biocompatibility of tio 2 nanotubes with different topographies

Biocompatibility of TiO2 nanotubes with different topographies

Yu Wang,1 Cuie Wen,2 Peter Hodgson,1 Yuncang Li1

1Institute for Frontier Materials, Deakin University, Geelong, Victoria 3217, Australia2Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia

Received 4 February 2013; revised 15 March 2013; accepted 25 March 2013

Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). 10.1002/jbm.a.34738

Abstract: The biological response of osteoblast cells to

implant materials depends on the topography and physico-

chemistry of the implant surface and this determines the cell

behavior such as shaping, adhesion and proliferation, and

finally the cell fate. In this study, titanium (Ti) was anodized

to create different topographies of titania nanotubes (TNTs)

to investigate the cell behavior to them. TNTs with and with-

out a highly ordered nanoporous layer on their top surface

were fabricated using two-step and one-step anodizing proc-

esses, respectively. The TNTs without a highly ordered nano-

porous layer on the top surface exhibited a rougher surface,

higher surface energy and better hydrophilicity than the

TNTs with such a layer. Osteoblast-like cells (SaOS2) were

used to assess the biocompatibility of the TNTs with different

topographies in comparison to bare cp-Ti. Results indicated

that TNTs can enhance the proliferation and adhesion of

osteoblast-like cells. TNTs without a highly ordered nanopo-

rous layer exhibited better biocompatibility than the TNTs

covered by such a nanoporous layer. Cell morphology obser-

vation using confocal microscopy and SEM indicated that

SaOS2 cells that were adhered to the TNTs without the

highly ordered nanoporous layer showed the longest filopo-

dia compared to TNTs with a highly ordered nanoporous

layer and bare cp-Ti. VC 2013 Wiley Periodicals, Inc. J Biomed Mater

Res Part A: 00A:000–000, 2013.

Key Words: TiO2 nanotubes, topography, surface energy,

biocompatibility

How to cite this article: Wang Y, Wen C, Hodgson P, Li Y. 2013. Biocompatibility of TiO2 nanotubes with different topogra-phies. J Biomed Mater Res Part A 2013:00: 000–000.

INTRODUCTION

Titanium (Ti) and some of its alloys have been widely usedfor orthopedic and dental implants because of their excel-lent mechanical properties and outstanding biocompatibil-ity.1 Many surface treatment methods, such as the sol–gelmethod,2 alkali and heat treatment,3 physical vapor deposi-tion (PVD),4 chemical vapor deposition (CVD)5 and so forth,have been used to modify the implant surfaces to promoteadhesion and proliferation of cells where the biomedicalimplants are in direct contact with tissues.

In recent years, Ti-based implants with nanoscale topog-raphies,6 especially titania (TiO2) nanotubes (TNTs),7,8 havereceived increasing attention in the area of biomedical appli-cations. It has been reported that TNTs exhibit outstandingbiocompatibility and depress adverse reactions by reducingthe adhesion of macrophages on the surface of the implants.9

Many studies have shown that the topography of TNTs signif-icantly influences the adhesion and proliferation of osteoblastcells.10–12 TNTs with diameter 70–100 nm are generallyaccepted for possessing excellent biocompatibility,13 althoughthere are still controversial results regarding the optimumTNT diameters for biomedical applications.10,14

In the anodization process, a series of parameters such asapplied voltage,15,16 water content,17 the pH of electrolyte,18

and the anodization time,19 affect the topography of TiO2

nanotubes. A two-step anodization method is popularly usedto synthesize multilayer nanotubes by adjusting the appliedvoltage in different anodization electrolytes.20–22 It has beenrecently reported that TNTs with a highly ordered nanopo-rous layer can be fabricated using a multistep anodizationprocess.23–25 However, most studies on this type of TNTsare focused on its photoelectrochemical abilities24 and so itsbiomedical performance is still not well understood.

The wetting ability of the TNTs plays a crucial role inthe biomedical applications as the surface energy of animplant influences the adhesion of cells to the surface.26,27

The wetting ability of biomedical implants determines theadsorption of DNAs and proteins.28 However, the relation-ships among the topographies of TiO2 nanotubes, surfaceenergy and the adhesion and proliferation of osteoblast cellsare scarcely reported to date.

In this study, two kinds of TNTs with different topogra-phies were fabricated by adjusting anodization parametersto investigate the effect on the biocompatibility and celladhesion on TNTs. The microstructure and in vitro perform-ance of the two different topographies were investigated.Wetting characteristics and the surface energy of the TNTslayer were evaluated using the Owens-Wendt (OW) method.

Correspondence to: Y. Li; e-mail: [email protected] grant sponsor: Australian Research Council (ARC); contract grant number: DP110101974

VC 2013 WILEY PERIODICALS, INC. 1

The biocompatibility of TNTs with and without a highly or-dered nanoporous layer was assessed using osteoblast-likecells (SaOS2). The relationships between the topography,the surface energy and the biocompatibility of TNTs weredetermined.

EXPERIMENTS

Cp–Ti plates (Grade 2, purity > 99.9 wt %) with sizes of10 3 10 3 0.5 mm3 were used as starting materials. The Tiplates were ground with 600 and 1200 grit silicon carbidepapers, and then ultrasonically cleaned in distilled water,ethanol and acetone for 5 min progressively. TNTs with ahighly ordered nanoporous layer (termed TNTs-1) were fab-ricated using a two-step anodization process. First, Ti plateswere anodized in an ethylene glycol based electrolyte with0.25 wt % NH4F and 2 wt % distilled water (type 1 electro-lyte) at a voltage of 30 V (DC power supply, BK 9124, BKPrecision, US) for 1 h. The as-anodized samples then wereultrasonicated in acetone for 10 min. After that, the Ti sam-ples were anodized in the type-1 electrolyte at 30 V for 3 hto obtain TNTs-1 samples. TNTs without a nanoporous layer(termed TNTs-2) were only anodized in ethylene glycol elec-trolyte with 0.25 wt % NH4F and 10 wt % distilled water(type-2 electrolyte) at 30 V for 3 h. After anodizing, all as-anodized samples were annealled at 500�C for 3 h in a muf-fle furnace with a preheating rate of 5�C min21.

The phase structure of the TNTs was analyzed usingX-ray diffraction (TF-XRD, X’pert pro-MPD, PANalytical, theNetherlands). The microstructure was observed using Scan-ning Electron Microscopy (SEM, Supra 55 VP, Zeiss, Ger-many). Atomic force microscopy (AFM, Cypher, AsylumResearch, USA) was used to measure the absolute roughness(Ra) and to observe the surface topographies. The contactangle was measured using a contact angle tester (KSV Cam101, KSV Instruments, Finland). All samples were stored inthe testing room for 24 h in advance to minimize the effectsof temperature and humidity. Drops of ultrapure distilledwater and ethylene glycol were delivered onto the specimensurface by a syringe giving the same drop size (5 lL). Thecontact angle was measured after 10 s and repeated at leastfive times for each group of samples. The surface energywas calculated using a theory of permanent dipole interac-tions between the solid and liquid according to the Owens–Wendt (OW) method given by29,30

ð1þ cos uÞgL ¼ 2ðffiffiffiffiffiffiffiffiffiffigdLg

dS

qþ

ffiffiffiffiffiffiffiffiffiffigpLg

pS

qÞ (1)

gS ¼ gds þ gp

s (2)

where the subscripts L and S represent the liquid and solid,respectively; the supscripts d and p represent the dispersivecomponent and polar component; u is the contact anglebetween a liquid droplet and solid surface; gL is the surfacetension of the liquid L; gS is the surface energy of the solid.The surface tension, dispersive component and polar com-ponent for water are 72.8, 21.8, and 51.0 mJ m22, and forethylene glycol 48.0, 29.0, and 19.0 mJ m22, respectively.30

According to Balaur et al.’s work,31 the OW approach can beapplicable when describing the wetting ability of the surfaceof TNTs, and the influence of nanotubes were thought to bethe surface roughness.

All samples for cell culture were sterilized in a mufflefurnace at 180�C for 3 h after the wash process describedabove. The samples were placed in a well in the cell cultureplate. SaOS2 cells (Barwon Biomedical Research, GeelongHospital, Victoria, Australia) were seeded on the surface ofTNTs that had different nanotopography and cp–Ti samplesusing a cell density of 1 3 104 cells per well (100 mm2).MTS assay was used to measure the in vitro proliferationof the SaOS2 cells. The cell morphology was observed usinga confocal microscopy (Leica SP5, Leica Microsystems,Germany) and SEM after cell culturing.

For the confocal microscopy observation, the cell-seededsamples after cell culture were fixed in paraformaldehyde,then permeabilized with triton-X 100 in phosphate-bufferedsaline (PBS) (Sigma–Aldrich, Australia) for 10-min each atroom temperature. The samples were then stained with 1%phalloidin and 40-6-diamidino-2-phenylindole for 40 min atroom temperature. Three washes by PBS were includedbetween each of the steps. The stained samples were storedin PBS at 4�C until required. The confocal microscopy obser-vations of the samples were conducted within a week ofstaining. For the SEM observation of the cell morphology,cells were dehydrated by immersing in buffer solution inwhich the ethanol concentrations were increased progre-sively every 10 min to 60, 70, 80, 90, and 100% andfollowed by chemical drying using hexamethyldisilazane(HMDS, Sigma–Aldrich, Australia) for 10 min. A gold layerwas deposited on the samples prior to SEM observation.

In all cases, one-way analysis of variance was employedto evaluate the significant difference in the data, and the sta-tistical difference was thought to be significant at p < 0.05.

RESULTS

Figure 1 shows the topographies of TNTs-1 and TNTs-2observed using AFM. TNTs-1 showed a flat surface with bothconcave and convex structures [Fig. 1(b)]. TNTs-2 exhibited acone-like pattern structure [Fig. 1(c)] whilst cp–Ti showed atypical pattern of metal surface after sanding [Fig. 1(a)]. Theroughness (Ra) of cp-Ti, TNTs-1, and TNTs-2 were 111.22 6

3.42, 92.01 6 5.22, and 182.48 6 11.57 nm, respectively[shown in Fig. 1(d)]. TNTs-2 exhibited the roughest surface.

Figure 2 shows the topographies of TNTs with and with-out a nanoporous layer (TNTs-1 and TNTs-2). It can be seenthat the top of TNTs-1 was evenly covered by a highlyordered nanoporous layer with a thickness of about 20 nm[Fig. 2(a,e)]. The diameter of the nanopores in this layerwas �100 nm. The diameter of the nanotubes below thenanoporous layer was �70 nm. The length of TNTs-1 was�5 lm. The diameter and length of TNTs-2 were �100 nmand 4 lm, respectively [Fig. 2(b,f)]. The surface of TNTs-2showed a cone-like pattern of TNTs as shown in Figure1(c). As it was covered by a nanoporous layer, the surfaceof TNTs-1 was flatter than that of TNTs-2 [Fig. 2(c,d)].

2 WANG ET AL. BIOCOMPATIBILITY OF TiO2 NANOTUBES WITH DIFFERENT TOPOGRAPHIES

To understand the formation of the highly ordered nano-porous layer on the top of TNTs when fabricating TNTs-1using a two-step anodization process, Ti plate was first ano-dized in type-1 electrolyte using a voltage of 30 V for 1 h toform a TNT layer on it. The as-anodized sample then wasultrasonicated in acetone for 10 min to remove the layer ofTNTs on the top of the Ti substrate. Figure 3(a) shows thesurface of the Ti substrate after removing the TNTs layer. Itcan be seen that a layer with highly ordered concaves(termed the barrier layer) was formed on Ti substrate. Thislayer consists of amorphous titanium dioxide.32 The TNTslayer conjunct with the barrier layer was carefully peeledoff from the Ti substrate to observe its microstructure. Fig-ure 3(b) shows the TNTs layer, conjunct with the barrierlayer, viewed from the bottom. It can be seen that there wasthe barrier layer [indicated by an arrow in Fig. 3(b)]between TNTs and the Ti substrate after anodization. Thebarrier layer showed highly ordered concaves, and provideda template for the second time anodization process. Thus,after removing the TNTs layer formed in the previous anod-ization process, the Ti substrate with such barrier layer wasagain anodized in the type-1 electrolyte at 30 V for 3 h.TNTs were produced underneath the surface with highlyordered concaves to obtain TNTs-1 with a thickness of�20 nm (shown in Fig. 2).

The XRD patterns of TNTs-1 and TNTs-2 before and af-ter annealing are shown in Figure 4. It can be seen thatboth TNTs demonstrated the same phase structure beforeannealing and exhibited the same phase transformationafter annealing. The main phase of both of the as-anodizedTNTs was Ti with an amorphous TiO2 structure. Afterannealing, an anatase phase appeared in TNTs.

Figure 5 shows the water droplets on the surfaces ofcp–Ti and the annealed TNTs. The shapes of the water drop-lets on surfaces of TNTs-1 and TNTs-2 appeared flatter thanthose on cp–Ti, which indicated that TNTs were much morehydrophilic than cp–Ti. The water contact angles of cp–Ti,TNTs-1 and TNTs-2 were 68.36� 6 2.67� , 32.09� 6 1.08�,and 11.06� 6 0.79�, respectively. The ethylene glycol contactangles of cp-Ti, TNTs-1, and TNTs-2 were 62.61� 6 2.55�,23.04� 6 2.26�, and 17.12� 6 1.73�, respectively.

The surface energy of cp–Ti, TNTs-1, and TNTs-2 wascalculated according to the OW method and listed inTable I. The surface energy increased significantly aftersynthesizing the TiO2 nanotube layer on Ti substrates. Thesurface energy of cp–Ti was 26.78 6 0.21 mJ m22, whichwas significantly lower than that of TNTs. TNTs-2 exhibiteda higher surface energy (58.19 6 2.32 mJ m22), comparedto TNTs-1 (45.56 6 1.13 mJ m22).

A biocompatibility assessment was carried out using anin vitro cell culture for the TNTs-1 and TNTs-2 while cp–Tiwas used as the control group. The cell adhesion density ofosteoblast-like cells to TNTs-1 and TNTs-2 after cell cultur-ing for 7 and 14 days is shown in Figure 6. It can be seenthat the cell numbers on both TNTs increased with theextending of cell culture time, and were significantly higherthan that on the substrate of cp–Ti, indicating that bothTNTs are of higher biocompatibility than cp–Ti andpreferred for cells adhesion. The cell numbers on TNTs-2were slightly higher than those of TNTs-1 after cell culturingfor 7 and 14 days.

The morphology of the SaOS2 cells on TNTs-1 andTNTs-2 after culturing for 1, 7, and 14 days was observedusing confocal microscopy (Fig. 7) and SEM (Fig. 8). Cells

FIGURE 1. AFM images show topographies of (a) cp-Ti, (b) as-anodized TNTs-1 and (c) as-anodized TNTs-2 and (d) their surface roughness.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2013 VOL 00A, ISSUE 00 3

grew and spread on all samples healthily. After cell cultur-ing for 1 day, the cells on TNTs-1 and TNTs-2 were flatterand showed greater spreading extension than those on cp–Ti, which meant that the cells preferred to adhere to andgrow on the surface of the TNTs rather than on the cp–Ti

[Fig. 7(a,d,g)]. The SaOS2 cells attached and spread well onthe surfaces of both TNTs after culturing for 7 and 14 days[Fig. 7(b,c,e,f,h,i)]. The number of SaOS2 cells on the cp–Tiwas lower than those on both TNTs. This is consistent withthe results of the MTS assay (Fig. 6). A morphology study of

FIGURE 2. Top views of TNTs-1 at (a) high and (c) low magnifications and TNTs-2 at (b) high and (d) low magnification, and cross-sectional

views of (e) TNTs-1 and (f) TNTs-2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


the cells further confirmed that the TNTs both with andwithout highly nanoporous layers had greater biocompati-bility than cp–Ti. High-resolution images of the filopodia af-ter cell culturing for 7 days are shown in Figure 8. Theextension of cells on the surface of both TNTs was clearlygreater than on the cp–Ti samples. As shown in Figure8(b,c), filopodia anchored soundly to the nanotubes on thesurface of TNTs-1 and TNTs-2.

DISCUSSION

A serial anodization reactions33 occur when pure Ti isimmersed in electrolyte with F2 ions applied with an elec-trical field according to Eqs. ((3)) and ((4)) given by:

Ti12H2O! TiO214H114e2 (3)

TiO216F214H1 ! ½TiF6�2212H2O (4)

There are two main electric-assisted reactions in the for-mation of TNTs.34 Equation (3) described the formation ofTiO2. The second process of field-assisted dissolution ofTiO2 was dependent on the reaction with F2 ions accordingto Eq. (4). At the early stage of the anodization processusing an ethylene glycol based electrolyte with F2 ions andwater, a compact oxide layer (random top layer) forms onthe mouths of TNTs.35 Meanwhile, a transition layer contain-ing F- ions is also produced on the bottom of TNTs, which isthe barrier layer with highly ordered concaves.36 The move-ment of F2 ions is driven by the electrical field and by thedifference in concentration. In the one-step anodization pro-cess without interruption, a barrier layer is formed betweenthe top TNTs layer and the substrate (Fig. 3), thus achievinga balance between the etching process with F2 ions and theformation of TiO2. In the two-step anodization process, afterthe first anodization process, the ultrasonic washing in ace-tone removes the random top TNTs layer and the highly or-dered barrier layer is kept on the Ti substrate [Fig. 3(a)]. Inthe second anodization, the driven force for the F2 ions inthe barrier layer decreases, as this layer is composed of

passive TiO2, which is a semiconductor. The barrier layerwith highly ordered concaves is used as a template for thesecond anodization. Thus, the highly ordered nanoporouslayer is fabricated on top of the TNTs to obtain TNTs-1during the second anodization.

The content of distilled water plays a vital role in theprocess of TiO2 formation and in the diffusion of the[TiF6]

22.33 The TiO2 layer can be etched and dissolved at ahigh speed in electrolyte 2, and this can further influencethe morphology and structure of the TiO2 nanotubes. First,the nanoporous layer can be etched off thoroughly, so thatthe upper mouths of TNTs-2 were totally open without ananoporous layer. Second, the alignment of nanotubes inTNTs-2 was not as compact stacked as TNTs-1 as shown inthe cross-sectional images [Fig. 2(e,f)]. This was becausemore water in the electrolyte results in a higher diffusionrate of ions in the formation of TiO2 nanotubes.

FIGURE 3. SEM images showing (a) the barrier layer on the Ti substrate after removing the TNT layer and (b) the barrier layer conjunct with the

TNTs layer viewed from the bottom. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 4. XRD patterns of (1) annealed TNTs-1, (2) annealed TNTs-2,

(3) as-anodized TNTs-1 and (4) as-anodized TNTs-2. [Color figure can

be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

ORIGINAL ARTICLE


Cells contact and spread on the surface of biomedicalmaterials that absorb water and proteins from cell culturefluid, rather than a bare surface.37 The wetting property iscrucial for protein adsorption, cell adhesion and spread,which is significantly influenced by the status of the surfacesuch as the topography and surface energy.38 TiO2 nano-tubes with diameters from 20 to 100 nm are usually hydro-philic due to their nanotubular structures.31 An annealingprocess transfers the amorphous TiO2 to anatase structureand improves the hydrophilicity,33 resulting in an increasein surface energy. In this study, the TNTs-1 exhibited alarger contact angle with a water droplet compared to theTNTs-2 samples. This would be because that nanotubes onTNTs-2 showed a much looser distribution than those onthe TNTs-1 [Fig. 1(b,c)], which gave the surface of the TNTs-2 a greater roughness than the TNTs-1 [Fig. 1(d)] and morespace for water to penetrate.

The cell adhesion phase is surface anchoring through fi-bronectin and vitronectin for the formation of focal pointsat cell membrane integrins.39 Filopodia and finger-like pro-trusions are then produced to enable sensing of the opti-mum anchorage and spreading on the surface. Thetopography of TNTs affected not only the adhesion but alsothe shape of the cells. As shown in previous research, TNTswith diameters ranging from 70 to 100 nm have resulted inelongated cellular morphology13 and have further improvedthe proliferation of cells. In this study, the filopodia of cellson TNTs-2 were significantly longer than those on theTNTs-1 and cp-Ti. This suggests that TNTs-2 with higherroughness supplies larger volume and surface for theabsorption of water, nanoparticles and proteins, whichattracted anchorage of filopodia, and cells prefer the surfaceof the TNTs-2. Furthermore, TNTs possessed rougher andsharper convex edges compared to the cp-Ti samples(Fig. 2). The osteoblast cells were positively charged withCa21,40 and the sharp edges of the nanotubes were negative

charged and attracted cells, which enhances the adhesion ofcells to the implant surface.12 In the present study, the via-bility of cells on the surface of TNTs was greater than thoseon the cp–Ti.

It has also been reported that cell adhesion can beenhanced by increasing both the wettability and the surfaceenergy.41 Mesenchymal stem cells showed a worse biologi-cal performance on a super hydrophobic surface.42 In thepresent study, the lower water contact angle on the surfacesof TNTs possessed a better hydrophilic ability than cp–Ti.Furthermore, the TNTs layer with the higher surface energysignificantly improved the in vitro proliferation of osteo-blast-like cells, as indicated by the MTS results in Figure 6.As the surface energy of TNTs-2 was higher than that ofTNTs-1, the cell number on TNTs-2 was slightly higher thanthat on TNTs-1 after culturing for 7 and 14 days. Theresponse of SaOS2 cells to TNTs depended on their basicphysical and chemical features. Based on the above discus-sion, it can be concluded that the topography and surfaceenergy play important roles in the adhesion and prolifera-tion of SaOS2 cells.

In a comparison of the morphological differencebetween TNTs-1 and TNTs-2 (Fig. 2), TNTs-2 demonstratedmuch sharper edges than TNTs-1. As shown in Figure 8,SaOS2 cells in TNTs-1 interacted closely with each otherthrough the filopodia. SaOS2 cells maintained a round shapeon the highly ordered nanoporous layer, and the cell filopo-dia did not extend far away. However, on TNTs-2, more

FIGURE 5. Water droplets on the surfaces of (a) pure Ti, (b) TNTs-1, and (c) TNTs-2.

TABLE I. Surface Energy of Ti and TNTs

cd mJ m22 cp (mJ m22) g (mJ m22)

Pure Ti 9.78 6 0.07 17.00 6 0.16 26.78 6 0.21TNTs-1 18.58 6 .15 29.98 6 1.09 45.56 6 1.13TNTs-2 10.45 6 0.42 47.74 6 2.36 58.19 6 2.32

Data are shown in the format of mean 6 standard error. FIGURE 6. Cell adhesion after cell culturing for 7 and 14 days.


FIGURE 7. Confocal images showing cell growth on cp–Ti (a, d, and g), TNTs-1(b, e, and h) and TNTs-2 (c, f, and i) after cell culturing for 1, 7,

and 14 days, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 8. Filopodia on the surfaces of (a) cp-Ti, (b) TNTs-1, and (c) TNTs-2 after cell culturing for 7 days (inserted images at low magnification).

ORIGINAL ARTICLE


filopodia attached to the TNTs rather than interacting withothers [Fig. 8(c)]. As shown in Figure 8(b,c), the cells on thesurfaces of TNTs-1 and TNTs-2 spread through protoplasmicprocesses and stimulated more filopodia connecting withTNTs, which indicated better bonding with the surface thanbare cp-Ti; and TNTs-1 and TNTs-2 displayed better bioac-tivity than cp-Ti.

CONCLUSIONS

Titania nanotubes (TNTs) were fabricated both with andwithout a highly ordered nanoporous layer on top surfaceusing both two-step and one-step anodizing processes. Theeffects of TNTs with different topographies on osteoblast-like cell behaviors such as shaping, adhesion, proliferationand the cell fate were observed. The roughness and surfaceenergy of the TNTs without the highly ordered nanoporouslayer on the top surface were higher than those of the TNTswith such a layer. TNTs enhanced the proliferation andadhesion of osteoblast-like cells. TNTs without a highly or-dered nanoporous top layer exhibited better biocompatibil-ity than TNTs covered by such a layer. Cell morphologyobservation indicated that the SaOS2 cells that adhered tothe TNTs without a highly ordered nanoporous layer exhib-ited a longer filopodia than both TNTs with a highly orderednanoporous layer and cp–Ti.

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ORIGINAL ARTICLE


biocompatibility of tio 2 nanotubes with different topographies

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