)2·3H2O) according to the distinct diffrac-tion spots (JCPDS # 42-1452, Fig. 5d). The results indicate that thedegree of crystallinity of CSH nanostructures follows the trend: MAO-HT120 coating N MAO-HT24 coating N MAO-HT4 coating.

Overall, MAO-HT4, MAO-HT24 and MAO-HT120 coatings all com-prise two layers, exhibiting hierarchical features compared to MAOcoating with single-scale surface topography. The outer layer is charac-terized to be CSH nanostructures and the inner layer is composed ofTiO2-based matrix. The current work reveals that the morphology, de-gree of crystallinity and the type of crystalline phase of CSH nanostruc-tures can be precisely tuned by optimizing HT conditions. MAO-HTmethod can be considered as an effective strategy to develop micro/nano hierarchical surface topography and simultaneously modify thesurface chemistry of titanium-based substrate.

), MAO-HT24 (c) and MAO-HT120 (d) coatings.

Fig. 5. TEM images and SAED patterns of the nanoplate and nanoleaf scratched from MAO-HT24 (a and b) and MAO-HT120 coatings (c and d), respectively.

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3.2. The nucleation and growth of CSH nanostructures

In the previous sections, we proved that different morphologies ofCSH nanostructures can be tuned in situ on MAO coating based on theHT condition. Therefore, it is considered to be necessary to elucidatethe nucleation mechanism and growth behavior of these nanostruc-tures. It has been reported that the elements such as Ca, Si and Na usu-ally exist as constituent to amorphous compounds in MAO coating [39].During the subsequent HT process, these elements could be released tothe aqueous environment [36]. Therefore, ICP-OES has been used tomonitor the release of those ions upon HT treatment for different pe-riods. It is interesting to notice that there is a decrease in Ca concentra-tion at 24 h of HT process (Fig. 6), revealing that there exists a re-precipitation process of elements. The re-precipitating rate of Ca isfaster than its releasing rate at 24 h, while the re-precipitating rate ofSi is always slower than its releasing rate at all time points (Fig. 6).

Fig. 6. Element concentrations of Ca and Si released fromMAO coating to aqueous solutionduring HT process.

Thus, it is reasonable to speculate that the nucleation process of CSHnanostructures follows a release and re-precipitationmechanism (sche-matically shown in Fig. 7) [36]. Under HT condition, the elements suchas Ca and Si can be released into the aqueous environment and thus in-crease the ion concentrations of Ca2+ and SiO3

2− (Fig. 7a). Meanwhile,the TiO2 surface is attacked by OH− ions in the alkaline environmentand transforms to `Ti–O− surface according to the following reaction:

`Ti–O–Ti`+2OH−→2 `Ti–O−+H2O.Subsequently, the negatively charged`Ti–O− surface attracts posi-

tively charged Ca2+ ions to the interface (Fig. 7b). Consequently, a pos-itively charged surface is created through the accumulation of Ca2+

ions. The positively charged interface subsequent attracts negativelycharged ions, such as OH− and SiO3

2− to the interface (Fig. 7c). Finally,the enrichment of Ca2+, SiO3

2− and OH− ions at the interface can inducethe heterogeneous nucleation of CSH on the surface of TiO2 matrix(Fig. 7d). In this way, the CSH nanostructures can nucleate in situ on ti-tania surface during HT process.

The growth process of CSH nanostructures is also accompaniedby continually releasing and re-precipitating of elements (Fig. 6).The initially formed CSH shows rod-like morphology and amorphousnature after HT for 4 h (Fig. 7d). With prolonged HT duration (24 h),the amorphous nanorod grows to polycrystalline nanoplate(Ca6Si3O12·H2O, Fig. 7e). Till 120 h, the nanoplate finally grows tomonocrystalline nanoleaf (gyrolite, Ca4Si6O15(OH)2·3H2O, Fig. 7f).Therefore, Ca6Si3O12·H2O and amorphous CSH could be consideredas intermediate CSH phases during the HT synthesis of gyrolite(Ca4Si6O15(OH)2·3H2O). In addition, CSH nanostructures tend togrow in length andwidth, but not in thicknesswith prolonged HT dura-tion, as having been discussed in Section 3.1 (Fig. 7g). Gyrolite(Ca4Si6O15(OH)2·3H2O) synthesized by HT technique has been report-ed to exhibit lamellar or plate-like morphology, which is differentfrom the leaf-like morphology in this study [40,41]. It is considered tobe caused by the in situ nucleation of CSH on coating surface. As oneend of the nanoleaf bonds to TiO2matrix, the other end tends to narrowdown gradually in order to minimize the surface energy. Moreover, theatomic ratios of Ca and Si (Ca/Si) of the nanoplate and nanoleaf are 2.0and 0.67 respectively, according to their chemical formulas. It indicates

Fig. 7. Schematics showing the nucleation and growth mechanisms of CSH nanostructures during HT process.

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that more Si ions could re-precipitate to CSH nanostructures than Ca2+

during 24 h to 120 h.

3.3. Contact angle measurement

The wettability of MAO and TiO2/CSH hierarchical coatings wasquantified by static contact angle measurement. It has been widely ac-cepted that the surface wettability could influence the initial proteinsadsorption, which would subsequently affect cellular and tissue reac-tions [42]. It has been reported that hydrophilic surfaces tend to en-hance the early stages of cell adhesion, proliferation, differentiationand bone mineralization compared to hydrophobic surfaces [43]. Com-monly, a contact angle smaller than 65° is considered as a hydrophilicsurface, larger than 65° as hydrophobic one [44]. In the current work,all coatings are characterized to be hydrophilic (Fig. 8). As shown inFig. 8, it is found that the hydrophilicity of various coatings follows thetrend: MAO-HT120 coating ≈ MAO-HT24 coating N MAO-HT4 coating.It indicates that the superposition of specific nanostructures to coating

Fig. 8. Static contact angles of various coatings.

surface could be an effective strategy to modulate the surface wettabil-ity of biomaterial.

3.4. Protein adsorption

One of the first biological events to occur when an implanttransplanted is the adsorption of water molecules, proteins and lipidsfrom the blood to the surface of the device [45]. The adsorbed proteinlayer on implant surface subsequently mediates the interactions be-tween cells and implant, which may finally affect the outcome of im-plantation [46]. In this work, fluorescence microscopy, which has beenreported as a reliablemethod to study the alteration tendency of proteinadsorption [47], was used for semi-quantitative detection of theadsorbed amounts of fibronectin (FN) on various coating surfaces. FNis chosen as protein model because of its abundant in blood whichmay play an important role in mediating cell/material interactionsby providing binding sites for integrin receptors on cytomembrane[48,49]. As shown in Fig. 9, the amounts of adsorbed FN on all hierarchi-cal coatings are found to be larger than that on MAO coating. Theamount of adsorbed FN on various coating surface follows the followingorder: MAO-HT120 coating N MAO-HT24 coating N MAO-HT4 coating.The amount of adsorbed FN increases up to 48% and 113% on MAO-HT24 and MAO-120 coatings compared to MAO coating, respectively.It has been reported that nanostructured TiO2 and HA coatings couldsignificantly enhance the protein adsorption compared to the counter-part without nanostructures [12,50]. Likewise, the enhanced proteinadsorption of hierarchical coatings can be attributed to the increasedsurface area. Based on thedimensional characteristic of different coatingmorphology, nanoleaf might have the largest surface area, thennanoplate, and finally nanorod. It is also possible that crystallinecoatings of the nanoleaf and nanoplate as shown by SAED patternFig. 5 (b and d), is preferential for protein adsorption on their lattice in-terfaces. The results shows that the superposition of CSHnanostructuresto MAO coating surface could significantly enhance protein adsorption.The enhanced FN adsorption on hierarchical coatings is considered to bebeneficial for cell/material interactions.

Fig. 9. Fluorescent images (a–d) and the calculated fluorescence intensities (e) of the adsorbed FN onvarious coatings; Data are presented as themean±SD, n=6, ⁎p b 0.05 and ⁎⁎p b 0.01compared with MAO coating, &p b 0.05 and &&p b 0.01 compared with MAO-HT4 coating, #p b 0.05 and ##p b 0.01 compared with MAO-HT24 coating.

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3.5. Surface mineralization

The apatite-inducing ability of an implant is known to play an im-portant role in osseointegration, herein the coatings were investigatedfor their apatite-inducing ability. In the current work, MAO and varioushierarchical coatings were subjected to urea thermal decomposition-mediated mineralization process. This mineralization process has beenreported to be driven by a gradual increase of the pH of an acidic aque-ous solution of HA by ammonium hydroxide, generated from controlledthermal decomposition of urea, in order to induce supersaturation ofthemineralization solution and subsequent heterogeneous mineral nu-cleation and growth [37,38]. As shown in Fig. 10, it is observed thatnumerous spherical mineral nodules were formed on all coating sur-faces. The EDS spectrum reveals that the nodules contain element P(Fig. 10a, shown by white arrows), which does not exist in all coatings(Table 1), indicating that the nodules are composed of apatite. More-over, the morphology of the nodules is similar to those in references[37,38]. Liu et al. have demonstrated the nodules formedby thismethodto be HA by high-resolution TEM (HRTEM) and SAED analyses [38]. Inorder to quantify the nucleation and growth of HA crystals during themineralization process, the density and size of the nodules were

calculated. As shown in Fig. 10e, the densities of the nodules on hierar-chical coatings are higher than that on MAO coating. The density of HAnodules (crystal number/mm2) is found to be increased by 115%, 210%and 270% on MAO-HT4, MAO-HT24 and MAO-HT120 coatings, respec-tively compared to HA nodules on MAO coating. It suggests that theapatite-inducing ability of various coatings follows the followingorder: MAO-HT120 coating N MAO-HT24 coating N MAO-HT4coating N MAO coating. The size of nodules formed on various coatingsurfaces showed a reversed trend compared to the nucleation rate(Fig. 10f). It indicates that higher nucleation rate usually correspondsto lower growth rate of crystals because those two processes happensimultaneously.

The differences on apatite-inducing ability of various coatings areconsidered to be caused by their different surface chemistry. It hasbeen widely accepted that functional groups, such as Si–OH, Ti–OH,and Ta–OH, could effectively induce the formation of apatite on theirsurfaces by electrostatic interactions [51,52]. As the surface chemistryof MAO coating is mainly determined by the TiO2 matrix, the exposedSi–OH groups on MAO coating surface are less than those on hierarchi-cal coatings during the mineralization process. Meanwhile, the apatite-inducing ability of Ti–OH groups has been reported to be weaker than

Fig. 10. SEM images (a–d), the calculated density (e) andsize (f) ofmineral nodules formedonvarious coating surfaces after themineralization process; Data are presented as themean±SD,n = 6, ⁎p b 0.05 and ⁎⁎p b 0.01 compared with MAO coating, &p b 0.05 and &&p b 0.01 compared with MAO-HT4 coating, #p b 0.05 and ##p b 0.01 compared with MAO-HT24 coating.

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that of Si–OH groups [39]. Therefore, MAO coating shows the weakestapatite-inducing ability among all. The surface chemistry of the hierar-chical coatings is determined by their nanostructured outer-layers. AsCSH are mainly composed of Ca, Si, O and H, they contribute in the en-hancement of apatite nucleation due to the abundant Si–OH groups[34]. It has been discussed that more Si ions could re-precipitate toCSH during 24 to 120 h compared to Ca ions in Section 3.2, indicatingthe amount of Si–OH groups of hierarchical coatings increases withprolonged HT duration. Therefore, MAO-HT120 coating shows thegreatest apatite-inducing ability among all. Another possible explana-tionmight be correlatedwith themorphological features of each coatingwhich correspond to different dimensions and subsequently differentsurface area. Similar to FN adsorption results in Section 3.4, the abilityof the nanoleaf to have many nucleation sites for HA deposition wasfound higher than nanoplate, then nanorod which might be attributedto the difference in the dimensionalmorphology of CSH on each coating.

The present findings suggest that TiO2/CSH hierarchical coatings,with controllable surface topography and chemistry and enhanced hy-drophilicity, FN adsorption and apatite-inducing ability, are of greatpromise to be applied as bioactive coatings for titanium-based implant.However, further studies on the in vitro and in vivo performances ofthese coatings are still needed.

4. Conclusions

In this study, TiO2/CSH hierarchical coatings have been fabricated ontitanium substrate through MAO-HT approach. Various CSH nanostruc-tures, such as nanorod-, nanoplate- and nanoleaf-like structures, couldbe hydrothermally prepared on MAO coating surface. The hierarchicalcoatings revealed the formation of nanorod-, nanoplate- and nanoleaf-like structures upon increasing HT duration from 4 h, 24 h to 120 h, re-spectively. It is suggested that the morphology, degree of crystallinityand crystal phase of CSH nanostructures can be controlled by optimizedHT conditions. Compared to MAO coating with single-scale surfacetopography, TiO2/CSH hierarchical coatings exhibit enhanced hydrophi-licity, FN adsorption and apatite-inducing ability. In addition, the hydro-philicity, FN adsorption and apatite-inducing ability of various coatingsbasically all follow the trend: MAO-HT120 coating N MAO-HT24coating N MAO-HT4 coating N MAO coating. The results indicate thatthe superposition of nanostructures to biomaterial surface could be aneffective way to modify the surface properties of biomaterial for moreefficient biological response. Moreover, the combination use of MAOand HT is considered to be a promising surface modification strategyto simultaneously modify the surface topography and chemistry oftitanium-based implant.

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Acknowledgments

The authors are grateful for the financial support from the NationalNatural Science Foundation of China (51361130032, 51472139) andDoctor Subject Foundation of the Ministry of Education of China(20120002130002).

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