corrosion test, cell behavior test, and in vivo study of gradient tio2 layers produced by compound...

Corrosion test, cell behavior test, and in vivo study ofgradient TiO2 layers produced by compoundelectrochemical oxidation

Lin Zhu,1 Xun Ye,2 Guangxin Tang,3 Nanming Zhao,1 Yandao Gong,1 Yuanli Zhao,2 Jizong Zhao,2

Xiufang Zhang1

1Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and MembraneBiotechnology, Tsinghua University, Beijing 100084, China2Department of Neurosurgery, Tiantan Hospital, Capital University of Medical Sciences, Beijing 100050, China3Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China

Received 2 September 2005; revised 8 December 2005; accepted 25 January 2006Published online 30 May 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30745

Abstract: This paper describes efforts to improve implantbiocompatibility and durability by applying a hybrid tech-nique using composite oxidation. Pure titanium was used asthe substrate material. A porous oxide film as the outer layerwas produced by micro-arc oxidation and a dense oxide filmas the inner layer was produced by pre-anodic oxidation. Inthis study, physicochemical characteristics, corrosion test,cell attachment behavior, and in vivo studies were used tocompare this gradient layer with untreated titanium. Theresults revealed that the gradient layer was composed of twolayers of oxide films which were made up of rutile andanatase and the surface was porous with calcium and phos-phor. The corrosion resistance of the gradient layer wasimproved remarkably, which was about three times the

values for titanium and two times the value for the denselayer. The cell–material interaction study indicated that L929cells seeded and cultured on the gradient layer appeared toattach well and the rate of proliferation was the greatest. Thestudy in vivo showed that the gradient layer had goodbiocompatibility. This gradient layer provides a materialwith high corrosion resistance, bioactivity, and biologicalproperties suitable for tissue engineering applications.© 2006 Wiley Periodicals, Inc. J Biomed Mater Res 78A:515–522, 2006

Key words:biocompatibility; corrosion; titanium oxide; com-posite

INTRODUCTION

Titanium (Ti) and its alloys are frequently employedfor dental and orthopedic implants because of theirlow toxicity, low corrosion rate, and favorable me-chanical properties.1,2 However, the response of thehost to the implant is not always favorable. There aresome adverse foreign body reactions caused by releaseof metal ions from the implants. Furthermore, tita-nium together with its natural oxide thin film is

known to be bio-inert. It is difficult to achieve chem-ical bond with tissues and form new bone on its sur-face at an early stage after implantation, which maketitanium to display little bioactivity and indolence invivo.3–5

To overcome these drawbacks, an ongoing goal inbioengineering is the modification of the Ti implantsurface. Various bioceramic layers have been used,including TiO2, calcium phosphates such as hydroxy-apatite,6–8 and silica-based glasses such as Bio-glass.9,10

Titania film has been studied for a long time as awell-known material used in a wide variety of appli-cations. Recently, its use has been extended to medicalapplications, such as an active surface for orthopaedicor dental implants.11,12 Titania consists mainly of threepolymorphs: rutile, anatase, and brookite. With differ-ent methods and conditions, single or multiple poly-morphs can be formed with various morphologies. Ithas been suggested that titania has different proper-ties depending on its microstructure. A relatively po-

Correspondence to: X. Zhang; e-mail: [email protected]

Contract grant sponsor: The National Natural ScienceFoundation of China; contract grant number: 30400099

Contract grant sponsor: The National Basic Research Pro-gram of China (also called 973 Program, No. 2005CB623905)

Contract grant sponsor: Tsinghua-Yue-Yuen Medical Sci-ences Fund

Contract grant sponsor: Tsinghua University Basic Re-search Fund

© 2006 Wiley Periodicals, Inc.

rous oxide film was found to facilitate the incorpora-tion of mineral ions,13 which may be important in theinteractions between titanium implants and biologicalenvironments. A dense oxide film was known to beeffective as a chemical barrier against the in vivo re-lease of metal ions from the implants.14

Many processes for producing oxide films on tita-nium and its alloys have been reported recently, suchas ion plating, sputtering, chemical vapor deposition(CVD), plasma spraying, sol–gel, and ion implanta-tion.15–17 In these methods, the process called electro-chemical oxidation method can form ceramic-likefilms on metal surfaces with complex geometry at lowtemperature. Additionally, the thickness and chemicalcomposition of layers can be well controlled throughadequate conditions of the process.18

The previous studies usually produced single oxidefilms that could not take advantage of titania ade-quately.16,18 Therefore, the present study attempts tosynthesize a structural gradient layer on titanium toimprove the biocompatibility using a composite elec-trochemical oxidation method combining pre-anodicoxidation and micro-arc oxidation treatment. A po-rous oxide film as the outer layer was produced bymicroarc oxidation and a dense oxide film as the innerlayer was produced by pre-anodic oxidation. The un-treated titanium and titanium coated with only adense oxide film were used as control. The phase,composition, and microstructure of the three kinds ofspecimens were measured. Then L929 cells were cul-tured on these specimens and the cell spreading andproliferation behaviors were investigated. Finally, thespecimens were implanted into rabbits to investigatethe biocompatibility.

MATERIALS AND METHODS

Dulbecco’s minimum essential medium (DMEM), fetalbovine serum (FBS), and trypsin were purchased fromGIBCO (Grand Island, NY). All other reagents were of ana-lytical grade.

Preparation of dense titania and porous titanialayers on titanium

Preparation of pure titaniun

A rectangular Ti (grade 1) sample (10 mm � 10 mm � 0.4mm) was used as substrate. The surfaces of the sampleswere ground to 1200 particles using silicon carbide sandpa-per. This was followed by cleaning with distilled water andacetone, and then by immersion in a mixture of aqueous HFand HNO3 acids (the volume ratio of HF/HNO3 equaled to1:3) to remove the native oxide. The samples were then

rinsed with distilled water and air dried prior to pre-anodicoxidation and micro-arc oxidation treatments.

Preparation of dense titania layer by preanodicoxidation

For the preanodic oxidation treatment, a pure titaniumplate was put into the electrolyte, which was composed ofoxyacid and deionized water. The titanium plate was ananode, and the stainless steel electrobath was a cathode. Theparameters in the preoxidation treatment were as follows:the applied voltage was 150–200 V; the current density was40 mA/cm2; and the duration time was 2 min.

Preparation of porous titania layer by micro-arcoxidation

After the preanodic oxidation treatment, the sample wasquickly immersed into microarc oxidation electrolyte, whichwas composed of phosphate and calcium salt and deionizedwater. In the microarc anode oxidation treatment, a prean-odic oxidized titanium plate was used as an anode, while thestainless steel electrobath was still employed as a cathode.The applied voltage was 200–320 V. The current density ofthe microarc anodic oxidation was kept at 30 mA/cm2 for 10min.

Physicochemical characteristics

The microstructure of the layer was determined by anX-ray diffraction apparatus (XRD, model D8 Advance,Bruker), with Ni-filtered Cu radiation generated at 30 kVand 30 mA as the X-ray source.

The morphology of the film was observed using a scan-ning electron microscope (SEM; KYKY-2800, Chinese Acad-emy of Science, Beijing, China) with energy-dispersive spec-troscopy analysis (EDS) instrument.

Scratch test

The bond strength of the film was tested with an auto-matic scratch tester (WS-97, China). A spherical Rockwell Cdiamond stylus of 200 �m with a progressive load from 0 to100 N was used. All scratch traces were observed by ste-reomicroscopy. The critical loads (Lc) at which the sharppeak occurred were recorded. Ten samples were selectedand the value was determined by averaging the data.

Corrosion test

Because implants stay in contact with body fluid andtissues for long periods of time, the corrosion resistance willinfluence the use of materials. Like other metals, titanium

516 ZHU ET AL.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

also releases metal ions from the implants in the body.Usually, in the study of corrosion valuation, an added cur-rent is used to polarize the surface of metal to obtain acorresponding polarization curve. The polarization curvecan be used to obtain the polarization resistance and corro-sion current, which represent the ability to resist corrosionby Tafel’s equation.19

In the present investigation, the electrochemical polariza-tion technology was used to test the corrosion resistance ofuntreated titanium and two layers immersed in simulatedbody fluid (SBF) over a period of time. The principle of themethod, measuring techniques, and data analysis are de-scribed in detail elsewhere.19

The polarization measurements were made using an elec-trochemical analysis system (CHI608A, Shanghai Instru-ments), which was connected to a three-electrode electro-chemical cell. A platinum foil was used as counter electrodeand a saturated calomel electrode (SCE) with a salt bridgewas used as a reference electrode. The treated and untreatedtitanium specimens were used as the working electrodes.The starting voltage was �300 mV and the rate of scanningwas 0.5 mV s�1. The temperature of the SBF was (25 � 1)°C.Data registration and analysis were performed on a com-puter.

Cell attachment test

Cell line and growth of the cell line on specimens

The mouse fibroblast cell line L929 was purchased fromthe Institute of Virology Academia Sinica, Beijing. The cellline was incubated in a CO2 incubator supplied with 5% CO2

at 37°C. The culture medium was Dulbecco’s modified eaglemedium (DMEM) supplemented with 10% fetal calf serumand 0.1% penicillin–streptomycin solution. For experiments,cells were digested by trypsinase to form cell suspensions.The cell suspensions were seeded on all specimens, whichwere placed in 24-well plates at a density of 4 � 104 cells in1 mL of medium. The cells were cultured in the same way.The above seeded specimens were then inoculated in theCO2 incubator (5% CO2, 37°C) and the complete mediumwas changed every 3 days.

Scanning electron microscopy

The L929 cells cultured on specimens were washed 3times after 3 days of culture with phosphate buffered saline(PBS), followed by fixing with 3% glutaraldehyde in PBS for12 h at 4°C. The specimens were then washed 3 times withPBS, followed by postfixation with 1% osmium tetroxide indistilled water for 1 h at 4°C. After fixation, the specimenswere dehydrated in increasing concentrations of ethanol(from 50%, 70%, 80%, 90%, 95%, to 100%), followed bylyophilization and then observation by SEM.

Methylthiazol tetrazolium analysis

After cell culturing for 1, 3, and 7 days, the proliferation ofL929 cells was determined by methylthiazol tetrazolium(MTT) assay, in which the mitochondrial dehydrogenaseactivity of viable cells was measured spectrophotometri-cally.20 Briefly, 1 mL of serum-free medium and 100 �L ofMTT solution (5 mg/mL in PBS) were added to each well.After 4 h incubation at 37°C, the MTT solution was removedand the insoluble formazane crystal was dissolved in di-methyl sulfoxide (DMSO). About 100 �L of solution fromeach well was aspirated and poured into a 96-well plate forabsorbance measurement at 570 nm. The absorbance wasdirectly proportional to cell viability. MTT assay was firstperformed on a directly counted L929 cell series (0.2 � 105,0.4 � 105, 0.8 � 105, 1.6 � 105, 3.2 � 105, 8�105), and theabsorbency values were plotted against the counted cellnumbers to establish a standard calibration curve. Viable cellnumbers on scaffolds were then determined from the stan-dard curve on the basis of their MTT absorbency.

In vivo study

Young New Zealand white rabbits ( approximately 12 weeksold and weighing 2500 g) were used for this experiment. Forthe experiment, 12 rabbits were anesthetized with pentobarbi-tal (35 mg/kg). After the skin was prepped and sterilized withiodine and ethanol, three dots were selected in subcutaneousdorsal pockets for untreated titanium and titanium with twokinds of layers. For each dot, a 1.5 cm incision was cut to makea small pouch with a diameter of 10 mm. Three specimens(untreated titanium and the two coated samples) with a diam-eter of 10 mm and a depth of 1 mm were implanted into thepouches. After insertion of the specimens, the wounds werecarefully closed in layers. In the experiment, all surgical inci-sions healed without evidence of infection or other complica-tions. The rabbits were killed to harvest the implanted speci-mens in groups of three at 14, 30, 60, and 90 days after surgery.The harvested specimens were fixed in 10% formaldehyde,then dehydrated and embedded with paraffin. The specimenswere cut into 5 �m-thick slices for hematoxylin/eosin (H&E)staining.

RESULTS

Layer characteristics

Figure 1(a–c) shows SEM micrographs of the twocoated samples. The surface morphology of dense ti-tania layer is shown in Figure 1(a). A dense and com-pact thin film was formed on the titanium surface bypre-anodic oxidation treatment. Particles with submi-cron sizes were homogeneously distributed on thefilm. Figure 1(b) shows the surface morphology of thegradient layer, which was processed by micro-arc ox-idation of the pre-anodic oxidized titania film. The

GRADIENT OXIDE FILM-COATED TITANIUM LAYERS 517


surface of the layer had microprotrusions with holes.The diameter of the holes was 1–3 �m, and they werewell separated and homogeneously distributed overthe sample. There were some white flakes of amor-phous apatite scattered on the surface.

Figure 1(c) shows the EDS analysis results at thesame visual fields shown in Figure 1(a). The EDSspectrum of the dense layer showed that the depositedfilm was composed of Ti and O, without other ele-ments. Figure 1(d) shows that the EDS analysis resultat the same visual fields shown in Figure 1(b). TheEDS spectrum of the gradient layer revealed that thedeposited film was made up of Ca, P, Ti, and O. Theatomic ratio of Ca/P was 1.82.

Figure 1(f) shows the cross section of the layers aftercomposite oxidation treatment. The inner layer was a1-�m-thick dense oxide film made by pre-oxidation.The outer layer was 15 �m thick and was composed ofporous anatase and rutile TiO2, and was formed bymicroarc oxidation treatment. There were no evidentinterfaces between the substrate and the dense layer,or between the porous layer and the dense layer.

The XRD patterns of the two layers are shown inFigure 2. The dense layer indicated that the thin film

was TiO2 with peaks for the anatase phase. The gra-dient layer exhibited that in spite of the titanium, thefilm was TiO2 with peaks for the anatase and rutilephase. The diffraction peaks of both types were sharp,which indicated that the states of anatase and rutilewere good.

Table I shows the result of adhesion strength of thesurface TiO2 layers on the matrix. Because the denselayer was too thick, the credible data could not beobtained from the results. Only the adhesion strengthof the gradient layer is shown in the table. The loadingforce of gradient layer was 40.2 � 2.7 N, which wasmore than that of the other layers produced by plasmaspraying reported previously.21,22

Corrosion test

Figure 3 shows the samples’ ability to resist corro-sion. The more the polarization resistance is, the betterthe ability to resist corrosion. The polarization resis-tance of untreated titanium was much lower than theothers on the first day and decreased with time. Thepolarization resistance of dense layer decreased re-markably within 5 days but underwent a minorchange thereafter at a significantly reduced rate. Thepolarization resistance of gradient layer was also re-

Figure 1. Scanning electron micrograph and EDS spectrumof two layers. (a, c) Represent SEM and EDS of dense layer.(b, d) Represent gradient layer. (e) Shows the cross section ofthe layer after composite oxidation treatment.

Figure 2. XRD characterization of dense layer after prean-odic oxidation (a) and gradient layer after microarc oxida-tion (b).

TABLE IThe Thickness of Two Layers and the Adhesion Strength

of the Gradient Layer on Titanium

Sample Thickness (�m) Load Force (N)a

Dense layer 1 –Gradient layer 16 40.2 � 2.7

aBecause the dense layer was too thick, the load forcecould not be obtained.

518 ZHU ET AL.


duced initially but the rate was smaller than that ofdense layer. After 3 days, the resistance became stableand retained this state thereafter. Finally the polariza-tion resistance of the porous layer was the highest ofall and approximately two times more than that ofuntreated titanium, which indicated that the gradientlayer provided the best resistance to corrosion.

Cell attachment test

Cell attachments, proliferation, and differentiationover time on a material are the indications of biocom-patibility of the material and determine the suitabilityof the material for tissue engineering applications.L929 cell is one of the most common cells which areused to estimate the biocompatibility of the materials.The proliferation of L929 cells on both untreated tita-nium and the two layers was assessed using MTTassay. Figure 4 shows the number of cells attached tountreated titanium and the two layers after 1, 3, and 7days of cell culture. The number of cells on all thespecimens increased with time. The cells on gradientlayer were the most among all the specimens through-out the period of the study. Although the number ofcells initially attached to the surface of the gradientlayer was a little more than the others on day 1, astatistically significant increase in proliferation wasmeasured for L929 cells on the gradient layer than onthe others after 3 and 7 days of culture, respectively(p � 0.01).

Figure 5(a–c) shows SEM micrographs of L929 cellson the untreated titanium, dense layer and gradientlayer after 3 days of culture. L929 cells grew on allspecimens but more cells were observed on the gradi-ent layer than the others throughout the period of

study. Figure 5(d–f) details the morphology of indi-vidual L929 cells on all specimens after 3 days ofculture. The cells on all specimens showed an elon-gated shape and were anchored to the surface bydiscrete filopodia, but there was some difference be-

Figure 3. Changes of polarization resistance over time foruntreated titanium and two layers immersed in simulatedbody fluid.

Figure 4. Cell proliferation on untreated titanium, denselayer, and gradient layer as a function of time (n � 4). (*p �0.05, **p � 0.01).

Figure 5. SEM micrographs of L929 cells cultured on un-treated titanium, dense layer, and gradient layer after 3days. (a–c) Represent untreated titanium, dense layer, andgradient layer, respectively. (d–f) Details the morphology of(a–c).



tween untreated titanium and the two layers. The cellson the dense and gradient layers had bipolar or mul-tipolar shapes with highly extended filopodia, whichwas the shape of normal L929 cells. However, themorphology of some cells on untreated titanium didnot spread as well as that on the two layers, and theyhad few filopodia which extended badly. This factindicated that untreated titanium had worse biocom-patibility than the others for the growth of L929 cells.

In vivo study

Figure 6(a–c) shows in subcutaneous dorsal embed-ment the response of the tissue surrounding the spec-imens at 14 days after implantation. After 14 days ofimplantation it was observed that fibrous capsulesformed in all harvested specimens because of hyper-plasia of connective tissue. Mature and immature fi-broblasts were distributed in this layer with few lym-phocytes spread around. This was due to foreign body

reaction, and so the thickness of the layer representedthe biocompatibility of the specimen. The thinner theformative fibrous layer was, the more biocompatiblethe specimen was. Besides the formation of capillaryvessels around the fibrous capsule, it was also foundthat muscular atrophy did not occur. Figure 6(d–f)shows the response of tissue after 90 days. Comparedwith 14 days, the fibrin in the layer was orderly andhad become compact. The results for each sampleshowed that the number of blood vessels in the fibrouscapsules decreased and the tissue around the speci-mens remained normal without muscular atrophy.Between 14 and 90 days, there was little change in thethickness of fibrous capsules formed around the un-treated titanium and the dense layer. However, thefibrous layer formed around gradient layer decreasedto about 50 �m. During the period of study the layerformed around the gradient layer was the thinnest ofall the specimens, indicating that the gradient layerwas the most biocompatible.

DISCUSSION

Titanium and its alloys have been used extensivelyas hard tissue replacements, but there are many prob-lems, such as low combination strength, little bioac-tivity, and indolence. Compared with titanium, titaniahas better biocompatibility. In this study, a structuralgradient titania layer was formed on titanium using acomposite electrochemical oxidation method, result-ing in improved biocompatibility. By using micro-arcoxidation treatment, a porous uneven film wasformed. Therefore, the porous and titania film pro-duced in this study is expected to be of significance foruse in medical implants.

There are several polymorphs of titania of whichrutile and anatase are the most common. Rutile isconsidered as the stable form of titania. Anatase ismetastable and converts to rutile at high temperaturesaccompanying grain growth. In pre-oxidation treat-ment, because of the short treatment time and lowvoltage, the temperature was not high enough to makeanatase convert to rutile. Then, there was only ananatase phase without any other phases. In the micro-arc oxidation treatment, the high voltage made thetemperature of the surface increase, which created theconditions necessary for the formation of rutile. Be-cause the maximal voltage was less than 320 V, not allof the anatase could convert to rutile. So after micro-arc oxidation, a mixture of anatase and rutile wasformed. The diffraction peaks of both types weresharp, which indicated that the state of anatase andrutile was good.

After pre-anodic oxidation treatment, a dense andcompact thin film on the titanium surface was formed

Figure 6. Optical micrographs of specimens harvested af-ter implantation and stained with H&E in subcutaneousdorsal embedment. (a–c) Represent untreated titanium,dense layer, and gradient layer, respectively after 14 daysand (d–f) after 90 days. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

520 ZHU ET AL.


because the intensity of the electric charge on theelectrode was high. In micro-arc oxidation, the denseoxide film acted as an anode. Because the electriccapability of the dense layer was not as good as un-treated titanium, the intensity of the electric chargedecreased. So a porous surface with holes was formedafter micro-arc oxidation. Furthermore, the phosphatein the electrolytes moved to the surface of the anodeby way of an electric field and absorbed calcium ions.So the formed film contained calcium and phosphor.The chemical reactions for two oxidation processeswere the same: Ti � 4OH� � TiO2 � 2H2O � 4e�, andthe calcium and phosphor existed in the porous layeras ions.

Corrosion relates not only to the biocompatibility ofmetallic biomaterials but also to the fracture. Titani-um’s resistance to corrosion is attributed to the thinsurface oxide film formed passively. However, thepassive film is continuously dissolved in aqueous so-lutions from the microscopical viewpoint,23 whichcauses the dissolution of metal. So it is necessary toimprove the corrosion resistance of titanium by man-ual layer. In the present investigation, all specimenswere immersed in SBF over a period of time and thecorrosion resistance was tested by electrochemical po-larization technology. The polarization resistance oftitanium was very low at first and became even lowerthereafter. This fact meant that the passive film waseasily destroyed and could not protect titanium frombeing dissolved. So the titanium was continuallyeroded by the SBF and released metal ions. The polar-ization resistance of dense layer was approximatelytwo times greater than that of untreated titanium onthe first day and decreased at a rapid rate over severaldays. Because of the protection of the dense oxidefilm,14 this specimen exhibited great ability to resistcorrosion initially. However, the thinness of the film(about 1 �m) allowed it to be easily dissolved and itlost its ability to protect the titanium. This resulted incorrosion of metal and diminishment of resistance.The polarization resistance of the gradient layer was alittle higher than that of the dense layer at first. There-after, the resistance decreased at a slow rate comparedwith that of the dense layer. After 3 days the resistancebecame stable and retained this state throughout theperiod of study. The gradient layer was composed ofa thick porous film (about 15 �m) and an inner densefilm. This compound layer demonstrated excellentcorrosion resistance and could provide protection toresist erosion. So the corrosion resistance of the gradi-ent layer was the best of all three specimens and thepolarization resistance was approximately two timesmore than that of untreated titanium at the end.

In the present study, the biocompatibility of allspecimens was tested by culturing L929 cells on thesurface of each specimen. The significant difference ofcells adhering and spreading on the materials depends

on the differences in the chemical composition, topog-raphy of the specimen, and surface roughness.24,25

Compared with smooth titanium, the dense and po-rous oxide films increased the roughness of the twolayers. Furthermore, both dense layer and gradientlayer included anatase which is one of the phases oftitania. Anatase has unique properties and advantagesfor medical applications. Anatase exhibits stronger in-teractions between metal and support compared withrutile, and the surface of anatase can absorb OH� andPO4

3� in body fluid, which is favorable for depositingbone-like apatite.3–5,14,26 So the attachments, spread-ing, and proliferation of L929 cells cultured on the twolayers were better than that of untreated titanium.Compared with the dense layer, the gradient layer hada porous surface and included calcium and phospho-rus, which were propitious to the growth of cells.27,28

As a result, L929 cells proliferated more rapidly on thegradient layer than on the dense layer.

In the study of subcutaneous dorsal embedment,fibrous capsule could be observed around each spec-imen. This was due to the adverse foreign body reac-tion caused by degradation products or dissolutionions from the implant.29 After 14 days of implantation,the average thicknesses of the fibrous capsules were281, 194, and 169 �m for the untreated titanium, thedense layer, and the gradient layer, respectively. Thecorrosion test revealed that the corrosion resistance ofthe gradient layer was the best of all three specimens.This meant that under the same conditions, theamount of metal ions dissolved was the lowest for thetitanium with a gradient layer. So the fibrous capsuleformed around the gradient layer was the thinnest.Because of the good biocompatibility of the gradientlayer, the body adapted to foreign objects rapidly,which made the fibrous layer decrease markedly.Then after 90 days the thickness of the fibrous capsuleformed around the gradient layer decreased to about50 �m, and the others changed little.

CONCLUSIONS

We have constructed a structural gradient layer ontitanium using a composite electrochemical oxidationmethod. A porous oxide film as the outer layer wasproduced by micro-arc oxidation and a dense oxidefilm as the inner layer was produced by pre-anodicoxidation. The specimen with a gradient layer showedsignificantly improved biocompatibility as comparedwith the untreated titanium and the single-layereddense TiO2 layer sample. The gradient layer was com-posed of two layers of oxide films, which were madeup of rutile and anatase, and the surface was porouswith Ca and P. There were no evident interfaces be-tween the substrate and the dense layer, or between



the porous layer and the dense layer by SEM analysis.The cell–material interaction study indicated that L929cells seeded on the gradient layer and cultured ap-peared to attach well and the rate of proliferation wasthe greatest. The gradient layer markedly improvedthe corrosion resistance, which was about three timesthe value for untreated titanium or two times thevalue for the dense layer after 10 days of immersion inSBF. The in vivo study showed that the gradient layerhad good biocompatibility. These encouraging resultssupport the potential applications of the gradient ox-ide film-coated titanium as an improved alternative tountreated titanium for tissue engineering applications.

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corrosion test, cell behavior test, and in vivo study of gradient tio2 layers produced by compound...

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