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Colloids and Surfaces B: Biointerfaces 89 (2012) 40– 47

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

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he influence of UV irradiation on the biological properties of MAO-formed ZrO2

iqi Zhanga, Kunzheng Wanga,∗, Chuanyi Baia, Xiaobin Lib, Xiaoqian Danga, Chen Zhanga

Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, ChinaDepartment of Orthopedic Surgery, the Central Hospital of Henan Province, Zhengzhou 450000, China

r t i c l e i n f o

rticle history:eceived 5 March 2011eceived in revised form 20 August 2011ccepted 24 August 2011vailable online 31 August 2011

eywords:AO-formed ZrO2

ltravioletiocompatibilityioactivity

a b s t r a c t

Zirconium and its alloys are thought to be ideal materials for dental and orthopedic implants. However,the surface of native zirconium is bio-inert. It has been reported that micro-arc oxidation (MAO) is aconvenient and effective method to improving the biocompatibility and bioactivity of the zirconiumsurface, and ultraviolet (UV) irradiation can improve the bioactivity of the MAO-formed ZrO2 withoutaltering its surface morphology, grain size and phase component. The aim of the present study was toevaluate the influence of UV irradiation on the biocompatibility and bioactivity of MAO-formed ZrO2.Two types of samples were established. Those formed by MAO were labeled as MAO ZrO2 samples,while those that underwent UV irradiation after MAO treatment were labeled as MAO-UV ZrO2 sam-ples. In the in vitro study, osteoblasts were seeded on the surfaces of the MAO and MAO-UV samplesand were then studied by inverted phase contrast microscopy, scanning electron microscope (SEM) and

mplant MTT (3-(4.5-dimethyl-2-thiazolyl)-2.5-diphenayl-2H-tetrazolium bromide) testing. While in the in vivostudy, the samples were implanted into calvarias of New Zealand white rabbits and were then evalu-ated by histology and shear strength analysis. The results indicated that the MAO-UV surfaces showedbetter biocompatibility, faster new bone formation and firmer bonds with bone than the MAO surfaces.Therefore, UV irradiation may be an optimal second-stage treatment that can improve the properties ofMAO-formed ZrO2.

. Introduction

In recent years, zirconium and its alloys have been foundo be promising alternative materials for dental and orthopedicmplants because of their excellent mechanical properties andiocompatibility [1–6]. However, the thin oxide layer on nativeirconium, which is closely related to the biocompatibility ofirconium, is bio-inert, and generally encapsulated by fibrousissues without producing any chemical bond with bone aftermplantation [7–9].

An excellent implant material that can induce a bone-likepatite layer on its surface in vivo without any local inflamma-ory reactions should have good bioactivity and biocompatibility10,11]. In addition, the bone-like apatite layer should provide

tight chemical bond between the material and bone, and pro-

ote new bone formation around the material at the early stage of

mplantation.Micro-arc oxidation (MAO), also known as plasma electrolytic

xidation or anodic spark oxidation, is a convenient technique for

∗ Corresponding author. Tel.: +86 029 87679678; fax: +86 029 87679678.E-mail address: xjtuzzq@163.com (K. Wang).

927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2011.08.020

© 2011 Elsevier B.V. All rights reserved.

forming an oxide ceramic coating on the surface of non-ferrousmetals such as Ti, Mg and their alloys. This oxide coating has beenproven to be porous and firmly adherent to these materials [12–15].Yan and Han [16] reported that MAO could form porous zirco-nia films that firmly adhere to zirconium and the bond strengthwas strongest at an applied voltage of 350–450 V (>57.4 ± 2.1 MPa).They tested the material in simulated body fluid (SBF) and foundthat the zirconia films induced apatite formation within 3 days andexhibited good bioactivity.

Ultraviolet (UV) irradiation is an economical technique forsurface treatment, which has been reported to be able to gen-erate a hydrophilic surface on TiO2 [17–19] and transform Zrdiscs from hydrophobic to hydrophilic status [20]. Accordingto Han et al. [21], UV irradiation can significantly improve thebioactivity of MAO ZrO2 without altering its surface morphol-ogy, grain size or phase component, as a result of improvingits hydrophilicity by generating abundant Zr–OH groups on thesurface.

Inspired by the above reports, we wanted to know whether UVirradiation would make MAO ZrO2 more suitable for cell growth

and being implanted into a living body for a long time. Therefore,we designed the present study to investigate the cell responseson the MAO ZrO2 and MAO-UV ZrO2 surfaces as well as the boneformation after implantation.

faces B: Biointerfaces 89 (2012) 40– 47 41

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. Materials and methods

.1. Sample preparation

Commercially pure zirconium (99.9%, Material Depart-ent, Xi’an Jiaogtong University, Xi’an, China) was processed

nto discs (Ø5 mm × 1 mm) for in vitro tests and cubes3.2 mm × 3.2 mm × 2.0 mm) for in vivo tests. All sample sur-aces were polished with abrasive papers twice (800# aluminumxide/silicon carbide for the first time, and 1000# aluminumxide/silicon carbide for the second time), and then ultrasonicallyleaned with acetone and distilled water for three times.

For the MAO treatment, a pulsed DC power supply (400 V) wasmployed, and a zirconium sample was used as the anode while atainless steel plate was taken as the cathode in the electrolytic cell.he electrolyte was composed of 0.15 M �-glycerophosphate dis-dium salt pentahydrate (�-C3H7O6PO4Na2), 0.2 M Ca(OH)2 and

M glycerine (C3H8O3). The condition for MAO treatment waset based on Han’s reports [16,21]. The applied voltage, pulse fre-uency, duty ratio and duration time were fixed at 400 V, 100 Hz,6% and 5 min, respectively.

After MAO, the samples were irradiated by UV light in distilledater for 2 h at room temperature. The UV light was provided by

high-pressure mercury lamp of 1000 W, which generates light in00–600 nm range with a maximum intensity at 365 nm [21]. Theamples irradiated by UV light after MAO were labeled as MAO-UVamples.

According to Han’s reports [16,21,22], the MAO ZrO2 is com-osed of m-ZrO2 (monoclinic ZrO2) as a predominant phase and arace of t-ZrO2 (tetragonal ZrO2). The surface appears porous, with

pore size of around 3 �m and a porosity of around 30%. Comparedith the MAO ZrO2 film, the MAO-UV ZrO2 does not exhibit obvious

hange in phase component, surface morphology and grain size.

.2. Contact angle study

Wettability of the samples was measured by contact angle tests. 10 �l droplet of deionized water (pH 6.8–7.2) was suspended

rom the tip of a microliter syringe. The syringe tip was advancedlowly toward the sample surface to make sure the droplet con-acted with the sample gently. Images were collected with a contactngle meter (CA-X, Kyowa Interface Science, Tokyo, Japan) 1 minfter the droplets completely contacted with the samples. The con-act angles between the droplets and the samples were measuredrom the magnified images. All the reported contact angles in thisaper are the averages of 10 measurements taken at different loca-ions of the sample surfaces.

.3. In vitro cell response assay

.3.1. Cell seeding24-Well plates (n = 6) were used for cell seeding. For each plate,

discs of each kind of materials (MAO samples, MAO-UV samplesnd pure Zr as the positive control) were attached on the bottomf the wells, and 6 blank wells were used as negative control. Auantity of 2 × 105 fifth generation rabbit osteoblasts (The Fourthilitary Medical University, Xi’an, China) were seeded in each well

n Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) con-aining 10% fetal bovine serum (FBS, Gibco, USA) in a humidifiedtmosphere of 5% CO2. All the sample discs were sterilized at 180 ◦Cor 30 min before cell seeding.

.3.2. Optical microscope observationCells were analyzed by an inverted phase contrast microscope

Motic AE31 Germany) 1, 4, 7 days after seeding. The light source

Fig. 1. Contact angles of deionized water droplets on the MAO and MAO-UV sam-ples.

was put on the edge of samples so that morphology of the cellsattached on the sample surfaces could be observed.

2.3.3. SEM analysisScanning electron microscope (SEM. TM-1000, Hitachi, Tokyo,

Japan) analysis was conducted 1 day and 5 days after cell seeding.For SEM, samples were cleaned with phosphate buffer (PBS), soakedin a 3% glutaraldehyde solution for 24 h, dehydrated in an ascendingseries of ethanol (30–100%), and then dried in a critical point dryer(HCP-2, Hitachi, Tokyo, Japan). An ion sputter coater (E102, Hitachi,Tokyo, Japan) was used to sputter a thin layer of Pt on the samplesunder a vacuum pressure of 0.1 Pa for 3 min.

2.3.4. MTT testingMTT (3-(4.5-dimethyl-2-thiazolyl)-2.5-diphenayl-2H-

tetrazolium bromide) tests were carried out 1, 3, 5 and 7 days aftercell seeding. MTT was prepared in advance as a solution of 5 mg/mlin PBS (pH 7.1–7.3). During the test, samples were transferredinto a new 24-well culture plate containing 100 �l MTT solutionand incubated for 4 h at 37 ◦C in a 5% CO2 incubator. Then 650 �ldimethylsulfoxide ((CH3)2SO, DMSO) was added. The 24-well platewas shaken for 10 min and then the samples were transferredinto 96-well plates. After an overnight incubation, the opticaldensity (OD), by which the cell relative proliferation rate (CRPR)was measured, was assayed by an enzyme-linked immunosorbentassay (ELISA) reader at 490 nm and was compared with that of thenegative control.

2.4. In vivo test

2.4.1. Animal models

The in vivo test in present study was supervised by the Ethics

Committee of Xi’an Jiaotong University. Ten-month-old white NewZealand rabbits (n = 52) weighing 3–3.5 kg were used. After the ani-mals were anesthetized with 3% pentobarbital sodium (1 ml/kg IV),

42 Z. Zhang et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 40– 47

Fig. 2. Representative images of optical microscope observation. Magnification 400×. (A) MAO sample, (B) MAO-UV sample and (C) Zr sample. After 1 day culture, cellsattached on the MAO and MAO-UV surfaces have bright cytosol, clear margin and normal shape. On Zr, no cell is attached on Zr surface. After 4 days culture, cells attached onM distriA becaue

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AO and MAO-UV surfaces have clear margin and lucid cytosol, and are uniformlyfter 7 days culture, cells on MAO and MAO-UV surfaces are bright, and deformeddges and have toxic granulations within.

hree holes (Ø = 4.5 mm) were drilled in the calvarium of each rab-it, and were implanted with different cubic samples (MAO ZrO2,AO-UV ZrO2 and pure Zr). Since 3.2 mm × 3.2 mm is the largest

ize of a square that can fit into a circle of Ø = 4.5 mm (3.2 ×√

2 =.5), the cubic samples would stay stable in the holes. There wereour equal arc-shaped bone defects (BDs) around each cube, whichould be used to measure new bone formation. The operationsere performed under aseptic conditions, after which a single dose

f antibiotic was given (0.25 g Cefazolin, IM). The animals werellowed to take food 1 day after operations. No complications oreaths occurred in the postoperative period.

.4.2. Histology analysisSix animals were sacrificed 4 and 8 weeks post operation respec-

ively with an overdose of anesthetic. The implants and theirurrounding bone tissues were removed and washed in saline solu-ion, then fixed in 4% paraformaldehyde for 24 h to be processed forackaging. The specimens were dehydrated in an ascending seriesf ethanol (30–100%) for 24 h and then packed in 2 cm × 5 cm gly-olmethacrylate resins (Institute of Orthopaedics of Chinese PLA,ijing Hospital, Xi’an, China). After packaging, a microtome for hard

issue (Leica1600, Leica, Wetzlar, Germany) was used to section thealvaria tangentially at a thickness of 50 �m. The slides were soakedn tri-distilled water for 2 h, stained with haematoxylin for 30 min,nd then rinsed with tri-distilled water for 30 min. After that, the

buted, while those attached on Zr surface have blurry margin and gathered locally.se of over-crowdedness caused by fast proliferation, while those in C show blurry

slides were washed with ponceau-azaleine solution and 1% aceticacid solution, and observed under a normal transmitted light LeicaLaborlux microscope (Leica, Wetzlar, Germany).

2.4.3. Shear strength analysisTen animals were sacrificed 2, 4, 8 and 12 weeks, respec-

tively after operation with an overdose of anesthetic, and thewhole calvarium of each animal was removed and fixed in 4%paraformaldehyde for 24 h. The bonding force of the implant–boneinterface was quantified by a mechanical push-out test using a ser-vohydraulic testing machine (MTS8850, MTS, MN, USA). The leastforce, perpendicular to the calvaria, for pushing samples out waslabeled as shear force. We did not cut the three material samplesseparately so as to avoid micromotion which might result in errors.Then the shear strength was determined with the following equal-ity:

� = P4×0.0032×H

where � denotes the shear strength, P denotes the shear force, andH denotes the combined thickness of the implant and bone. Since

the combined thickness might be non-uniform, we measured twopoints randomly on each of the four quadrants of the circle, and theaverage value of a total of eight points was labeled as the combinedthickness.

faces B: Biointerfaces 89 (2012) 40– 47 43

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Fig. 3. Representative images of SEM after 1 day culture. Magnification 250×. (A)MAO sample, (B) MAO-UV sample and (C) Zr sample. Cells on MAO showed round

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Z. Zhang et al. / Colloids and Sur

.5. Statistical analysis

Data are expressed as means ± standard deviation, and normal-ty of the data distribution was assessed with the Shapiro–Wilkest. ANOVA followed by the Fisher’s test was conducted to assessifferences among groups using SPSS 16.0 (SPSS Inc., Chicago, IL,SA). Statistical significance was set at a p-value less than or equal

o 0.05.

. Results

.1. Contact angle study

The contact angles of water droplets on the MAO and MAO-UVamples were 94.3 ± 8.4◦ and 15.7 ± 2.3◦ (Fig. 1), respectively. It isidely agreed that a contact angle smaller than 65◦ indicates theaterial has a hydrophilic nature [23].

.2. Optical microscope observation

It was found that some cells, rounded or oval, attached to theurfaces of MAO and MAO-UV samples after 1 day culture, whichad bright cytosol and clear margin, showing good ability of refrac-ion. In contrast, no cell attachment was observed on the pure Zrurface (Fig. 2A1–C1). After 4 days culture, more oval cells attachedo the surfaces of the MAO and MAO-UV samples. In contrast, cellsttached to the Zr surfaces were few, presenting prismatic or poly-on shapes and blurry edges, indicating that they were not in goodealth (Fig. 2A2–C2). After 7 days culture, the MAO-UV surfacesere densely filled with cells that were sharply demarcated, uni-

ormly distributed and normally shaped, while the cells on the MAOurfaces exhibited a similar morphology but were less dense. Cellsn the Zr surfaces were sparse and in abnormal shapes, gatheredocally, and had toxic granulation within (Fig. 2A3–C3).

.3. SEM study

Fig. 3 shows the SEM picture of cells after 1 day culture (250×).ells on MAO surfaces showed round shape and clear margin, whilehose on MAO-UV surfaces showed pseudopodia, and stretched to areater extent. In contrast, cells on Zr showed rough margin, whichs a sign of apoptosis.

Fig. 4 shows a SEM picture of cells after 5 days culture. It waseen that the cells on the MAO surface were linked by pseudopo-ia and surrounded by ECM, which suggested a regular secretoryunction of them. The surface of the MAO-UV sample was coveredith ECM, indicating that the cells here secreted abundant ECM and

ould grow and proliferate well. On the Zr surface, cells underwentpoptosis and became cauliflower-shaped.

.4. MTT analysis

Fig. 5 shows the relationship of culture time and relative

ell proliferation rate (CRPR). It was found that the CRPRs ofhe MAO and MAO-UV samples were not statistically differ-nt from that of the negative control at all 4 time pointsp > 0.05). For each time point, the CRPR of MAO-UV samples

able 1ell relative proliferation rate of materials based on MTT analysis (mean ± standard devia

1 day 3 days

Negative control 100% 100%

MAO 98.01 ± 0.93% 96.30 ±

MAO-UV 101.25 ± 0.36% 97.43 ±

Zr 80.33 ± 0.89% 74.27 ±

shape and clear margin, while those on MAO-UV showed polygon shape and pseu-dopodia. On Zr, cells showed rough margin.

was a little higher than that of the MAO ones, but there was no

significant difference (p > 0.05), while the CRPR of the Zr sam-ples was significantly lower than that of the negative control(p < 0.05).

tion, n = 6).

5 days 7 days

100% 100%0.56% 98.74 ± 0.57% 97.24 ± 0.17%0.58% 100.40 ± 0.65% 98.00 ± 0.28%0.81% 70.44 ± 0.33% 69.04 ± 0.17%

44 Z. Zhang et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 40– 47

Fig. 4. Representative images of SEM after 5 days culture. Magnification 1500×. (A) MAO sample, (B) MAO-UV sample and (C) Zr sample. Cells on MAO ZrO2 have ECM andpseudopodia among them. The surface of MAO-UV sample is covered with abundant ECM. On the Zr sample, cells are cauliflower-shaped, indicating cell apoptosis.

Fig. 5. Relationship of cell relative proliferation rate (CRPR) and culture time. There was no significant difference in CRPR between MAO or MAO-UV samples and negativecontrol (p > 0.05) at all 4 time points, while Zr showed a significantly lower CRPR (p < 0.05).

Z. Zhang et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 40– 47 45

Table 2Relationship between cell relative proliferation rate and cytotoxicity grade (fromUSP).

Relative cell proliferation ratio (100%) Cytotoxicity grade

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Table 1 shows the CRPR of each material sample compared withhe negative control. According to United States PharmacopoeiaUSP, Table 2), the cytotoxicity of MAO-UV ZrO2 was ranked at grade–1, while that of MAO ZrO2 was at grade 1 and Zr was at grade. This indicated that the material, MAO-UV ZrO2, might be moreuitable for implantation into a living body for a long time thanAO ZrO2.

.5. Histological analysis

In present study, bone defects filled percentage (BDFP) was usedo measure the speed of new bone formation, and Image Pro Plus.0 (IPP, Media Cybernetics, MD, USA) was employed to determineDFP. Fig. 6 shows the histological staining of samples 4 weeksfter the operations. The BDFPs of MAO, MAO-UV and Zr samplesere 45.37 ± 5.26%, 59.28 ± 5.87% and 28.63 ± 4.13%, respectively.

ig. 7 shows the histological staining of samples 8 weeks post oper-tion. At this time point, the BDFPs of MAO, MAO-UV and Zr were2.81 ± 6.20%, 89.57 ± 7.33% and 61.98 ± 4.77%, respectively.

At the time points of 4 weeks and 8 weeks, the BDFPs of MAO-V were 13.91% and 16.76% higher than that of MAO, respectively,nd the differences were of statistical significance (p < 0.05).

.6. Shear strength analysis

Table 3 presents a summary of the shear strength tests and Fig. 8ives the results of the shear strength analysis. Zr/bone had lowerhear strength than MAO/bone and MAO-UV/bone at all 4 timeoints (2, 4, 8 and 12 weeks). Compared with that of MAO/bone, thehear strength of MAO-UV/bone was 1.04%, 2.52%, 6.65% and 6.63%igher respectively at all 4 time points, and showed significantifferences with that of MAO/bone at 8 and 12 weeks (p < 0.05).

. Discussion

In present study, we evaluated the biological properties of UVrradiated MAO ZrO2 by analyzing cell morphology, secretion ofCM, MTT, new bone formation and bond shear strength. The

able 3ummary of shear strength (mean ± standard deviation, n = 10).

Material Time Maximumshear strength(MP)

Minimumshear strength(MP)

Mean shearstrength (MP)

MAO 2 w 1.04 0.88 0.96 ± 0.054 w 1.35 1.05 1.19 ± 0.118 w 3.76 3.04 3.31 ± 0.26

12 w 4.06 3.44 3.77 ± 0.19

MAO-UV 2 w 1.08 0.78 0.97 ± 0.094 w 1.51 1.07 1.22 ± 0.128 w 3.98 3.17 3.53 ± 0.22

12 w 4.51 3.75 4.02 ± 0.24

Zr 2 w 1.01 0.64 0.91 ± 0.114 w 1.34 1.03 1.17 ± 0.108 w 3.27 2.64 2.96 ± 0.19

12 w 3.63 3.16 3.37 ± 0.15

Fig. 6. Bone defects after 4 weeks of implantation.

results indicated that compared to the MAO surfaces, the MAO-UVsurfaces showed better biocompatibility, faster new bone forma-tion, and firmer bonds with bone, but did not show significantlyless cytotoxicity. It means the MAO-UV surfaces might be moresuitable for implantation than the MAO ones. This is because byUV irradiation, the hydrophilicity of MAO ZrO2 is improved, whichmakes cells attach to the implant surface more easily. According toHan et al. [21], this improved hydrophily is resulted from the abun-dant Zr–OH groups generated by UV irradiation. In our study wehave found that the Zr–OH groups may keep on existing in livingbodies and their effects of speeding up new bone formation and

enhancing the bond of bone–material will increase after implanta-tion, especially after 8 weeks.

Cells are sensitive to the physical and chemical characteris-tics of the material surfaces with which they interact, and simple

46 Z. Zhang et al. / Colloids and Surfaces B

Fig. 7. Bone defects after 8 weeks of implantation.

Fig. 8. Relationship of mean shear strengths and implantation time.

: Biointerfaces 89 (2012) 40– 47

modifications of the material, variations in surface texture or topog-raphy can produce significant changes in cellular morphology[24–29]. Thus, cell morphology is an important parameter in deter-mining the cytocompatibility of an artificial material. In Wael’sreport, osteoblasts stretched to a greater extent on UV-treated zir-conia surfaces than untreated surfaces. In present study, we provedthat UV irradiation could improve the biocompatibility of MAOZrO2 as well. In fact, after MAO treatment, the material surfacesare no longer metal but a kind of biological ceramics which is com-posed of Ca/P and ZrO2. This indicates that UV treatment is effectiveon both metal and biological ceramics.

Generally, an excellent implant surface should have propertiessuch as good biocompatibility and bioactivity, with as little biotox-icity as possible [30–32]. Particularly for orthopedic and dentaluse, in addition, the surface should be able to accelerate new boneformation, and bond with bone steadily and tightly at the wholeperiod of implantation [33,34]. These properties are influencedby the physical structure (e.g. roughness, porosity or uniformity)and chemical composition of the surface [35–38]. By now, it iswidely agreed that bone tissues attach to an implant surface in twoways: osteoconduction, which is mainly determined by the phys-ical structure of the surface, and osteoinduction, which is mainlydetermined by the chemical composition [39,40].

In present study, MAO was used to produce a coating whichhad Ca/P in it and was porous. In this process, both the physicalstructure and chemical composition of the Zr surface were altered.However, in the process of UV irradiation, the surface of MAO ZrO2was just irradiated by UV light in distilled water without any chem-ical element added in. In this process, no physical structure waschanged on the material surface, which indicates that UV irradi-ation can improve the biocompatibility of MAO-formed materialsurface in a chemical way but with no element added in. We definethe treatments like UV irradiation as “catalyzer-like treatments”.

The “catalyzer-like treatments” should match the following con-ditions: (a) no physical structure of a surface is changed; (b) notany chemical element is added in the surface; (c) the biologicalproperties of the surface can be altered significantly. This type oftreatments can help us make further improvement to the surfacesalready used in clinic.

Our questions are: Can we alter lipotropy by UV irradiation inorganic solvent? And can other such “catalyzer-like treatments”as ultrasonic, alternating current electricity, liquid nitrogen frozenand magnetic field also improve some surfaces as well? These willbe our next work. We believe that the “catalyzer-like treatments”will have a broad prospect.

5. Conclusion

After irradiated by UV, MAO ZrO2 surfaces obtain better biocom-patibility, which makes the material more suitable for cell growing.In addition, MAO-UV ZrO2 can induce faster new bone formationand firmer bond with bone than MAO ZrO2.

Conflict of interest

All authors have no conflicts of interest.

Acknowledgments

This work was supported by the National Hi-tech Project 863,National Science Foundation of China (No. 2006AA03Z447), and thesupport of Xi’an Jiaotong University is acknowledged.

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